Handbook of Olfaction and Gustation, Second Edition (Neurological Disease and Therapy)

Handbook of Olfaction and Gustation Second Edition Revised and Expanded edited by

Richard L. Doty University of Pennsylvania Philadelphia, Pennsylvania, U.S.A.

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NEUROLOGICAL DISEASE AND THERAPY

Advisory Board

Louis R. Caplan, M.D.

William C. Koller, M.D.

Professor of Neurology Harvard University School of Medicine Beth Israel Deaconess Medical Center Boston, Massachusetts

Mount Sinai School of Medicine New York, New York

John C. Morris, M.D.

Bruce Ransom, M.D., Ph.D.

Fnedman Professor of Neurology Co-Director, Alzheimer's Disease Research Center Washington University School of Medicine St Louis, Missouri

Warren Magnuson Professor Chair, Department of Neurology University of Washington School of Medicine Seattle, Washington

Kapil Sethi, M.D.

Mark Tuszynski, M.D., Ph.D.

Professor of Neurology Director, Movement Disorders Program

Associate Professor of Neurosciences Director, Center for Neural Repair University of California-San Diego

Medical College of Georgia Augusta, Georgia

La Jolla, California

1. Handbook of Parkinson's Disease, edited by William C. Koller 2. Medical Therapy of Acute Stroke, edited by Mark Fisher 3 Familial Alzheimer's Disease Molecular Genetics and Clinical Perspectives, edited by Gary D. Miner, Ralph W. Richter, John P 8/ass, Jimmie L. Valentine, and Linda A. Winters-Miner 4 Alzheimer's Disease. Treatment and Long-Term Management, edited by Jeffrey L. Cummmgs and Bruce L Miller 5. Therapy of Parkinson's Disease, edited by William C Koller and George Paulson 6. Handbook of Sleep Disorders, edited by Michael J Thorpy 1 Epilepsy and Sudden Death, edited by Claire M. Lathers and Paul L Schraeder 8. Handbook of Multiple Sclerosis, edited by Stuart D Cook 9. Memory Disorders Research and Clinical Practice, edited by Takehiko Yanagihara and Ronald C. Petersen 10. The Medical Treatment of Epilepsy, edited by Stanley R. Resor, Jr, and Henn Kuti 11. Cognitive Disorders Pathophysiology and Treatment, edited by Leon J Thai, Walter H Moos, and Elkan R Gamzu 12. Handbook of Amyotrophic Lateral Sclerosis, edited by Richard Alan Smith 13. Handbook of Parkinson's Disease' Second Edition, Revised and Expanded, edited by William C Koller 14 Handbook of Pediatric Epilepsy, edited by Jerome V Murphy and Fereydoun Dehkhargham 15 Handbook of Tourette's Syndrome and Related Tic and Behavioral Disorders, edited by Roger Kurlan 16 Handbook of Cerebellar Diseases, edited by Richard Lechtenberg 17 Handbook of Cerebrovascular Diseases, edited by Harold P. Adams, Jr. 18 Parkmsonian Syndromes, edited by Matthew B Stem and William C Koller 19 Handbook of Head and Spine Trauma, edited by Jonathan Greenberg 20. Brain Tumors' A Comprehensive Text, edited by Robert A Morantz and John W. Walsh 21. Monoamine Oxidase Inhibitors in Neurological Diseases, edited by Abraham Lieberman, C. Warren Olanow, Moussa B H Youdim, and Keith Tipton 22 Handbook of Dementing Illnesses, edited by John C Moms 23. Handbook of Myasthema Gravis and Myasthenic Syndromes, edited by Robert P Lisak 24. Handbook of Neurorehabilitation, edited by David C Good and James R Couch, Jr. 25. Therapy with Botulinum Toxin, edited by Joseph Jankovic and Mark Hallett 26. Principles of Neurotoxicology, edited by Louis W. Chang 27 Handbook of Neurovirology, edited by Robert R. McKendall and William G Stroop 28 Handbook of Neuro-Urology, edited by David N Rushton 29. Handbook of Neuroepidemiology, edited by Philip B. Gorelick and Milton Alter 30 Handbook of Tremor Disorders, edited by Leslie J Findley and William C. Koller 31. Neuro-Ophthalmological Disorders. Diagnostic Work-Up and Management, edited by Ronald J Tusa and Steven A. Newman 32. Handbook of Olfaction and Gustation, edited by Richard L Doty

33. Handbook of Neurological Speech and Language Disorders, edited by Howard S. Kirshner 34. Therapy of Parkinson's Disease Second Edition, Revised and Expanded, edited by William C. Kollerand George Paulson 35. Evaluation and Management of Gait Disorders, edited by Barney S. Spivack 36. Handbook of Neurotoxicology, edited by Louis W. Chang and Robert S. Dyer 37 Neurological Complications of Cancer, edited by Ronald G. Wiley 38. Handbook of Autonomic Nervous System Dysfunction, edited by Amos D. Korczyn 39. Handbook of Dystonia, edited by Joseph King Ching Tsui and Donald B. Calne 40. Etiology of Parkinson's Disease, edited by Jonas H. Ellenberg, William C. Koller, andJ. William Langston 41. Practical Neurology of the Elderly, edited by Jacob I. Sage and Margery H. Mark 42. Handbook of Muscle Disease, edited by Russell J. M. Lane 43. Handbook of Multiple Sclerosis: Second Edition, Revised and Expanded, edited by Stuart D. Cook 44. Central Nervous System Infectious Diseases and Therapy, edited by Karen L. Roos 45. Subarachnoid Hemorrhage: Clinical Management, edited by Takehiko Yanagihara, David G. Piepgras, and John L. D. Atkmson 46. Neurology Practice Guidelines, edited by Richard Lechtenberg and Henry S. Schutta 47. Spinal Cord Diseases: Diagnosis and Treatment, edited by Gordon L Engler, Jonathan Cole, and W. Louis Merlon 48. Management of Acute Stroke, edited by Ashfaq Shuaib and Larry B. Goldstem 49. Sleep Disorders and Neurological Disease, edited by Antonio Culebras 50. Handbook of Ataxia Disorders, edited by Thomas Klockgether 51. The Autonomic Nervous System in Health and Disease, David S. Goldstein 52. Axonal Regeneration in the Central Nervous System, edited by Nicholas A. Ingoglia and Marion Murray 53 Handbook of Multiple Sclerosis. Third Edition, edited by Stuart D Cook 54. Long-Term Effects of Stroke, edited by Julien Bogousslavsky 55. Handbook of the Autonomic Nervous System in Health and Disease, edited by C. Liana Bolts, Julio Licinio, and Stefano Govoni 56. Dopamme Receptors and Transporters: Function, Imaging, and Clinical Implication, Second Edition, edited by Anita Sidhu, Marc Laruelle, and Philippe Vernier 57. Handbook of Olfaction and Gustation: Second Edition, Revised and Expanded, edited by Richard L. Doty 58. Handbook of Stereotactic and Functional Neurosurgery, edited by Michael Schulder 59. Handbook of Parkinson's Disease: Third Edition, edited by Rajesh Pahwa, Kelly E. Lyons, and William C. Koller

Additional Volumes in Preparation

Clinical Neurovirology, edited byAvi Nath and Joseph R. Berger

Foreword

In the rise of modern neuroscience during the last century, the great sensory systems—vision above all, but also the somatosensory systems and audition—played the leading roles in the elucidation of principles underlying the neural mechanisms of perception. Work on the senses of taste and smell lagged behind, hampered by the difficulties of controlling the stimuli in precise ways and by the belief that these senses were of minor importance to humans. No more! That era of chemosensory darkness ended during the past two decades because of illumination from new studies at many levels of these systems. Gene families that express receptors for chemical stimuli have been identified. Membrane mechanisms of stimulus transduction and second messenger signaling have been revealed. Topographic patterns of the convergence of axons from the sensory cells onto higher levels in the sensory pathways have been mapped. A variety of methods have revealed that different odors elicit different activity patterns, which constitute virtual “odor images.” In the olfactory system, as in the other great sensory systems, stimulus space (in this case, the multidimensional space of odor molecules) has now been mapped into two-dimensional neural space. The synaptic microcircuits in the olfactory bulb have attracted a new generation of electophysiologists from other fields. Using patch recordings, calcium imaging and advanced microscopy, they are analyzing the dendritic and synaptic properties of the microcircuits that process the odor images as the basis for perception. In addition to this revitalization of electrophysiological studies of the chemical senses, the neuroscience community has been attracted to the extraordinary plasticity of these systems, evidenced, in part, by the ongoing turnover of taste and olfactory cells, and the constant generation of new interneurons from the anterior migratory stream at the base

of the brain. These systems are therefore on the cutting edge of current research on stem cells and neurogenesis in the brain. New psychophysical studies challenge the traditional view of human olfaction as weak, and suggest instead that our ability to perceive low levels of odorants may be as good or better than that of macrosmats such as rodents and carnivores. Moreover, such studies have expanded our understanding of the complexity of the chemical senses, and, along with a plethora of basic science studies, have demonstrated that these senses are intimately involved in a wide range of medical disorders. Indeed, the olfactory system may provide early indicators for disease states such as neurodegeneration and schizophrenia. We now have a better understanding of the significance of the olfactory and gustatory systems for such critical human behaviors as infant nutrition, the prevalence of obesity in developed countries, and the strong links between the chemical senses and emotion and memory. All of these developments and many more are covered in the second edition of this widely recognized book, the largest compendium of data on the chemical senses published to date. Richard Doty’s introduction provides a masterly overview of the rapidly evolving events in these fields, and the ensuing chapters provide a wealth of information on topics ranging from basic anatomy, physiology, and clinical disorders of the chemical senses to advances in functional imaging, molecular neurobiology, human and animal psychophysics, and even olfactory system cybernetics. As the fields of gustation and olfaction continue their strong growth, there will be an increasing need for a source to which one can go for orientation to the broad range of research involved and critical assessments of progress, problems, and opportunities. This new edition fills those needs iii

iv

Foreword

superlatively for a wide range of readers: neuroscientists, organic chemists, toxicologists, biomedical engineers, psychologists, and a variety of clinicians, as well as the interested layperson. Gordon M. Shepherd, M.D., D. Phil. Professor of Neuroscience Yale University New Haven, Connecticut, U.S.A.

Preface

Since the publication of the first edition of the Handbook of Olfaction and Gustation in 1995, advances in chemosensory science have been staggering. Indeed, during this period the chemical senses have become a central element of the field of modern neuroscience, largely because of their regenerative capacities, integral association with stem cell research, and unique transduction processes. As a consequence of the proliferation of commercially available olfactory tests, olfaction is now routinely and quantitatively evaluated in most major medical centers, as well as within the food, beverage, cosmetic, and energy (e.g., gas works) industries. Of particular relevance to the physician is the fact that decreased smell function is likely the first clinical manifestation of Alzheimer’s disease (AD) and idiopathic Parkinson’s disease (PD). Indeed, accurate assessment of olfaction can aid in the “preclinical” identification of individuals at risk for these disorders. Such assessment can also aid in differential diagnosis, since diseases often misdiagnosed as AD or PD (e.g., major affective disorder, progressive supranuclear palsy) are unaccompanied by meaningful olfactory loss. The second edition of the Handbook represents the largest collection of basic, clinical, and applied knowledge on the chemical senses ever compiled in one volume, with contributions from over 80 of the world’s leading researchers. The material in this up-to-date treatise has been tailored to be of value to a wide range of medical specialists, as well as to basic scientists working in academics, industry, and government. Because the information is presented in a straightforward manner, this volume can serve as a textbook for graduate students, medical students, and

postdoctoral fellows from numerous disciplines. The chapters are conveniently arranged into three major sections corresponding to olfaction, gustation, and other chemosensory systems and, with the exception of the last section, are subdivided into (A) anatomy and neurobiology, (B) functional measurement, ontogeny, and genetics, and (C) clinical applications and perspectives. Unlike the first edition, this edition contains an author index that makes it possible for researchers and others to quickly find references and sections based on individual contributions. As in the first edition, historical perspective and clinical relevance have been emphasized, but not at the expense of basic science. The book has been expanded from 38 to 48 chapters, so as to take into account major growth in a number of fields, including neuroscience, functional imaging, cybernetics, toxicology, structure–activity assessment, molecular biology, and animal behavior. I am grateful to the contributors, who have been a model of objectivity and scholarship in the development of their chapters, and who have graciously taken into consideration my often extensive editorial suggestions and criticisms. I am also indebted to the staff of Marcel Dekker, Inc., particularly Jinnie Kim, Assistant Acquisitions Editor, and Ann Pulido, Production Editor, who have patiently and painstakingly worked with me to ensure a volume of the highest quality. Without the support of the following grants from the National Institutes of Health, this work would have never been accomplished: PO1 DC 00161, RO1 DC 04278, RO1 DC 02974, and RO1 AG 27496. Richard L. Doty

v

Contents

Foreword Gordon M. Shepherd Preface Richard L. Doty Contributors Introduction and Historical Perspective I. A.

Richard L. Doty

iii v xi xv

OLFACTION Anatomy and Neurobiology

1.

Anatomy of the Human Nasal Passages Dean M. Clerico, Wyatt C. To, and Donald C. Lanza

1

2.

Morphology of the Mammalian Olfactory Epithelium: Form, Fine Structure, Function, and Pathology Bert Ph. M. Menco and Edward E. Morrison

17

3.

Olfactory Mucosa: Composition, Enzymatic Localization, and Metabolism Xinxin Ding and Alan R. Dahl

51

4.

Molecular Neurobiology of Olfactory Transduction Cheil Moon and Gabriele V. Ronnett

75

5.

Neurogenesis in the Adult Olfactory Neuroepithelium Alan Mackay-Sim

93

6.

Developmental Anatomy of the Olfactory System Meng Inn Chuah, James E. Schwob, and Albert I. Farbman

115

7.

Anatomy and Neurochemistry of the Olfactory Bulb Igor L. Kratskin and Ottorino Belluzzi

139

8.

Central Olfactory Structures Thomas A. Cleland and Christiane Linster

165

9.

Sensory Physiology of Central Olfactory Pathways Donald A. Wilson and Regina M. Sullivan

181

vii

viii

Contents

B. Functional Measurement, Ontogeny, and Genetics 10.

Psychophysical Measurement of Human Olfactory Function, Including Odorant Mixture Assessment Richard L. Doty and David G. Laing

203

11.

Electrophysiological Measurement of Olfactory Function Gerd Kobal

229

12.

Functional Neuroimaging of Human Olfaction Noam Sobel, Bradley N. Johnson, Joel Mainland, and David M. Yousem

251

13.

Structure–Odor Relationships: A Modern Perspective Luca Turin and Fumiko Yoshii

275

14.

Olfactory System Cybernetics: Artificial Noses Krishna C. Persaud

295

15.

Olfaction and the Development of Social Behavior in Neonatal Mammals Richard H. Porter and Benoist Schaal

309

16.

Genetics of Olfactory Perception Nancy L. Segal and Tari D. Topolski

329

17.

Mammalian Pheromones: Fact or Fantasy? Richard L. Doty

345

18.

Psychophysical Evaluation of Olfaction in Nonhuman Mammals Lloyd Hastings

385

19.

Methods for Determining Odor Preferences in Nonhuman Mammals Richard L. Doty

403

20.

Olfactory Memory Aras Petrulis and Howard Eichenbaum

409

C. Clinical Applications and Perspectives 21.

Nasal Patency and the Aerodynamics of Nasal Airflow: Measurement by Rhinomanometry and Acoustic Rhinometry, and the Influence of Pharmacological Agents Richard E. Frye

439

22.

Clinical Disorders of Olfaction Claire Murphy, Richard L. Doty, and Heather J. Duncan

461

23.

Odor Perception in Neurodegenerative Diseases Richard L. Doty

479

24.

Olfactory System Neuropathology in Alzheimer’s Disease, Parkinson’s Disease, and Schizophrenia Gregory S. Smutzer, Richard L. Doty, Steven E. Arnold, and John Q. Trojanowski

503

Contents

25.

Multiple Chemical Intolerance Claudia S. Miller

26.

The Olfactory System and the Nasal Mucosa as Portals of Entry of Viruses, Drugs, and Other Exogenous Agents into the Brain Harriet Baker and Mary Beth Genter

ix

525

549

27.

Influence of Environmental Toxicants on Olfactory Function Lloyd Hastings and Marian L. Miller

575

28.

Evaluation of Olfactory Deficits by Structural Medical Imaging Cheng Li, Richard L. Doty, David W. Kennedy, and David M. Yousem

593

29.

Plasticity Within the Olfactory Pathways: Influences of Trauma, Deprivation, Stem Cells, and Other Factors Joel Maruniak

30.

II.

Head Injury and Olfaction Richard M. Costanzo, Laurence J. DiNardo, and Evan R. Reiter

615

629

GUSTATION

A.

Anatomy and Neurobiology

31.

Saliva: Its Role in Taste Function Robert M. Bradley and Lloyd M. Beidler

639

32.

Morphology of the Peripheral Taste System Martin Witt, Klaus Reutter, and Inglis J. Miller, Jr.

651

33.

Central Taste Anatomy and Neurophysiology Edmund T. Rolls and Thomas R. Scott

679

34.

Molecular Physiology of Gustatory Transduction Timothy A. Gilbertson and Robert F. Margolskee

707

35.

Gustatory Neural Coding David V. Smith and Thomas R. Scott

731

36.

Development of the Taste System: Basic Neurobiology Charlotte M. Mistretta and David L. Hill

759

B. 37.

Functional Measurement, Ontogeny, and Genetics Contemporary Measurement of Human Gustatory Function Marion E. Frank, Thomas P. Hettinger, Michael A. Barry, Janneane F. Gent, and Richard L. Doty

783

x

Contents

38.

Human Perception of Taste Mixtures Hendrik N. J. Schifferstein

805

39.

The Ontogeny of Human Flavor Perception Judith R. Ganchrow and Julie A. Mennella

823

40.

Genetics of Human Taste Perception Adam Drewnowski

847

41.

Psychophysical Evaluation of Taste Function in Nonhuman Mammals Alan C. Spector

861

C.

Clinical Applications and Perspectives

42.

Nutritional Implications of Taste and Smell Richard D. Mattes

881

43.

Conditioned Taste Aversions Kathleen C. Chambers and Ilene L. Bernstein

905

44.

Clinical Disorders Affecting Taste: Evaluation and Management Steven M. Bromley and Richard L. Doty

935

45.

Head Injury and Taste Richard M. Costanzo, Laurence J. DiNardo, and Evan R. Reiter

959

III.

OTHER CHEMOSENSORY SYSTEMS

46.

The Vomeronasal Organ Peter A. Brennan and Eric B. Keverne

967

47.

Trigeminal Chemosensation Richard L. Doty and J. Enrique Cometto-Munˇiz

981

48.

The Structure and Function of the Nervus Terminalis Marlene Schwanzel-Fukuda and Donald W. Pfaff

Author Index Subject Index

1001

1027 1103

Contributors

Meng Inn Chuah, Ph.D. Department of Anatomy and Physiology, University of Tasmania, Hobart, Australia

Steven E. Arnold, M.D. Smell and Taste Center and Departments of Psychiatry and Neurology, University of Pennsylvania, Philadelphia, Pennsylvania, U.S.A.

Thomas A. Cleland, Ph.D. Department of Neurobiology and Behavior, Cornell University, Ithaca, New York, U.S.A.

Harriet Baker, Ph.D. Department of Neurology and Neuroscience, The Burke Medical Research Institute, Weill Medical College, Cornell University, White Plains, New York, U.S.A.

Dean M. Clerico, M.D. Valley ENT, Forty Fort, Pennsylvania, U.S.A.

Michael A. Barry, Ph.D. Division of Neurosciences, Department of Oral Diagnosis, School of Dental Medicine, University of Connecticut Health Center, Farmington, Connecticut, U.S.A.

J. Enrique Cometto-Muñiz, Ph.D. Chemosensory Perception Laboratory, Department of Surgery (Otolaryngology), University of California, San Diego, La Jolla, California, U.S.A.

Lloyd M. Beidler, Ph.D. Department of Biological Science, Florida State University, Tallahassee, Florida, U.S.A.

Richard M. Costanzo, Ph.D. Department of Physiology, Virginia Commonwealth University, Richmond, Virginia, U.S.A.

Ottorino Belluzzi, Ph.D. Department of Biology, University of Ferrara, Ferrara, Italy

Alan R. Dahl, Ph.D. Ohio, U.S.A.

Ilene L. Bernstein, Ph.D. Department of Psychology, University of Washington, Seattle, Washington, U.S.A.

Battelle Memorial Institute, Columbus,

Laurence J. DiNardo, M.D. Department of Otolaryngology Head and Neck Surgery, Virginia Commonwealth University, Richmond, Virginia, U.S.A.

Robert M. Bradley, M.D.S., Ph.D. Department of Biologic and Materials Science, School of Dentistry, University of Michigan, Ann Arbor, Michigan, U.S.A. Peter A. Brennan, Ph.D. Department of Animal Behaviour, University of Cambridge, Cambridge, United Kingdom

Xinxin Ding, Ph.D. Wadsworth Center, New York State Department of Health, and School of Public Health, State University of New York at Albany, Albany, New York, U.S.A.

Steven M. Bromley, M.D. Smell and Taste Center, University of Pennsylvania, and Department of Neurology, Thomas Jefferson University, Philadelphia, Pennsylvania, U.S.A.

Richard L. Doty, Ph.D. Smell and Taste Center and Department of Otorhinolaryngology: Head and Neck Surgery, University of Pennsylvania, Philadelphia, Pennsylvania, U.S.A.

Kathleen C. Chambers, Ph.D. Department of Psychology, University of Southern California, Los Angeles, California, U.S.A.

Adam Drewnowski, Ph.D. Nutritional Sciences Program, School of Public Health and Community Medicine, University of Washington, Seattle, Washington, U.S.A. xi

xii Heather J. Duncan, Ph.D. Department of Internal Medicine, University of Cincinnati College of Medicine, Cincinnati, Ohio, U.S.A. Howard Eichenbaum, Ph.D. Department of Psychology, Boston University, Boston, Massachusetts, U.S.A. Albert I. Farbman, D.M.D., Ph.D. Department of Neurobiology and Physiology, Northwestern University, Evanston, Illinois, U.S.A. Marion E. Frank, Ph.D. Division of Neurosciences, Department of Oral Diagnosis, School of Dental Medicine, University of Connecticut Health Center, Farmington, Connecticut, U.S.A. Richard E. Frye, M.D., Ph.D. Department of Neurology, Children’s Hospital, Boston, Massachusetts, U.S.A. Judith R. Ganchrow, Ph.D. Institute of Dental Sciences, The Hebrew University–Hadassah School of Dental Medicine Founded by the Alpha Omega Fraternity, Jerusalem, Israel Janneane F. Gent, Ph.D. Department of Epidemiology and Public Health, Yale University, New Haven, Connecticut, U.S.A. Mary Beth Genter, Ph.D. Department of Environmental Health, University of Cincinnati, Cincinnati, Ohio, U.S.A. Timothy A. Gilbertson, Ph.D. Department of Biology, Utah State University, Logan, Utah, U.S.A. Lloyd Hastings, Ph.D. Smell and Taste Center and Department of Otorhinolaryngology: Head and Neck Surgery, University of Pennsylvania, Philadelphia, Pennsylvania, U.S.A. Thomas P. Hettinger, Ph.D. Division of Neurosciences, Department of Oral Diagnosis, School of Dental Medicine, University of Connecticut Health Center, Farmington, Connecticut, U.S.A. David L. Hill, Ph.D. Department of Psychology, University of Virginia, Charlottesville, Virginia, U.S.A.

Contributors Gerd Kobal, M.D., Ph.D. Department of Pharmacology and Toxicology, University of Erlangen, Erlangen, Germany Igor L. Kratskin, M.D., Ph.D. Smell and Taste Center and Department of Otorhinolaryngology: Head and Neck Surgery, University of Pennsylvania, Philadelphia, Pennsylvania, U.S.A. David G. Laing, Ph.D. Centre for Advanced Food Research, University of Western Sydney, Sydney, Australia Donald C. Lanza, M.D. Department of Otolaryngology and Communicative Disorders, The Cleveland Clinic Foundation, Cleveland, Ohio, U.S.A. Cheng Li, M.D. Smell and Taste Center, and Department of Otorhinolaryngology, Head and Neck Surgery, University of Pennsylvania, Philadelphia, Pennsylvania, U.S.A. Christiane Linster, Ph.D. Department of Neurobiology and Behavior, Cornell University, Ithaca, New York, U.S.A. Alan Mackay-Sim, Ph.D. Centre for Molecular Neurobiology, Griffith University, Brisbane, Queensland, Australia Joel Mainland, Ph.D. Wills Neuroscience Institute and Department of Psychology, University of California, Berkeley, California, U.S.A. Robert F. Margolskee, M.D., Ph.D. Department of Physiology and Biophysics, Howard Hughes Medical Institute, The Mount Sinai School of Medicine, New York, New York, U.S.A. Joel Maruniak, Ph.D. Department of Biological Sciences, University of Missouri, Columbia, Missouri, U.S.A. Richard D. Mattes, Ph.D., R.D. Department of Foods and Nutrition, Purdue University, West Lafayette, Indiana, U.S.A. Bert Ph. M. Menco, Ph.D. Department of Neurobiology and Physiology, Northwestern University, Evanston, Illinois, U.S.A. Julie A. Mennella, Ph.D. Monell Chemical Senses Center, Philadelphia, Pennsylvania, U.S.A.

Bradley N. Johnson, M.D. Department of Bioengineering, University of California, Berkeley, California, U.S.A.

Claudia S. Miller, M.D. Department of Family Practice and Community Medicine, University of Texas Health Science Center, San Antonio, Texas, U.S.A.

David W. Kennedy, M.D. Department of Otorhinolaryngology: Head and Neck Surgery, University of Pennsylvania, Philadelphia, Pennsylvania, U.S.A.

Inglis J. Miller, Jr., Ph.D. Department of Neurobiology and Anatomy, Wake Forest University School of Medicine, WinstonSalem, North Carolina, U.S.A.

Eric B. Keverne, M.A., Ph.D., D.Sc., F.R.S. Department of Animal Behaviour, University of Cambridge, Cambridge, United Kingdom

Marian L. Miller, Ph.D. Department of Environmental Health, University of Cincinnati College of Medicine, Cincinnati, Ohio, U.S.A.

Contributors

xiii

Charlotte M. Mistretta, Ph.D. Department of Biological and Materials Sciences, School of Dentristry, University of Michigan, Ann Arbor, Michigan, U.S.A.

James E. Schwob, M.D., Ph.D. Department of Anatomy and Cellular Biology, Tufts University School of Medicine, Boston, Massachusetts, U.S.A.

Cheil Moon, Ph.D. The Johns Hopkins University School of Medicine, Baltimore, Maryland, U.S.A.

Thomas R. Scott, Ph.D. College of Sciences, San Diego State University, San Diego, California, U.S.A.

Edward E. Morrison, Ph.D. Department of Anatomy, Physiology, and Pharmacology, Auburn University, Auburn, Alabama, U.S.A.

Nancy L. Segal, Ph.D. Department of Psychology, California State University, Fullerton, California, U.S.A.

Claire Murphy, Ph.D. Department of Psychology, San Diego State University, and Department of Surgery (Otolaryngology), University of California, San Diego, School of Medicine, San Diego, California, U.S.A. Krishna C. Persaud, Ph.D. Department of Instrumentation and Analytical Science, University of Manchester Institute of Science and Technology, Manchester, United Kingdom Aras Petrulis, Ph.D. Department of Psychology, Georgia State University, Atlanta, Georgia, U.S.A. Donald W. Pfaff, Ph.D. Laboratory of Neurobiology and Behavior, The Rockefeller University, New York, New York, U.S.A. Richard H. Porter, Ph.D. Laboratoire de Comportement Animal, Unité de Physiologie de la Reproduction et des Comportements, Institut National de la Recherche Agronomique/Centre National de la Recherche Scientifique, Nouzilly, France Evan R. Reiter, M.D. Department of Otolaryngology–Head and Neck Surgery, Virginia Commonwealth University, Richmond, Virginia, U.S.A. Klaus Reutter, Ph.D. Anatomical Institute, University of Tübingen, Tübingen, Germany Edmund T. Rolls, D. Phil., D.Sc. Department of Experimental Psychology, University of Oxford, Oxford, United Kingdom Gabriele V. Ronnett, M.D., Ph.D. Department of Neuroscience, The Johns Hopkins University School of Medicine, Baltimore, Maryland, U.S.A.

David V. Smith, Ph.D. Department of Anatomy and Neurobiology, University of Tennessee Health Science Center, Memphis, Tennessee, U.S.A. Gregory S. Smutzer, Ph.D. Smell and Taste Center and Department of Otorhinolaryngology, University of Pennsylvania, Philadelphia, Pennsylvania, U.S.A. Noam Sobel, Ph.D. Wills Neuroscience Institute and Department of Psychology, University of California, Berkeley, California, U.S.A. Alan C. Spector, Ph.D. Department of Psychology, University of Florida, Gainesville, Florida, U.S.A. Regina M. Sullivan, Ph.D. Department of Zoology, University of Oklahoma, Norman, Oklahoma, U.S.A. Wyatt C. To, M.D. Department of Otolaryngology and Communicative Disorders, The Cleveland Clinic Foundation, Cleveland, Ohio, U.S.A. Tari D. Topolski, Ph.D. Department of Health Services, University of Washington, Seattle, Washington, U.S.A. John Q. Trojanowski, M.D., Ph.D. Smell and Taste Center, Center for Neurodegenerative Disease Research, Department of Pathology and Laboratory Medicine, University of Pennsylvania, Philadelphia, Pennsylvania, U.S.A. Luca Turin, Ph.D. Department of Physiology, University College, London, United Kingdom Donald A. Wilson, Ph.D. Department of Zoology, University of Oklahoma, Norman, Oklahoma, U.S.A.

Benoist Schaal, Ph.D. Centre Européen des Sciences du Goût, Dijon, France

Martin Witt, M.D., Ph.D. Department of Anatomy, University of Technology Dresden, Dresden, Germany

Hendrik N. J. Schifferstein, Ph.D. Department of Industrial Design, Delft University of Technology, Delft, The Netherlands

Fumiko Yoshii, Ph.D. Graduate School of Science and Technology, Niigata University, Niigata, Japan

Marlene Schwanzel-Fukuda, Ph.D. Laboratory of Neurobiology and Behavior, The Rockefeller University, New York, New York, U.S.A.

David M. Yousem, M.D. Department of Radiology, The Johns Hopkins University School of Medicine, Baltimore, Maryland, U.S.A.

Introduction and Historical Perspective Richard L. Doty University of Pennsylvania, Philadelphia, Pennsylvania, U.S.A.

I.

INTRODUCTION

“smoke.” Fire, with its dangerous and magical connotations, must have become associated early on with religious activities, and pleasant-smelling smoke was likely sent into the heavens in rituals designed to please or appease the gods. Importantly, food and drink became linked to numerous social and religious events, including those that celebrated birth, the attainment of adulthood, graduation to the status of hunter or warrior, and the passing of a soul to a better life. The goal of this introduction is to provide a brief historical overview of (1) the important role that tastes and odors have played in the lives of human beings throughout millennia and (2) key observations from the last four centuries that have helped to form the context of modern chemosensory research. Recent developments, which are described in more detail elsewhere in this volume, are briefly mentioned to whet the reader’s appetite for what is to follow. Although an attempt has been made to identify, rather specifically, major milestones in chemosensory science since the Renaissance, some important ones have undoubtedly been left out, and it is not possible to mention, much less discuss, even a small fraction of the many studies of this period that have contributed to our current fund of knowledge. Hopefully the material that is presented provides some insight into the basis of the present Zeitgeist. The interested reader is referred elsewhere for additional perspectives on the history of chemosensory science (e.g., Bartoshuk, 1978, 1988; Beidler, 1971a,b; Boring, 1942; Cain, 1978; Cloquet, 1821; Corbin, 1986; Doty, 1976; Douek, 1974; Farb and Armelagos, 1980; Farbman, 1992; Frank, 2000; Gloor, 1997; Harper et al., 1968; Harrington

All environmental nutrients and airborne chemicals required for life enter our bodies by the nose and mouth. The senses of taste and smell monitor the intake of such materials, not only warning us of environmental hazards, but determining, in large part, the flavor of our foods and beverages. These senses are very acute; for example, the human olfactory system can distinguish among thousands of airborne chemicals, often at concentrations below the detection limits of the most sophisticated analytical instruments (Takagi, 1989). Furthermore, these senses are the most ubiquitous in the animal kingdom, being present in one form or another in nearly all air-, water-, and land-dwelling creatures. Even bacteria and protozoa have specialized mechanisms for detecting environmental chemicals— mechanisms whose understanding may be of considerable value in explaining their modes of infection and reproduction (Jennings, 1906; Russo and Koshland, 1983; van Houten, 2000). While the scientific study of the chemical senses is of relatively recent vintage, the role of these senses in the everyday life of humans undoubtedly extends far into prehistoric times. For example, some spices and condiments, including salt and pepper, likely date back to the beginnings of rudimentary cooking, and a number of their benefits presumably were noted soon after the discovery of fire. The release of odorants from plant products by combustion was most likely an early observation, the memory of which is preserved in the modern word perfume, which is derived from the Latin per meaning “through” and fumus meaning xv

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and Rosario, 1992; Johnston et al., 1970; Jones and Jones, 1953; McBurney and Gent, 1979; McCartney, 1968; Miller, 1988; Moulton and Beidler, 1967; Mykytowycz, 1986; Ottoson, 1963; Pangborn and Trabue, 1967; Parker, 1922; Pfaff, 1985; Piesse, 1879; Simon and Nicholelis, 2002; Smith et al., 2000; Takagi, 1989; Wright, 1914; von Skramlik, 1926; Zippel, 1993). II. A BRIEF HISTORY OF PERFUME AND SPICE USE The relatively rich history of a number of ancient civilizations, particularly those of Egypt, Greece, Persia, and the Roman Empire, provides us with examples of how perfumes and spices have been intricately woven into the fabric of various societies. Thousands of years before Christ, fragrant oils were widely used throughout the Middle East to provide skin care and protection from the hot and dry environment, and at least as early as 2000 B.C. spices and fragrances were added to wine, as documented by an inscription on a cuneiform text known as the Enuma elish (Heidel, 1949). In Egypt, incense and fragrant substances played a key role in religious rites and ceremonies, including elaborate burial customs, and whole sections of towns were inhabited by men whose sole profession was to embalm the deceased. As revealed in the general body of religious texts collectively termed the “Book of the Dead”—a number of which predate 3000 B.C. (Budge, 1960)—the Egyptians performed funeral ceremonies at which prayers and recitations of formulas (including ritualistic repeated burning of various types of incense) were made, and where the sharing of meat and drink offerings by the attendees occurred. Such acts were believed to endow the departed with the power to resist corruption from the darkness and from evil spirits that could prevent passage into the next life, as well as to seal the mystic union of the friends and loved ones with the dead and with the chosen god of the deceased. The prayers of the priests were believed to be carried via incense into heaven and to the ears of Osiris and other gods who presided over the worlds of the dead (Budge, 1960). As noted in detail by Piesse (1879), the ancient Greeks and Romans used perfumes extensively, keeping their clothes in scented chests and incorporating scent bags to add fragrance to the air. Indeed, a different scent was often applied to each part of the body: mint was preferred for the arms, palm oil for the face and breasts, marjoram extract for the hair and eyebrows, and essence of ivy for the knees and neck. At their feasts, Greek and Roman aristocrats adorned themselves with flowers and scented waxes and added the fragrance of violets, roses, and other flowers to their wines. As would be expected, perfume shops were

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abundant in these societies, serving as meeting places for persons of all walks of life (Morfit, 1847). In Grecian mythology, the invention of perfumes was ascribed to the Immortals. Men learned of them from the indiscretion of Aeone, one of the nymphs of Venus; Helen of Troy acquired her beauty from a secret perfume, whose formula was revealed by Venus. Homer (eighth century B.C.) reports that whenever the Olympian gods honored mortals by visiting them, an ambrosial odor was left, evidence of their divine nature (Piesse, 1879). Interestingly, bad odors were a key element of a number of myths, including that of Jason and the Argonauts (Burket, 1970). As a result of having been smitten with the wrath of Aphrodite, the women of Lemnos developed a foul odor, which drove their husbands to seek refuge in the arms of Thracian slave girls. The women were so enraged by their husbands’ actions that one evening they slew not only their husbands, but all the men of the island. Thereafter, Lemnos was a community of women without men, ruled by the virgin queen Hypsiple, until the day when Jason and the Argo arrived, which ended the period of celibacy and returned the island to heterosexual life. Perfumes were not universally approved of in ancient Greece. Socrates, for example, objected to them altogether, noting, “There is the same smell in a gentleman and a slave, when both are perfumed,” and he believed that the only odors worth cultivating were those that arose from honorable toil and the “smell of gentility” (Morfit, 1847). Nevertheless, the use of perfumes became so prevalent in ancient Greece that laws were passed in Athens in the sixth century B.C. to restrain their use. Despite this prohibition, however, their use grew unabated, and the Greeks added greatly to the stock of fragrant plants from the East that made up the core of the perfume industry. Perfume and incense had religious significance to the followers of Zoroaster, the Persian religious leader of the sixth century B.C., who offered prayers before altars containing sacred fires to which wood and perfumes were added five times each day (Piesse, 1879). It is noteworthy that, to this day, sandalwood fuels the sacred fires of the Parsees (modem Zoroastrians) in India, and that similar rituals were required of the early Hebrews, as indicated by the following instructions from God to Moses (Exodus 30:1, 7–9, 34–38): And thou shalt make an altar to burn incense upon: of shittim wood shalt thou make it. And Aaron shall burn thereon sweet incense every morning: when he dresseth the lamps, he shall burn incense upon it [the altar]. And when Aaron lighteth the lamps at even, he shall burn incense upon it, a perpetual incense before the Lord throughout your generations. Ye shall offer no strange incense thereon, nor burnt sacrifice, nor

Introduction and Historical Perspective

meat offering; neither shall ye pour drink offering thereon. And the Lord said unto Moses, take unto thee three sweet spices, stacte, and onycha, and galbanum; these sweet spices with pure frankincense; of each shall there be a light weight. And thou shalt make it a perfume, a confection after the art of the apothecary, tempered together, pure and holy: And thou shalt beat some of it very small, and put of it before the testimony in the tabernacle of the congregation, where I will meet with thee: it shall be unto you most holy. And as for the perfume which thou shalt make, ye shall not make to yourselves according to the composition thereof: it shall be unto thee holy for the Lord. Whosoever shall make like unto that, to smell thereto, shall even be cut off from his people. Given such instructions from God and the Christian emphasis on cleansing the soul of evil spirits, as well as the fact that Christ himself, after his crucifixion, had been embalmed in pleasant-smelling myrrh, aloe, and spices (John 19:39–40), it is perhaps not surprising that bad smells came to signify the unholy at various times in Christian history. Indeed, St. Philip Neri reportedly found the stench emanating from heretics so great that he had to turn his head (Summers, 1926). One of the more interesting, and tragic, uses of bad smells was to identify witches and warlocks in Europe in the late 1500s. Remy, a distinguished appointee of Charles III to the Provosts of Nancy (a court that judged all criminal cases for some 72 villages in the Nancy region of France), wrote the following in his classic 1595 monograph Demonolatry: In the Holy Scriptures the Devil is constantly referred to as Behemoth, that is to say, “the impure animal and the unclean spirit” (see S. Gregory, in Memorabilia, Matthew XII, Mark I and V, Job XI). It is not only because the Devil is, as all his actions and purposes show, impure in his nature and character that we should consider this name to be aptly applied to him; but also because he takes immoderate delight in external filth and uncleanliness. For often he makes his abode in dead bodies; and if he occupies a living body, or even if he forms himself a body out of the air or condensation of vapours, his presence therein is always betrayed by some notable foul and noisome stench. The gifts of the Demon are also fashioned from ordure and dung, and his banquets from the flesh of beasts that have died . . . for the most part [he] has for his servants filthy old hags whose age and poverty serve but to enhance their foulness; and these . . . he instructs in all impurity and uncleanliness. . . . Above all he cautions them not to

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wash their hands, as it is the habit of other men to do in the morning; for he tells them that to do so constitutes a sure obstruction to his incantations. This is the case whether it is the witches themselves who wash their hands, as we learn from the answer freely given to her examiners by Alexia Galaea of Betoncourt at Mirecourt in December 1584, and by countless others whose names I have not now by me; or whether it is the intended victims of their witchcraft who wash their hands, as was stated by Claude Fellet (Mersuay, February 1587) and Catharina Latomia (Haraucourt, February 1587). In contrast to the detection of witches and warlocks by stench was the verification of sainthood by a pleasant odor, the so-called “odor of sanctity.” If a saint had been an impostor, a nauseating smell, rather than a delectable one, was present upon exhumation of his body (Rothkrug, 1981). This concept bears a striking resemblance to the Greek myths of the pleasant odors left by the Olympian gods who visited mortals and may well stem from the same tradition. It should be noted, however, that cleanliness was not always the vogue for Christianity, as described by McLaughlin (1971) in a series of interesting accounts from the Middle Ages. Thus, in their repudiation of Roman values, early Christians often went unbathed. Every sensation offensive to humans was believed acceptable to God, and the custom of bathing the limbs and anointing them with oil was condemned. Monks shaved their hair, wrapped their heads in cowls to avoid seeing profane objects, and kept legs naked except in the extreme of winter. St. Jerome criticized a number of his followers for being too clean, and St. Benedict, a key administrator of the early church, pronounced solemnly that “to those that are well, and especially to the young, bathing shall seldom be permitted.” St. Agnes reportedly had never washed throughout her life, and a pilgrim to Jerusalem in the fourth century is said to have boasted that her face had gone unwashed for 18 years so as not to disturb the holy water used at her baptism. During the Middle Ages, perfumery and the widespread use of spices and flavoring agents was little known in Europe, being practiced mainly by Arabs in the East. Marco Polo, visiting the China of Kublai Khan (1216–1295), noted that pleasantly perfumed silk paper money was used for exchange within Khan’s kingdom (Boorstin, 1985). The dearth of smell in Europe was to change dramatically, however, as a major element of the Renaissance was the relentless search for perfumes and spices, a number of which were more valuable than silver or gold. The quest was not only for aesthetic delight; some of these agents made it possible, much like cooking itself, to exploit a wider and more diverse range of foodstuffs, including ones that otherwise

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were unsafe or had little gastronomic appeal. In this regard, it is of interest that at the siege of Rome in A.D. 408, Alaric, the victorious king of the Goths, demanded 3000 pounds of pepper as ransom for the city, and when the Genoese captured Caesarea in A.D. 1101, each soldier received two pounds of pepper as his share of the spoils (Verrill, 1940). Perfume was introduced, at least in a widespread sense, to medieval Europe by the crusaders. After the downfall of the Roman Empire, the perfume industry moved to the Eastern Roman Empire, and Constantinople became the perfume center of the world. Reportedly, Avicenna (A.D. 980–1036), the great Arab scientist, philosopher, and physician, discovered a way to extract and maintain the fragrances of plants and possibly invented rose water (Takagi, 1989). In part because of its conducive soil and climate, southern France proved to be a natural place for the cultivation of flowers for the perfume industry, an industry for which France gained world supremacy that continues to this day (Vivino, 1960). In 1190, King Philip II (Philip Augustus, r. 1180–1223) of France granted the first charter to a perfume maker. In 1370, Queen Elizabeth of Hungary was given a perfume formula based upon rosemary, which was the first recorded alcohol-based perfume. This perfume, known as “The Queen of Hungary’s Water” or simply “Hungary Water,” was in use for more than five centuries and may be the precursor to eau de cologne, which is said to have been invented around 1690 in Milan, Italy, by Jean-Paul Feminis, who later resided in Cologne. King Charles V (Charles the Wise, r. 1364–1380) planted large fields of flowers in France to obtain perfume materials, and Charles VIII (r. 1483–1498) was reportedly the first French monarch to appoint a court perfumer. The guild of glove and perfume-makers was established in Paris in 1656. Perfume was readily accepted as a substitute for bath in the court of Louis XIV (r. 1643–1715), as the palace at Versailles totally lacked plumbing. The court of Louis XV (r. 1715–1774) was well known for the extravagant uses of perfumes; indeed, it was named “the perfume court,” reflecting the daily application of scents to skin, fans, clothing, furniture. Perfumes lost their popularity in England for more than a century prior to the Victorian era, unlike the case in France, Italy, and Spain (Piesse, 1879). Related to this loss of popularity was an act, introduced into the English parliament in 1770, that warned women of the use of scents and other materials in the seduction of men (Piesse, 1879, p. 20): That all women, of whatever age, rank, profession, or degree, whether virgins, maids, or widows, that shall, from and after such Act, impose upon, seduce or betray into matrimony, any of his Majesty’s subjects, by the scents, paints, cosmetic washes, artificial

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teeth, false hair, Spanish wool, iron stays, hoops, high-heeled shoes, bolstered hips, shall incur the penalty of the law now in force against witchcraft and the like misdemeanors, and that the marriage, upon conviction, shall stand null and void. The influences of such attempts to ban perfumes in England were short-lived, as perfume vendors thrived, although the state taxed them and required them, in 1786, to have licenses. By 1800 approximately 40 companies were making perfumes in London. In the 19th century the revolution that occurred in organic chemistry ensured the continuance of perfume manufacturing in Britain; the first important successful synthetic odorant, coumarin, was prepared in 1863 by the British chemist Sir William Henry Perkin (Vivino, 1960). III. THE CHEMICAL SENSES AND EARLY MEDICINE The close association between odors, spices, and medicine was undoubtedly forged long before recorded history and was likely fostered not only by stenches associated with plagues and death, but by the utility of essential oils and spices in warding off insects and microbes. Indeed, one reason why perfumes and spices were major objects of international trade in the ancient world was their medicinal properties. According to Morris (1984), such properties may have been as important to early civilizations as the development of the x-ray or discovery of penicillin was to our own, as modern studies confirm that numerous essential oils and spices are very effective in controlling pathogens, including Staphylococcus and various tuberculosis bacilli. Apparently this observation first came to the attention of European scientists in the latter half of the 19th century, when the perfumery workers at Grasse, France, were found to have a much lower rate of cholera and tuberculosis than the rest of the European population. As noted by Morris (1984, p. 15): Essential oils have shown startling fungitoxic properties. Oil of clove is toxic to specific growths, and oil of geranium is effective against a broad range of fungi. Cymbopogon grasses, an Indian genus of aromatic grasses, have been found effective against Heuninthosporium oryzae, a source of food poisoning, Aspergillus niger, a cause of seborrheic dermatitis of the scalp, Absidia ramosa, a cause of otitis, and Trichoderma viride, another cause of dermatitis. Man has long guessed that these oils that the plant secreted to protect itself from insect, fungal, and microbial dangers could serve him as well. Thus it is that the story of perfumery is intimately linked to the story of

Introduction and Historical Perspective

pharmacy. Our ancestors could not formulate the germ theory of disease, but they assumed that whatever smelled clean and healthy must be of use in hygiene.* The history of hygiene and public health is closely associated with the view that odors were the source, indeed often the cause, of diseases and pestilence. The stenches that developed in the cities of Europe during the Middle Ages are unimaginable to us today. Conditions were so bad that, for example, the monks of White Friars in London’s Fleet Street complained that the smell from the Fleet River overcame all the frankincense burnt at their altars and killed many of their brethren (McLaughlin, 1971). Such problems were the backdrop of the spread of the plague epidemics that traversed Europe and England in the 12th to 17th centuries. As chronicled by Corbin’s (1986) fascinating account of the history of hygiene and odors in 18th-century France, health administration of that era was based on a catalog of noxious odors. Indeed, authorities sought to locate the networks of miasmas by “mapping the flux of smells that made up the olfactory texture of the city” (p. 55). The desire to localize odors and to eliminate them in an effort to ward off diseases may well have been one reason why so many odor classification schemes arose during the 18th century, including those of van Haller (1756), Linnaeus (1765), Lorry (1784/85), and Fourcroy (1798). Throughout this period, as well as in earlier times, infection was believed to be stemmed by wearing a perfume or by burning aromatic pellets in special perfume pans. Lemery’s Pharmacopee universelle (1697) cataloged the therapeutic value of aromatics and perfumes and suggested the prescription of “apoplectic balms” because “what is pleasing to the nose, being composed of volatile, subtle, and *Billing and Sherman (1998) provide empirical support for the hypothesis that the amount of spice in foodstuffs from various world cuisines is better explained on the basis of their antibacterial than their sensory properties. These investigators quantified the frequency of use of 43 spices in the meat-based cuisines of 136 countries for which traditional cookbooks could be found. A total of 4578 recipes from 93 cookbooks was examined, along with information on the temperature and precipitation in each country, the ranges of spice plants, and the antibacterial properties of each spice. As mean annual temperatures (an index of relative spoilage rates of unrefrigerated foods) increased, the proportion of recipes containing spices, number of spices per recipe, total number of spices used, and use of the most potent antibacterial spices all increased, both within and among countries. The estimated fraction of bacterial species inhibited per recipe in each country was positively correlated with annual temperature. Although alternative hypotheses were considered (e.g., that spices provide macronutrients, disguise the taste and smell of spoiled foods, or increase perspiration and thus evaporative cooling), the data did not support any of these alternatives.

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penetrating parts, not only affects the olfactory nerve, but is spread through the whole brain and can deplete its pituita and other overcourse humors, increasing the movement of animal spirits” (Corbin, 1986, p. 62). During outbreaks of the plague, defenses included the burning of incense, juniper, laurel leaves, cypress, pine, balm, rosemary, and lavender, although, if effective, they were only marginally so. Various plague waters, to be poured on handkerchiefs or into pomanders, were invented, including the original eau de cologne. Unpleasant agents were also believed to keep away the plague, and the members of many households crouched over their privies inhaling the fumes in attempts to avert the disaster (McLaughlin, 1971). Even in the late 1800s, smells were associated with illnesses, as exemplified by the belief that decaying organic matter in swamps produced malaria (mal
bad, aira
air). This theory, apparently initially proposed by Varro (116–28 B.C.) and Palladius (fourth century A.D.), was brought to the more modern stage by Morton (1697) and Lancisi (1717), but was abandoned after the French physician Alphonse Laveran (1881) described the responsible parasite and Sir Ronald Ross (1923) demonstrated, a few years later, its transmission by the female anopheline mosquito. In the history of medicine, both odors and tastes have been used at various times in the diagnosis of diseases (see Doty, 1981, for review). Even today, diabetes is diagnosed in some areas of the world on the basis of the patient’s acetonelike breath and sweet-tasting urine, although, in general, the use of odor and taste in diagnosis has become a lost art. In addition, certain smells and tastes were known to elicit symptoms of some diseases, including epilepsy and hysteria. A classic example is reported by the Roman historian Caius Plinius Caecilius Secundus (Pliny) in his Historia Naturalis (circa A.D. 50), where sulfur and burning bitumen (asphalt) were noted to induce seizures (Bailey, 1932), a phenomenon that has also been reported in more modern times (West and Doty, 1994). Alum (alumen), which contained aluminum, was used as a deodorant in the Roman empire, predating the use of aluminum salts as deodorants in the United States in the 1880s, as evidenced by the following quotation of Pliny, which extols its values (Bailey, 1932, p. 103): Liquid alumen has astringent, hardening, and corrosive properties. Mixed with honey, it heals sores in the mouth, pustules, and itchy eruptions. In the latter case, the treatment is applied in a bath to which honey and alumen have been added in the proportion of two to one. Alumen diminishes offensive odours of the axilla, and reduces sweating in general. To my knowledge, there are no pre-Renaissance treatises on chemosensory dysfunction per se, although descriptions of loss of olfactory function are found in the writings of the ancient Greeks and Romans. Perhaps the

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first description of anosmia was that by Theophrastus in the third century B.C. (Stratton, 1917, p. 84): . . . it is silly to assert that those who have the keenest sense of smell inhale most; for if the organ is not in health or is, for any cause, not unobstructed, more breathing is to no avail. It often happens that man has suffered injury [to the organ] and has no sensation at all. Although the early Greeks routinely used surgical intervention for the treatment of polyps and other intranasal obstructive problems (for review, see Wright, 1914), the first description of the use of surgery to specifically correct anosmia was apparently made during the Renaissance by Forestus (1591; cited in Lederer, 1959): If it [anosmia] is from ethmoidal obstruction, or from the humor discharged from catarrh, the latter must first be cured. If from the flesh growing from within the nose . . . it is to be cured by the surgeons by operative procedures, either with a cutting instrument, or cautery, or snare. Claudius Galenus (Galen; A.D. 130–200), whose writings had a major impact on Western medicine in general, attributed anosmia to obstruction of the foramina within the cribriform plate (an attribution made by a number of early Greeks, including Plato and Hippocrates). He correctly described the role of the nose in warming and filtering the air and alluded to empirical studies noting the permeability of the dura matter around the cribriform plate to both water and air (Wright, 1914). He believed that the organ for smell was located in the ventricles of the brain and that particles responsible for olfactory sensations passed through the foramina of the cribriform plate during inhalation. As discussed in more detail later, this compelling idea continued until the 18th century, when light microscopy revealed that the nasal secretions came from secretory cells within the epithelium. In terms of taste, he posited that the lingual nerve communicated gustatory sensations, in accord with modern perspectives (see Chapters 32 and 44). IV. THE RENAISSANCE AND THE BIRTH OF MODERN STUDIES OF TASTE AND SMELL As is evidenced in this book, major advances have been made in understanding the senses of taste and smell—advances that follow on the footsteps of a long tradition of scientific observations stemming from treatises written in the 16th century. Indeed, the sense of smell did not escape the attention of Leonardo da Vinci (1452–1519), who, in the Codex Atlanticus, presented nine diagrams next to one another in which he compared the behavior of light, the

force of a blow, sound, magnetism, and odor (Riti, 1974). Cardinal Gasparao Contarini (1482–1542), an alchemist, wrote about the elements and their combinations in five brief volumes published posthumously in 1548 by Ioannes Gaignaeus. The last of these was dedicated to flavors, odors, and colors. Contarini believed that there were eight flavors or tastes and argued that cooking food or preserving fruit can produce flavors not found in nature. He felt that the sense of smell was imperfect and noted that the names of flavors are often employed to explain the variety of odors. Andrea Vesalius devoted one and a half large pages to the sense of smell in his classic anatomy treatise De Humani Corporis Fabrica (1543), although he failed to observe the olfactory filaments. In 1566, Gryll published what may be the first work solely devoted to the sense of taste, and in 1581, Fernel listed nine types of basic taste qualities, including the seven of Aristotle and Galen (sweet, bitter, sour, salty, astringent, pungent, harsh) and “fatty” and “insipid,” the latter apparently reflecting the lack of other taste qualities (Bartoshuk, 1978). Casserius (1609) described the detailed structure of the tongue, and Malpighi (1664) and Bellini (1665) associated the sense of taste with lingual papillae. Taste buds were first identified on the barbels and skin of fishes by Leydig (1851) and later were described in mammals (Loven, 1868; Schwalbe, 1868). In 1587, Iohannes Camerarius presented a thesis to the University of Marburg entitled “Themata Physica de Odorum Natura et Affectionibus.” In this work, he discussed odor classification, the relationship between taste and smell, a mechanism for explaining the function of olfaction, the ability of smelling in water, and the effect of heat from the sun on odors (Kenneth, 1928). In 1673, Robert Boyle wrote an article, “Nature, Properties, and Effects of Effluvia,” in which he provides vivid and accurate observations on such topics as olfaction in birds, odor tracking in dogs, and the physical nature of the materials released from various odor sources. In his 1675 paper, “Experiments and Observations About the Mechanical Production of Odours,” he addresses some simple issues of odorant mixtures and observes that the quality and intensity of odors can be related. He provides, in his “Experiments and Considerations About the Profity of Bodies” (1684), perhaps the first description of intravascular olfaction or taste (i.e., the smelling or tasting of substances that are initially bloodborne): One of the notablest instances I ever met with of the porosity of the internal membranes of the human body, was afforded to me by that British nobleman, of whom our famous Harvey tells a memorable, not to say matchiless story. This gentleman, having in his youth by an accident, which that doctor relates, had a great and lasting perforation made in his thorax, at

Introduction and Historical Perspective

which the motion of his heart could be directly perceived, did not only out-live the accident, but grew a strong and somewhat corpulent man; and so robust, as well as gallant, that he afterwards was a soldier, and had the honour to command a body of an army for the King. This earl of Mount-Alexander . . . gave me the opportunity of looking into his thorax, and of discerning there the motions of the cone, as they call it, or mucro of the heart. . . . Having then made several inquiries fit for my purpose, his lordship told me, that, when he did, as he was wont to due from time to time, (though not every day) inject with a syringe some actually warm medicated liquor into his thorax, to cleanse and cherish the parts, he should quickly and plainly find in his mouth the taste and smell of the drugs, wherewith the liquor had been impregnated. And I further learned, that, whereas he constantly wore, upon the unclosed part of his chest, a silken quilt fluffed with aromatic and odoriferous powders, to defend the neighboring parts and keep them warm; when he came, as he used to do after several weeks, to employ a new quilt, the fragrant effluvia of it would mingle with his breath in expiration, and very sensibly perfume it, not, as I declared I suspected, upon the score of the pleasing exhalations, that might get up between his cloathes and his body, but that got into the organs of respiration, and came out with his breath at his mouth, as was confirmed to me by a grave and judicious statesman, that happened to be then present, and knew this general very well. From the 14th to mid-19th centuries, research on gustation was much more limited in scope than that on olfaction, although notable advances were made in taste research, including (1) the discovery that dissimilar metals, when placed on the tongue, produced an “electric taste” sensation (Sulzer, 1752; Volta, 1792), (2) the observation that taste sensations are localized to papillae (Malpighi, 1664; Bell, 1803), (3) the identification of the chorda tympani as the nerve that mediates taste in the anterior tongue (Bellingeri, 1818; see Bartoshuk, 1978), and (4) the demonstration that different regions of the oral cavity are differentially sensitive to different taste qualities (Horn, 1825). As noted above, the observation that taste buds exist within the papillae of the mammalian tongue and depend on an intact nerve supply came in the latter half of the 19th century (see Chapter 32) (Loven, 1868; Merkel, 1880; Schwalbe, 1867; Vintschgau and Hönigschmied, 1877), as did the painstaking mapping of the sensitivity of individual papillae to stimuli representing the four basic taste qualities (Öhrwall, 1891; Kiesow, 1894). One reason for the comparatively greater interest in ol-

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faction than in taste during the post-Renaissance period stemmed from the compelling, albeit erroneous, conceptual framework in which olfactory functioning, disease, and nasal secretion were viewed. For smelling to occur, odorous bodies had to enter the brain via the foramina of the cribriform plate—the same foramina through which body humors were believed to flow to produce nasal mucus. From this perspective, blockage or alterations in this passageway (e.g., by the changes in the viscosity of the humors) were closely related to diseases that caused (1) anosmia, (2) running noses, (3) high fever, and (4) general ill feeling. There is no doubt that the major conceptual chemosensory advance of this period, indeed perhaps of the entire modern era, was refutation of this ancient concept. The compelling nature of this theory and its adaptation to a more modern era is illustrated by Descartes’ (1644) description of how olfaction works (Haldane and Ross, 1955, p. 292): . . . two nerves or appendages to the brain, for they do not go beyond the skull, are moved by the corporeal particles separated and flying in the air—not indeed by any particles whatsoever, but only by those which, when drawn into the nostrils, are subtle and lively enough to enter the pores of the bones which we call the spongy, and thus to reach the nerves. And from the diverse motions of these particles, the diverse sensations of smell arise. Interestingly, convincing evidence for this notion continued to be amassed during this period, as the following passage from Thomas Willis (1681, p. 100) indicates: The Sieve-like Bone in divers Animals is variously perforated for the manifold necessity and difference of smelling. A Process from the Dura Mater and manifold nervous Fibres pass through every one of its holes, and besmear the inside of the Nostrils. But as the impressions of sensible things, or sensible Species, confined as it were by the undulation or waving of the animal Spirits, ascend through the passages of these bodies stretched out from the Organ towards the Sensory; so the humidities watring the same bodies, for as much as some they may be more superfluous than usual, may distil into the Nostrils through the same ways. For indeed such humors as are perpetually to be sent away from the brain, ought so copiously to be poured upon the Organs of Smelling, as we shall shew hereafter, when we shall speak particularly of the smelling Nerves; in the mean time, that there is such a way of Excretion opening into the Nostrils, some observations, taken of sick people troubled with Cephalick diseases, do further perswade.

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. . . A Virgin living in this City, was afflicted a long time with a most cruel Head-ach, and in the midst of her pain much and thin yellow Serum daily flowed out from her Nostrils; the last Winter this Excretion stopped for some time, and then the sick party growing worse in the Head, fell into cruel Convulsions, with stupidity; and within three days dyed Apoplectical. Her Head being opened, that kind of yellow Latex overflowed the deeper turnings and windings of the Brain and its interior Cavity or Ventricles. . . . I could here bring many other reasons, which might seen to perswade, that the Ventricles of the Brain, of the Cavity made by the complicature or folding up of its border, is a mere sink of the excrementitious Humor; and that the humors there congested, are purged out by the Nose and Palate. The idea of movement of humors from the brain to the nasal cavity was most likely supported by other types of evidence as well. For example, demonstrations that dyes (e.g., Indian ink), after injection into the subarachnoid space or the cerebral spinal fluid, travel to the nasal mucosa via the cribriform plate were made in the 19th century, and there is no reason to believe that such information was not available in earlier times (see Jackson et al., 1979, for review). It is not clear who deserves the credit for identifying the olfactory nerves in the upper nasal cavity, although, according to Wright (1914), the 7th-century Greek physician Theophilis gave one of the better anatomical accounts of their distribution, despite the potential political ramifications of going against Galen’s dictates. Graziadei (1971) credits Massa, in 1536, as having first demonstrated the olfactory nerves in humans, and Scarpa, in 1789, as having shown that the fine fila olfactoria actually end in the regio olfactoria (note, however, Scarpa’s 1785 article). Wright (1914), on the other hand, notes that the Italian Anatomist Alessandro Achillini, who died in 1512, had described their intranasal distribution. Regardless of who is responsible for their first description, there was considerable disagreement, which spanned over a century and a half, among authorities as to whether the processes that extended from the olfactory bulbs into the nasal cavity were, in fact, nerves. Indeed, even after they were generally accepted as nerves, debate lasted into the 1840s as to whether they mediated smell sensations. As reviewed in Chapter 47, Francois Magendie (1824) was the primary proponent of the idea that such sensations were mediated via the trigeminal nerve, whereas Sir Charles Bell believed that the olfactory nerves subserved such sensations (Shaw, 1833). As late as 1860, experiments appeared in the literature that addressed this point (e.g., Schiff, 1860), although the more authoritative general physiology and medical textbooks from the 1820s to the 1850s cor-

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rectly noted that the olfactory nerve mediates qualitative odor sensations and the trigeminal nerve somatosensory sensations (e.g., Good, 1822; Kirkes, 1849). Schneider (1655) and Lower (1670) are generally credited as being the first to show that nasal secretions arise from glands, rather than from secretions secreted through the cribriform plate. However, a century earlier Berenger del Carpi, who taught surgery at Bologna (1502–1527), broke the Hippocratic and Galenic tradition and denied that fluids passed through these foramina, suggesting that they actually passed through the sphenoid sinus (Wright, 1914). The evidence that nasal secretions came from glands, rather than through the cribriform plate, was clearly an important observation in the history of medicine. Collectively, the aforementioned studies placed the first nails in the coffin of the theory propagated largely by Galen’s works that the cribriform plate is pervious to odors and that the sense of smell lies within the ventricles of the brain. Other major studies before 1890, a number of which are now considered classic, contributed the remaining nails to this coffin and include, in chronological order, those by Hunter (1786), Todd and Bowman (1847), Schultze (1856, 1863), Ecker (1856), Eckhard (1858), Clarke (1861), Hoffman (1866), Martin (1873), Krause (1876), von Brunn (1875, 1880, 1892), Sidky (1877), Exner (1878), Ehrlich (1886), and Cajal (1889). The dawn of human chemosensory psychophysics also occurred in the 19th century, as illustrated by the previously mentioned studies in which specific taste qualities were painstakingly mapped on the tongue by Öhrwall (1891) and Kiesow (1894). Although Boring (1942) credits Fischer and Penzoldt (1886) as having measured the first absolute threshold to an odorant, Valentin, in 1848, described a procedure that assessed olfactory sensitivity that predated even Fechner’s publication of threshold methodology by a dozen years. Zwaardemaker (1925), who invented the important draw-tube olfactometer (see Chapter 10) and who performed sophisticated studies on a wide range of topics, including adaptation and cross-adaptation, credited Passy (1892) as having made an important step in the development of olfactometry. In essence, Passy dissolved a given amount of odorant in alcohol in a 1:10 ratio. This new solution was then again diluted in such a ratio, and this was repeated over and over to provide a series of dilution steps. For testing, a small amount of solution at each concentration was placed in liter bottles, which were heated slightly to evaporate the alcohol. Such bottles were then sampled from highest concentration to lowest concentration until no smell was discernible to the subject. Zwaardemaker, however, expressed concern that the alcohol diluent used by Passy might influence the perception of the test odorant. This potential problem was eliminated to a large degree in the successive dilution series described by

Introduction and Historical Perspective

Toulouse and Vaschide (1899) and Proetz (1924), where water and mineral oil, respectively, served as the diluents. As noted above, the more scholarly physicians of the 18th and 19th centuries were very much aware of the major types of olfactory disorders that we recognize today. Good (1822), for example, classified disorders of olfaction into the following categories: Parosmia acris (acute smell), Parosmia obtusa (obtuse or distorted smell), and Parosmia expers (anosmia or lack of smell). Good (1822, pp. 260–261) notes the following regarding Parosmia obtusa: The evil is here so small that a remedy is seldom sought for in idiopathic cases; and in sympathetic affections, as when it proceeds from catarrhs or fevers, it usually, though not always, ceases with the cessation of the primary disease. It is found also as a symptom of hysteria, syncope, and several species of cephalaea, during which the nostrils are capable of inhaling very pungent, aromatic, and volatile errhines, with no other effect than that of a pleasing and refreshing excitement. Where the sense of smell is naturally weak, or continues so after catarrhs or other acute diseases, many of our cephalic snuffs may be reasonably prescribed, and will often succeed in removing the hebetude. The best are those formed of the natural order verticillatae, as rosemary, lavender, and marjoram; if a little more stimulus be wanted, these may be intermixed with a proportion of the teucrium Marum; to which, if necessary, a small quantity of asarum may also be added: but pungent errhines will be sure to increase instead of diminishing the defect. Good’s observations concerning Parosmia expers were as follows: This species is in many instances a sequel of the preceding [Parosmis obtusa]; for whatever causes operate in producing the former, when carried to an extreme or continued for a long period, may also lay a foundation for the latter. But as it often occurs by itself, and without any such introduction, it is entitled to be treated separately. It offers us the two following varieties: Organica. Organic want of smell. From natural defict, or accidental lesion, injurious to the structure of the organ. Paralytica. Paralytic want of smell. From local palsy. The FIRST VARIETY occurs from a connate destitution of olfactory nerves, or other structural defect; or from external injuries of various kinds; and is often found as a sequel in ozaenas, fistula lachrymalis,

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syphilis, small-pox, and porphyra. The SECOND is produced by neglected and long continued coryzas, and a persevering indulgence in highly acrid sternutatories. Among the more detailed and vivid descriptions of cases of anosmia in the 19th-century literature are those of Ogle (1870). He describes in detail three cases of anosmia due to head injury in which taste function was intact—a case of anosmia associated with facial palsy, a viral-induced case of anosmia, and a case of anosmia due to obstruction—and three cases of unilateral olfactory loss that were related to aphasia, agraphia, and seizure attributable to brain lesions. The olfactory losses due to head injury were believed to be caused by the shearing of the olfactory filaments at the level of the cribriform plate from movement of the brain produced by the blow. In this explanation of the problem, Ogle notes (p. 266) that “the anterior brain rests directly upon the bones of the skull, and is not separated from them as is the case elsewhere by the interposition of cerebro-spinal fluid.” V. THE MODERN ERA: MAJOR 20TH-CENTURY ADVANCES IN OLFACTION AND GUSTATION The major progress in the field of chemosensory research that took place in the 20th century was due largely to contemporaneous advances in other fields of science. Included among such advances, which are not mutually exclusive, are (1) the development of analytical devices such as the gas chromatograph and mass spectrometer, (2) the refinement of experimental design and the development of statistical methodology, (3) advances in basic psychophysical techniques, (4) the invention of sensitive methods for recording minute electrical potentials from the nervous system, including recordings from single cells and isolated components of cell membranes, (5) the development of not only new histological stains, but radically novel histological procedures, such as those that utilize autoradiography, immunohistochemistry, and various tracing agents, (6) the development and application of biochemical techniques for assessment of endocrine and neurotransmitter receptor events, (7) the development and continued refinement of microimaging systems, including the electron microscope, (8) advances in tissue preparation procedures that optimize such imaging technology, such as osmium preparations and freeze fracture techniques, (9) the invention of computerized tomography (CT), magnetic resonance imaging (MRI), and other noninvasive tools useful for evaluating the structure of the brain in vivo, (10) the development of functional imaging

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techniques, such as positron emission tomography (PET), single photon emission computed tomography (SPECT), and functional MRI (fMRI), and (11) numerous other major advances in biology, including the development of the fields of animal behavior and, importantly, molecular biology and molecular genetics, where recombinant DNA techniques, for example, have been used to identify and confirm the roles of many proteins involved in olfactory transduction. Obviously, in this introduction it is possible to mention only a few of the many important observations made in this century that have contributed to the present research climate. The areas selected for exposition were chosen, in part, on the basis of the amount of research they have generated and are continuing to generate. A number of these events or areas of research are mentioned in more detail elsewhere in this volume. A.

Electrophysiological Studies

A major 20th-century milestone that had a significant impact on modern chemosensory science was the development of means for electrophysiologically recording nerve impulses from the olfactory and gustatory receptors and pathways. Although crude electrical recordings were obtained from the olfactory system in the late 19th century (e.g., Saveliev, 1892; Garten, 1900), the sophisticated equipment necessary for reliable recordings, including sensitive electrodes, was not available until well into the 20th century [e.g., the oscilloscope was invented in 1922 (Erlanger and Gasser, 1937)]. The earliest extracellular recordings of single-cell gustatory primary afferent nerve activity were made by Zotterman (1935) and Pfaffmann (1941). Kimura and Beidler (1961) and Tateda and Beidler (1964) were first to record from single cells within the taste bud. Recordings of olfactory nerve fiber bundles were made by Beidler and Tucker (1955); recordings of singlecell olfactory receptor activity from extracellular electrodes were obtained by Hodgson et al. (1955) and Gesteland et al. (1963, 1965). The first evidence for between-species differences in single-cell neural firing was presented by Beidler and Tucker. (1955) and Pfaffmann (1955), a point that was later exploited by Frank (1973) in the use of the hamster as a model for species, such as the human, that exhibit salient responsiveness to sweet-tasting stimuli (see Chapters 9 and 35). Other manifestations of this advance in technology include (1) the recording of multicellular summated potentials at the levels of the vertebrate olfactory mucosa [the electro-olfactogram (EOG) (Hosoya and Yoshida, 1937; Ottoson, 1956)] and insect antenna [the electroantennogram (Schneider, 1957a,b)], (2) recording of transduction currents in isolated olfactory receptor cells (e.g., Kurahashi

and Shibuya, 1987; Firestein and Werblin, 1989), (3) measurement of ion channel activity in restricted patches of olfactory or taste cell membranes (Nakamura and Gold, 1987; Kinnamon et a1.,1988); (4) topographic analysis of responses within the olfactory epithelium and olfactory bulb (e.g., Kauer and Moulton, 1974; Kubie and Moulton, 1980; Leveteau and MacLeod, 1966; Mackay-Sim et al., 1982; Mozell, 1964, 1966; Moulton, 1976), (5) the application of voltage-sensitive dyes for recording electrical changes in chemosensory neural tissue (Kauer, 1988), (6) the recording of olfactory- and taste-evoked potentials in higher brain regions (e.g., Funakoshi and Kawamura, 1968, 1971; Kobal and Plattig, 1978; Plattig, 1968/1969), and (7) electrophysiological mapping of local, as well as more global, olfactory and gustatory brain circuits (e.g., Emmers et al., 1962; Getchell and Shepherd, 1975; Komisaruk and Beyer, 1972; Mori and Takagi, 1978; Motokizawa, 1974; Nicoll, 1971; Norgren, 1970; Pfaffmann et al., 1961; Rall et al., 1966; Scott and Pfaffmann, 1972; Shepherd, 1971, 1972; Tanabe et al., 1973, 1975). Electrophysiological data were critical for overturning the long-held notion that the entire “limbic lobe,” including the cingulate and parahippocampal gyri and hippocampus, were the primary projection regions of the olfactory system (e.g., Foville, 1844; Zuckerkandl, 1888). Thus, Hasama (1934) and Allen (1943) employed electrophysiological methods to accurately localize and delineate the olfactory cortex to a circumscribed region of the piriform lobe in the rabbit. Kaada (1951) localized, using similar methods, analogous regions in the monkey. B.

Studies of Receptor Function

Considerable progress has been made in the last several decades in elucidating the initial events that occur when a stimulus molecule activates either a taste or olfactory receptor cell, as is evidenced by the studies reviewed in detail in Chapters 4 and 34. A number of these studies of receptor function have been performed solely at the genetic, molecular genetic, or biochemical level, while others have made use of modern patch-clamp and dye-physiological measures (e.g., Korsching, 2002), sometimes in combination with biochemical ones, to address conductance changes that occur in the cell membrane following receptor activation. In both gustation and olfaction, stimulants are initially absorbed by the mucus overlying the receptor cells. In some cases, soluble proteins within the mucus may assist in the transport of hydrophobic molecules to receptor regions or possibly aid in stimulus removal and/or deactivation (Lee et al., 1987; Pevsner et al., 1988a,b; Schmale et al., 1990; Pelosi, 2001). Such binding proteins have been found in humans (Briand et al., 2002), as well as in insects (Kaissling, 2001), where they have been said to represent “a

Introduction and Historical Perspective

major evolutionary adaptation regarding the terrestrialization of the olfactory system, converting hydrophobic odorants into hydrophilic ones by increasing their aqueous solubility” (Vogt et al., 1991, p. 74). After traversing the mucus, chemosensory stimuli interact in some fashion with the receptor cell membrane (usually by binding with membrane-bound receptors, although other mechanisms may also be involved; e.g., Na salts and some other tastants pass through and/or activate ion channels directly; see Chapter 34). In olfaction, Bronshtein and Minor (1977) provided the first scientific evidence that these interactions occur on the olfactory cilia, an observation supported by subsequent workers (e.g., Menco, 1977; Rhein and Cagan, 1980; Lowe and Gold, 1991; see Chapters 2 and 4). In the taste system, as well as in the vomeronasal organ, interactions occur on microvilli associated with apical portions of the sensory receptor cells (Avenet and Lindemann, 1987, 1990; Heck et al., 1984; see also Biasi et al., 2001). In taste buds the importance of the apical region was first stressed by Renqvist in 1919 (see Kinnamon and Cummings, 1992). Although occluding junctions among cells in the apical region of the taste bud restrict most stimuli to that region, low molecular weight molecules may permeate these junctions (e.g., Holland and Zampighi, 1991). Until the application of electron microscopy to taste buds in the 1950s (de Lorenzo, 1958; Engström and Rytzner, 1956a,b), taste buds were believed to contain cilia, not microvillae. A considerable body of evidence indicates that the interaction of the stimulus molecule with the receptor membrane opens or closes, directly or indirectly (i.e., via second-messenger systems), membrane channels, resulting in a change in the flux of ions and alteration of the cell’s resting potential. Taste receptor cells possess a number of ion channels identical to some of those found in neurons (i.e., voltage-gated Na, Ca2, and K channels, as well as Ca-mediated cation channels and amiloride-sensitive Na channels) and release a neurotransmitter that activates the first-order taste neuron (Roper, 1989; Lindemann, 2001). In the case of olfaction, the receptor cell is the firstorder neuron, so changes in membrane potential, if of sufficient magnitude, generate the action potential (Frings, 2001; for vomeronasal organ, see Liman, 2001). Empirical evidence for considerable diversity in olfactory receptors was presented by Buck and Axel (1991) (see also Young and Trask, 2002). Under the assumption that olfactory receptors have elements in common with a large superfamily of surface receptors that evidence seven transmembrane domains and linkage to guanine nucleotide– binding proteins (G proteins) and second-messenger systems, these investigators synthesized oligonucleotides that coded for conserved (i.e., nearly invariant) amino acid sequences found among receptors from sensory systems other

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than olfaction. These oligonucleotides were then used as molecular probes. Eighteen clones were found that coded for proteins with seven transmembrane domains within olfactory tissue, but not within brain, retina, or various nonneural tissues. The variability in the amino acid sequences was found to be in regions of the molecule believed to be important in the binding of ligands in other receptor proteins with seven transmembrane domains. Based on this information, Buck and Axel concluded that there is considerable diversity in the genes that code for olfactory receptors and that ⬃1000 receptors are likely present, although many of these genes are now known to be pseudogenes. Other workers have shown that receptor-encoding complement any DNA can be expressed in nonneuronal cells, which, when stimulated with appropriate odorants, generate second-messenger responses (implying the receptors recognize odorants and couple to G proteins in the host cells) (Raming et al., 1993). Interestingly, the same receptor gene family described by Buck and Axel (1991) may also encode sperm cell receptors possibly involved in chemotaxis during fertilization (Parmentier et al., 1992). More recently, G-protein–coupled receptors have also been shown to be involved in vertebrate vomeronasal (Herrada and Dulac, 1997) and taste (Hoon et al., 1999) reception, as well as in invertebrate taste and olfactory reception (Bargmann and Kaplan, 1998; Clyne et al., 2000; Vosshall et al., 2000). A number of vertebrate olfactory (Krautwurst et al., 1998; Malnic et al., 1999; Touhara et al., 1999; Zhao et al., 1998), vomeronasal (del Punta et al., 2002), and taste (Nelson et al., 2001) receptors have now been functionally identified (for invertebrates, see Carlson, 2001; Wetzel et al., 1999). For example, Zhao et al. (1998) used an adenovirusmediated gene transfer procedure to increase the expression of a specific receptor gene in an increased number of receptor neurons in the rat olfactory epithelium, demonstrating ligand-specific increases in EOG amplitude. Krautwurst et al. (1998) employed a polymerase chain reaction (PCR) strategy to generate an olfactory receptor library from which cloned receptors were screened for odorant-induced responsiveness to a panel of odorants, as measured by an assay sensitive to intracellular Ca2 changes. Several receptor types with ligand specificity were found, including one differentially sensitive to the () and () stereoisomers of citronella. In 1989, a G-protein with 88% sequence identity to conventional Gs, designated Gaolf or Golf, was isolated in olfactory cilia (Jones and Reed, 1989) and found to the predominant signaling G-protein in olfactory receptor cells. Interestingly, Golf has also been found elsewhere in the CNS. For example, it has recently been implicated in the regulation of dopamine and adenosine action in the striatum (Hervé et al., 2001). Although G-proteins other than Golf (e.g., Gi2 and Go) have been identified in olfactory re-

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ceptor cells, they appear not to be involved in early transduction events, presumably assisting in such processes as axonal signal propagation, axon sorting, and target innervation (Wekesa and Anholt, 1999). However, Gi2 and Go appear to play primary roles vomeronasal sensory transduction (Biasi et al., 2001). A taste tissue–specific G-protein perhaps analogous to Golf, called “gustducin,” appears to be important in taste transduction, although its involvement seems to be primarily for sweet- and sour-tasting stimuli. Mice in whom the gustducin gene has been deleted still are able to discern such stimuli, although only at much higher concentrations, suggesting that multiple taste pathways may be involved or that G-proteins can substitute for gustducin in the signaling process (Ruiz-Avila et al., 2001) (see Chapter 34). Early studies indicated that the enzyme adenylyl cyclase, which is usually coupled to a G-protein, is highly active in olfactory (Kurihara and Koyama, 1972; Pace et al., 1985) and gustatory (Striem et al., 1991) tissues. Adenylyl cyclase activity is increased, typically in the presence of GTP, in ciliary preparations by a number of olfactory ligands (Pace et al., 1985; Shirley et al., 1986; Sklar al., 1986). A positive correlation was found to exist between an odorant’s ability to activate adenylyl cyclase activity in a frog ciliary preparation and both its perceived odor intensity to humans (Doty et al., 1990) and the magnitude of the EOG response it produces in frog epithelia (Lowe et al., 1989), suggesting that a functional relationship exists between the amount of adenylate cyclase activated and the intensity of odor perception. A number of odorants and tastants increase, in a doserelated manner, intracellular cyclic adenosine 3,5monophosphate (cAMP) in olfactory and taste receptor cells, respectively (Bruch and Teeter, 1989; Pace et al., 1985; Pace and Lancet, 1986; Sklar et al., 1986), thereby triggering the opening of cAMP-gated cation channels (Nakamura and Gold, 1987). In the case of taste, cAMPmediated responses may be limited to sweet- and/or bittertasting agents, although other pathways, including the activation of the cyclic guanosine monophosphate (cGMP), may be involved as well. cAMP likely plays a role in the modulation of the sensitivity of olfactory receptor neurons, such as during adaptation (Leinders-Zufall et al., 1996). Although it was believed, in the case of olfaction, that a second transduction pathway occurs in vertebrates (namely, that associated with the activation of the enzyme phospholipase C to produce the second messenger inositol triphosphate or IP3) (Breer and Boekhoff, 1991), recent data suggest this may not be the case, at least in mice (Gold, 1999). The discordant studies have used knockout mice in which genes have been deleted that are responsible for (1) an olfactory-specific adenylyl cyclase (Wong et al., 2000), (2) olfactory-specific cyclic-nucleotide-gated ion channels

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(Brunet et al., 1996), or (3) both the cyclic-nucleotidegated ion channel and Golf (Belluscio et al., 1998). In the channel knockout mouse, EOG responses to all odorants tested were eliminated, including those previously believed to be mediated by the IP3 system (Brunet et al., 1996). To date, IP3-gated channels have not been demonstrated in mammalian olfactory nerve cells using patch clamp techniques (Firestein et al., 1991; Lowe and Gold, 1993). C. Studies of the Olfactory Pathways in Transport of Agents from the Nose to the Brain A very important empirical observation, made in the first half of the 20th century, was that the olfactory nerve can serve as a conduit for the movement of viruses and exogenous agents from the nasal cavity into the brain (see Chapters 3 and 26). This route is direct, since the olfactory neurons lack a synapse between the receptive element and the afferent path. The existence this pathway for viral infection of the brain has been recognized for some time, as evidenced a number of studies from the 1920s and 1930s (see Clark, 1929; Hurst, 1936). For example, mice intraperitoneally inoculated with louping ill virus showed the first signs of central nervous system (CNS) localization of the virus in the olfactory bulbs. Mice whose olfactory mucosa was cauterized with zinc sulfate were partly protected against such infection (Barnet and Lush, 1938). Poliomyelitis virus, placed in the noses of primates, travels to the olfactory bulbs via the axoplasm of the olfactory nerves, rather than along the nerve bundle sheaths (Bodian and Howe, 1941a,b). In a pioneering paper, Armstrong and Harrison (1935) reported that monkeys could be protected against intranasal inoculations of poliomyelitis virus by previous lavage of the nose with solutions of alum or picric acid (or both). Subsequent studies (e.g., Schultz and Gebhardt, 1936) found that zinc sulfate gave a longer-lasting and higher degree of protection from poliomyelitis, leading to the prophylactic spraying of noses of children with this agent during poliomyelitis outbreaks in the late 1930s (Peet et al., 1937; Schultz and Gebhardt, 1937; Tisdall et al., 1937). Unfortunately, such spraying produced long-lasting, presumably permanent, anosmia in some individuals (Tisdall et al., 1938). Related to the observation that the olfactory nerves are a major carrier of viruses is the fact that the receptive elements of the olfactory system are exposed, to a large degree, to the vagaries of the external environment, making them susceptible to damage from bacteria, viruses, toxins, and other foreign agents. As reviewed in detail in Chapters 3, 26, and 27, there is a wealth of evidence that the olfactory mucosa is rich in enzymes that presumably minimize the deleterious influences and uptake of most xenobiotic

Introduction and Historical Perspective

agents into the olfactory receptor cells, including cytochromes P-450, flavin-containing monooxygenase, and aldehyde dehydrogenases and carboxylesterases. D. The Discovery That Taste Buds and Olfactory Receptor Cells Regenerate Another important 20th-century development was the discovery that both the gustatory and olfactory receptor cells can regenerate. Beidler and Smallman (1965) provided the first scientific demonstration that the sensory cells of the taste bud are in a dynamic state of flux and are constantly being renewed, with the more recently formed cells of the periphery migrating centrally to act as receptors for very limited periods of time. The observation that olfactory receptor cells, which are derived from ectoderm and which serve as the first-order neurons, can regenerate after they are damaged was first noted in mice by Nagahara (1940) and later confirmed in primates by Schultz (1960). This observation is particularly significant in that it is in conflict with the long-held notion that neurons in the adult animal are irreplaceable (see next section) and suggests that the olfactory system may contain the key to producing neural regeneration in a variety of neural systems (Farbman, 1992). However, questions remain as to why metaplastic respiratory epithelium often invades the region of the damaged olfactory epithelium and why, when such metaplasia occurs, the epithelium in that region may never convert to olfactory epithelium. Studies in which the olfactory epithelia of rodents were exposed to airborne or systemically administered toxic agents may shed some light on this question. Thus, the type of repair seems to correlate with the degree or extent of the initial epithelial damage (Keenan et al., 1990). For example, when the basilar layer of the mucosa is completely damaged, then metaplastic replacement with a respiratory-like epithelium occurs. When the damage is not marked or the toxic insult is not sustained, regeneration, usually with fewer or irregularly arranged cells, occurs. Closely related to the discovery of regeneration within the olfactory epithelium is the important observation made by Andres (1966, 1969) that mitotic cells, young sensory cells, mature sensory cells, and dying cells coexist within the olfactory epithelium (see Farbman, 1992, for a review). This suggested to Andres the hypothesis that the olfactory receptor cells were continually being replaced. The notion that olfactory receptor cells were in a state of flux received subsequent support by others (see Chapters 2–6; Moulton et al., 1970; Thornhill, 1970; Graziadei and Metcalf, 1971) and led to the idea that they are relatively short-lived. Hinds et al. (1984), however, found that a number of the olfactory receptor cells of mice reared in a pathogen-free environment survived for at least 12 months and hypothesized that olfactory nerve cell turnover involves recently formed or

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immature receptor cells that fail to establish synaptic connections with the olfactory bulb. This hypothesis implies that environmental agents play an important role in dictating which elements of the receptor sheet become replaced and that the rate of regeneration of the olfactory receptor cells is not genetically predetermined, as previously supposed (see Mackay-Sim and Kittle, 1991) (Chapters 5 and 29). The observation that improvement in olfactory function after cessation of chronic cigarette smoking occurs over a period of years and is dose-related (Frye et al., 1990) suggests that either turnover of the olfactory epithelial cell complement takes a much longer time than previously supposed or growth of olfactory epithelium into damaged areas is relatively slow and dependent on the extent of prior trauma, or both. The study of the regeneration of the olfactory neurons has been greatly enhanced by the ability to culture the olfactory mucosa in vitro (for review, see Mackay-Sim and Chuah, 2000). This was first demonstrated in the culture of olfactory organs from embryonic mice (Farbman, 1977) and used to show the importance of olfactory bulb in promoting differentiation of the olfactory sensory neurons (Chuah and Farbman, 1983). The next major development came with the investigations of dissociated cultures from embryonic and newborn rats (Calof and Chikaraishi, 1989; Pixley, 1992a). This allowed the growth factors regulating olfactory neurogenesis to be explored in the developing olfactory epithelium (DeHamer et al., 1994; Mahanthappa and Schwarting, 1993) and in the adult (MacDonald et al., 1996; Newman et al., 2000). Recently, normal targeting of glomeruli by olfactory receptor axons has been demonstrated in mice lacking functional olfactory cycle nucleotide-gated channels (Lin et al., 2000) and in mice lacking most intrabulbar GABAergic interneurons (Bulfone et al., 1998). Thus, establishment of the topographical map from the receptor cells to the glomeruli seems to require neither normal neural activity in these pathways nor cues provided by the major neural cell types of the bulb. Human olfactory neuronal progenitors have now been grown in vitro (Wolozin et al., 1992). This has been exploited to study biochemical changes in Alzheimer’s disease (Wolozin et al., 1993). Primary cultures of human olfactory mucosa (Féron et al., 1998; Murrell et al., 1996) have lead to investigations into the etiology of schizophrenia (Féron et al., 1999), and the vitro growth of olfactory ensheathing cells (Chuah and Au, 1991; Pixley, 1992b; Ramon-Cueto and Nieto-Sampedro, 1992). The latter glial cells assist sensory neuron regeneration (Doucette, 1984) and have, in fact, been employed in cell transplantation therapy for the damaged nervous system (Li et al., 1998; Lu et al., 2001; Ramon-Cueto et al., 2000; Ramon-Cueto and Nieto-Sampedro, 1994).

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E. The Discovery That Some Olfactory Bulb Cells Regenerate A long-held dogma regarding the nature of the CNS of vertebrates is now known to be false; namely, that the adult brain does not exhibit neurogenesis (for review, see Gross, 2000). Although early studies found mitotic figures within the walls of the lateral ventricle (e.g., Allen, 1912; Globus and Kuhlenbeck, 1944; Olpalski, 1934; Rydberg, 1932), definitive evidence that such cells represented neurogenesis awaited the development of the tritiated thymidine technique, the electron microscope, and immunohistochemistry (Gross, 2000). In the 1960s, Altman and his associates published a series of classic studies based upon tymidine autoradiography demonstrating neurogenesis in several brain regions of young and adult rats, including the olfactory bulb (Altman, 1969), the neocortex (Altman, 1963, 1966a), and the dentate gyrus of the hippocampus (Altman, 1963; Altman and Das, 1965). Regarding the olfactory bulb, proliferating cells were found within the subventricular zone lining segments of the lateral ventricles. These cells were found to reach the core of the olfactory bulb via the rostral migratory stream. Subsequent studies have confirmed and extended these observations (e.g., Luskin, 1993; Lois, Garcia-Verdugo and Alvarez-Buylla, 1996; O’Rourke, 1996), noting that the precursor cells invade the granule and periglomerular layers of the bulb, where they differentiate into local interneurons. A major differentiation is into GABAergic granule cells—the most numerous cells of the bulb. These stem-cell–related phenomena, which are only now beginning to receive widespread attention within the chemical senses community, are of considerable significance, as they indicate that the plasticity of the olfactory system goes far beyond simply replacing damaged neuroepithelial cells, and that continual cell replacement may play an integral role in olfactory perception. It is now known, for example, that reducing the numbers of interneurons recently generated via this process impairs the ability of an animal to discriminate among odorants (Gheusi et al., 2000). Moreover, enriching the odorous environment of mice enhances such neurogenesis and improves odor memory (Rochefort et al., 2002). The degree to which such processes influence, or are influenced by, endocrine state and various social processes is not well known, although interestingly glucocorticoids decrease, and estrogens increase, the rate of such neurogenesis within the hippocampus (Gould and Tanapat, 1999; Tanapot et al., 1999). F.

Functional Imaging Studies

A significant and rapidly evolving modern development in the study of the chemical senses is that of functional imag-

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ing. It has long been known or suspected that brain circulation changes selectively with neuronal activity (e.g., Broca, 1879; Mosso, 1881; Roy and Sherrington, 1890; Fulton, 1928), but was not until the late 1950s and early 1960s that the development of the [131I]trifluoroiodomethane ([131I)CF31]CF31) method provided a potential and novel means for quantitatively examining the influences of sensory, cognitive, and motor processes on local blood flow within regions of the brain (Landau et al., 1955; Freygang et al., 1958; Kety, 1960; Sokoloff, 1961). This early work led in the development of the [14C]2-deoxy-D-glucose (2DG) autoradiographic method for determining regional glucose consumption in animals (Reivich et al., 1971; Kennedy et al., 1975; Sokoloff et al., 1977, Sokoloff, 1981, 1982), and set the foundation for modern human functional imaging studies. Reivich et al. (1979) introduced the [18F] flurorodeoxyglucose method for assessing regional glucose metabolism, and Lassen et al. (1963) and Ingvar and Risberg (1965) subsequently developed and applied a procedure in which regional blood flow measurements could be established in humans by using scintillation detectors arrayed over the surface of the scalp. The refinement of such approaches led to the practical development of positron emission tomography (PET) (Ter-Pogossian et al., 1975; Hoffman et al., 1976), which was made possible by the earlier invention of x-ray computed tomography (CT) in 1973 (Hounsfield, 1973). The coincidence of these techniques provided the capability of mapping the regions with increased blood flow or glucose metabolism to specific regions of the brain in three-dimensional coordinates. Magnetic resonance imaging (MRI) technology emerged contemporaneously with the latter developments (e.g., Lauterbur, 1973). Based upon a set of earlier principles (Block, 1946; Fox and Raichle, 1986; Lauterbur, 1973; Pauling and Coryell, 1936), Ogawa et al. (1990) were able to demonstrate that changes in blood oxygenation could be detected, in vivo, with MRI, setting the stage for the development of functional MRI (fMRI). This phenomenon, known as the blood oxygen level–dependent (BOLD) signal, reflects the fact that blood flow changes more than oxygen consumption does in an activated region, reflected by a reciprocal alteration in the amount of local deoxyhemoglobin that is present, thereby altering local magnetic field properties. Details of fMRI, as well as other imaging procedures, are presented in Chapters 12 and 37. The first study to employ functioning imaging in the chemical senses was that of Sharp et al. (1975). These investigators injected four rats intravenously with 2-DG and immediately placed them in a sealed glass jar containing glass wool saturated with pentyl acetate. After 45 minutes the animals were sacrificed, and sections of the bulbs were appropriately prepared and autoradiographed. Two regions of heightened optical density were noted bilaterally, which

Introduction and Historical Perspective

tended to be centered in the glomerular layer, with variable spread into the external plexiform and olfactory nerve layers. Subsequent studies more clearly defined the regions of apparent activation (e.g., Sharp et al., 1977; Stewart et al., 1979), and resulted in the identification of a unique set of glomeruli in weanling rats responsive to odorants in their mothers’ milk (Teicher et al., 1980). The first published human olfactory PET study was that of Zatorre et al. (1992). These investigators found that odorants increased regional cerebral blood flow (rCBF) bilaterally in the piriform cortex, as well as unilaterally in the right orbitofrontal cortex. The first taste study employing PET was that of Small et al. (1997). Increased rCBF was noted, in response to citric acid, bilaterally within the caudolateral orbitofrontal cortex, and unilaterally within the right anteromedial temporal lobe and the right caudomedial orbitofrontal cortex. The first published fMRI report on olfaction was that of Yousem et al. (1997), who demonstrated (1) odor-induced activation of the orbitofrontal cortex (Brodmann area 11), with a mild right-sided predominance (in accord with the earlier PET study of Zatorre et al., 1992) and (2) unexpected cerebellar activation. Sobel et al. (1998a) noted that olfactory stimulation activated lateral and anterior orbitofrontal gyri of the frontal lobe, and that sniffing behavior, regardless of whether an odor is present, induces piriform cortex activation. These investigators, following up on the unexpected observation of cerebellar activation by odorants, subsequently demonstrated concentration-dependent odorant activation in the posterior lateral cerebellar hemispheres and activation from sniffing alone in the anterior cerebellum, most notably the central lobule (Sobel et al., 1998b) (see Chapter 12). G. The Animal Behavior Revolution Another large area of research activity that must be mentioned as having had a profound impact on modern chemosensory research is that of animal behavior and behavioral endocrinology (see Chapters 17–20, 41, 46). This field, which grew in geometric proportions after World War II, is still a major contributor to chemosensory studies. In addition to providing detailed explications of the many rich and often complicated influences of chemical stimuli on wide range of invertebrate and vertebrate behaviors (including, in mammals, behaviors related to aggression, alarm, suckling and feeding, mating, predator–prey relationships, social status appraisal, territorial marking, and individual and species recognition), this field has provided important methodology for assessing olfactory, gustatory, and vomeronasal function in animals, including preference paradigms (e.g., Richter, 1939; Mainardi et al., 1965), classical conditioning paradigms (Pavlov, 1927), conditioned

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aversion paradigms (e.g., Garcia et al., 1955), habituation paradigms (e.g., Krames, 1970), sniff rate analysis paradigms (e.g., Teichner, 1966), and operant conditioning paradigms using positive or negative reinforcers (Skinner, 1938). Furthermore, behavioral studies have been critical in the demonstration of the close association between neuroendocrine and chemoreception systems in both vertebrates and invertebrates and are critical for demonstrating the effects of various gene manipulations on smell- or tastemediated behaviors. A number of the chapters in this volume directly relate to this vast literature and, in some cases, provide means for assessing responses of animals to odorants (e.g., Chapters 18, 19, 20, 27, and 40). The reader is referred to the many general reviews of this topic (Albone, 1984; Doty, 1974, 1975, 1976, 1980, 1986; Johnston, 2000; Johnston et al., 1970; Leon, 1983; Marchlewska-Koj, 1983; Meredith, 1983; Mykytowycz, 1970; Slotnick, 1990; Slotnick and Schellinck, 2002; Smith, 1970; Stevens, 1975; Vandenbergh, 1983; Wysocki, 1979). According to Stürckow (1970), the studies by Barrows (1907), von Frisch (1919), and Minnich (1921) were seminal for the development of studies of insect chemosensory behavior and physiology, even though earlier, more equivocal, studies had been performed (e.g., Hauser, 1880). Barrows (1907) devised the first insect olfactometer and found, in the pomice fly (Drosophila ampilophila), that different degrees of responding were obtained from different concentrations of chemical attractants. Von Frisch (1919) demonstrated that bees could be trained to fly to a fragrant odor using simple reinforcement and later found the location of the olfactory sensilla to be on the eight distal segments of the antennae (von Frisch, 1921, 1922). Minnich (1921, 1926, 1929) explored the responses of various body parts of butterflies, certain muscid flies, and the bee to taste solutions. For example, he found that they extended their proboscises when their tarsi or certain mouth parts were touched with a sugar solution. These and other studies led to electrophysiological studies of the chemoreceptive systems of insects by Dethier (1941), Boistel (1953), Boistel and Coraboeuf (1953), Kaissling and Renner (1968), and Schneider (1955, 1957a,b). A number of important studies published in the 1950s, 1960s, and early 1970s demonstrated a close association between olfaction, social behavior, and reproductive processes in rodents and other mammalian forms. Pioneering reports on this topic include those that showed that volatiles from male and female mice influence the timing of estrous cycles (Lee and Boot, 1955; Whitten, 1956; Whitten et al., 1968), that urine odor from unfamiliar male mice can block the pregnancy of female mice (Bruce, 1959; Bruce and Parrott, 1960), and that chemical stimuli can accelerate the onset of puberty in mice (Vandenbergh, 1969). Other important studies demonstrated that olfactory bul-

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bectomy, anesthetization, or damage to the olfactory receptor region or vomeronasal organ, alone or in combination, can dramatically influence mating behavior, depending on the species involved [e.g., in the male or androgenized female hamster, anesthetization or damage of these systems can eliminate male copulatory behavior (Doty and Anisko, 1973; Doty et al.,1971; Murphy and Schneider, 1970; Powers and Winans, 1973, 1975; Winans and Powers, 1974)]. Such phenomena have been demonstrated to one degree or another in a wide variety of mammals and have important implications for animal ecology, husbandry, and perhaps even human behavior. Other studies of this period that had a considerable impact on the field of mammalian social behavior include those that examined, in a systematic manner, sexual odor preferences. Godfrey (1958), for example, found that estrous female bank voles (Clethrionomys) preferred homospecific male odors over heterospecific male odors and that hybrids were discriminated against. Le Magnen (1952) demonstrated that adult male rats (Rattus norvegicus) prefer the odor of receptive females to nonreceptive ones, whereas prepubertal or castrated males do not (unless they have been injected with testosterone). Beach and Gilmore (1949) noted that sexually active male dogs, but not a sexually inactive male dog, preferred estrous to nonestrous urine. This and other work led to a number of carefully designed studies by Carr and associates in the 1960s, which sought to determine the influences of sexual behavior and gonadal hormones on measures of olfactory function. Carr and Caul (1962) demonstrated that both castrate and noncastrate male rats can be trained to discriminate between the odors of estrous and nonestrous females in a Y-maze test situation, implying that the preference phenomenon observed by Le Magnen (1952) was not due to castrationrelated influences on olfactory discrimination ability, per se. Carr et al. (1965) subsequently demonstrated the important role of sexual experience in producing strong preferences in male rats for estrous over diestrous odor and in female rats for noncastrate male odors over castrate male odors. These investigators also showed that sexually inexperienced females preferred male noncastrate odors if they were administered gonadal hormones that induced estrus.* These general findings have been observed in a wide range of species, although some species differences do exist and castration has been shown to mitigate the increase in detection performance of rats that follows repeated testing (Doty and Ferguson-Segall, 1989; for reviews, see Brown and Macdonald, 1985; Doty, 1974, 1976, 1986). *In an unpublished M.A. thesis, Keesey (1962) found that sexually experienced, but not sexually inexperienced, male rats preferred the odor of female urine collected during proestrus than that collected during diestrus.

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Animal behavior studies in the 1980s contributed significantly to the understanding of the function of vomeronasal organ which was described histologically in many species in the 19th century, but whose function was unknown (for review of the early literature, see Wysocki, 1979). In the mouse, removal of the vomeronasal organ eliminates the surge in luteinizing hormone (LH) and subsequent increase in testosterone that ordinarily follows exposure of male mice to an anesthetized novel female mouse or her urine. However, this does not occur following exposure to an awake female mouse, suggesting that several sensory cues can produce the LH surge (Coquelin et al., 1984; Wysocki et al., 1983). In both mice and hamsters, vomeronasal organ removal impairs male sexual behavior, particularly in animals that have had no prior adult contact with females (Meredith, 1986; Wysocki et al., 1986). In mice whose vomeronasal organs have been removed soon after birth, long-lasting influences on male sexual behavior in adulthood have been noted (Bean and Wysocki, 1985). Vomeronasal organ removal also greatly decreases aggression in male house mice, particularly those that have not had much fighting experience with other males (Bean, 1982; DaVanzo et al., 1983; Wysocki et al., 1986). There is now considerable evidence that the adult human has a vomeronasal lumen and at least a rudimentary vomeronasal gland, although no neural connections have been described and the weight of the evidence suggests it is vestigial (Doty, 2001; Smith and Bhatnagar, 2000). A significant event for the field of odor communication was the coining of the term “pheromone” in insects for “substances which are secreted to the outside by an individual and received by a second individual of the same species, in which they release a specific reaction, for example, a definite behavior or a developmental process” (Karlson and Lüscher, 1959, p. 55). This term, which unfortunately has been applied by some workers to nearly any chemical involved in chemosensory communication in a wide variety of species, has permeated most areas of biology. This term replaced an earlier term (ectohormone) and conjures up the idea that the social organization of animals is akin to the endocrine organization of an organism, with disparate parts being influenced by chemicals that circulate within the social milieu. For many insects, this notion seems quite appropriate, given the high degree of stereotypical behavior and evidence for comparatively simple stimuli that induce behavioral or endocrinological changes. However, for many vertebrates, particularly mammals, the pheromone concept is of questionable value, as the term itself has little operational utility and many behavioral and endocrine responses said to be mediated by pheromones are either learned, induced by stress, or not unique to olfaction, being mimicked by other types of sensory stimulation (see Chapter 17).

Introduction and Historical Perspective

Pioneering behavioral studies of mammalian taste function began in the 1930s, heralded by experiments that sought to explain so-called specific hungers, e.g., salt craving in patients with adrenal gland hypofunction. In seeking to determine whether alterations in taste function are responsible for increased NaCl intake of adrenalectomized rats, Richter (1936, 1939) developed the two-bottle taste test (see also Richter and Campbell, 1940). In this test, differential fluid intake from two bottles, one of which contains a tastant (e.g., a NaCl solution) and the other water alone, is recorded over a period of time. The lowest concentration of the tastant that produces a differential intake is taken as the threshold measure. Although this pioneering behavioral procedure provided a means for measuring a preference threshold, postingestional factors may alter the behavioral response, and such a threshold is conceptually different from a sensory threshold. Thus, a lack of preference between two solutions need not reflect an inability to discriminate between them [see Chapter 19 and Stevens (1975) for reviews of analogous procedures for olfaction]. Subsequent workers, including Carr (1952), Harriman and MacLeod (1953), Morrison (1967), and Morrison and Norrison (1966), utilized shock-avoidance paradigms or operant conditioning paradigms that provided positive reinforcement to establish NaCl threshold values—values that were much lower than those obtained using Richter’s procedure and which corresponded more closely to neural thresholds. Numerous modifications of behavioral procedures for assessing taste function in mammals have since been developed which incorporate general principles that evolved from these pioneering behavioral studies (e.g., Brosvic et al., 1985, 1989; Spector et al., 1990) (see Chapter 41). Analogous procedures have been developed in olfaction (e.g., Bowers and Alexander, 1967; Braun et al., 1967; Braun and Marcus, 1969; Eayrs and Moulton, 1960; Goff, 1961; Henton, 1969; Moulton, 1960; Moulton and Eayrs, 1960; Pfaffmann et al., 1958; Slotnick and Katz, 1974; Slotnick and Ptak, 1977) (for reviews, see Chapter 27 and Slotnick and Schellinck, 2002). Another noteworthy development in behavioral testing was that of the conditioned aversion paradigm (Garcia et al., 1955, 1974). In one variant of this technique, an animal is allowed to drink or smell a novel tastant or odorant and is then injected with an agent that produces nausea (e.g., lithium chloride). The animal quickly learns to avoid the novel stimulus as a result of a single aversive conditioning experience, even if the aversion occurs long after the presentation of the sensory stimulus. This procedure can be used to establish whether detection of a given stimulus is present and is particularly useful for assessing cross-reactivity of stimuli (i.e., the extent to which a stimulus has elements in common with other stimuli). One of the more novel applications of this technique was by Smotherman

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(1982), who demonstrated that the olfactory system of rats is functional in utero. In this study, unborn rat pups (gestation day 20) received in utero injections of apple juice and lithium chloride. After birth, these individuals showed evidence of having developed a conditioned aversion to the odor of apple juice. H.

Clinical Chemosensory Studies

Considerable progress in understanding chemosensory disorders has been made in the last few decades, as reviewed in detail in Chapters 21–30 and 40–45 of this volume. The proliferation of clinical studies has been fueled, in large part, by the widespread commercial availability of standardized psychophysical tests of olfactory function (e.g., Doty et al., 1984a, 2000, 2001). It is now widely appreciated that smell loss is markedly depressed in elderly persons (Doty et al., 1984b), and that the most common causes of permanent smell loss are (1) upper respiratory viral infections, (2) head trauma, and (3) nasal and sinus disease (e.g., Deems et al., 1991). Moreover, it appears that these disorders largely reflect damage to the olfactory neuroepithelium, as revealed by autopsy and biopsy studies (Douek et al., 1975; Hasegawa et al., 1986; Jafek et al., 1989, 1990; Moran et al., 1992). Most complaints of taste loss reflect the loss of olfactory function, and flavor sensations are largely derived from retronasal stimulation of the olfactory system during active deglutition (Mozell et al., 1969; Burdach and Doty, 1987). We now know that the olfactory system seems more susceptible to damage than the taste system, although damage to regional lingual afferents is particularly striking in old age (Matsuda and Doty, 1995), and taste sensitivity is directly related to the number of taste buds or papillae stimulated, regardless of whether stimulation is by chemicals or by electrical current (Doty et al., 2001; Miller et al., 2002; Zuniga et al., 1993). Moreover, it has become increasingly apparent that many medicines, including a number of antibiotics, antidepressants, antihypertensives, antilipid agents, and psychotropic drugs, can produce alterations of the taste system (e.g., severe dysgeusia), alone or in combination with alterations in the smell system (Schiffman, 1983; Schiffman et al., 1998, 1999a,b, 2000). Importantly, recent studies suggest that damage to one of the major taste nerves (e.g., one chorda tympani) may release inhibition on other taste nerves (e.g., the contralateral glossopharyngeal nerve), resulting in hypersensitivity to some tastants and the production of phantom dysgeusias (Lehman et al., 1995; Yanagisawa et al., 1998). A major advance in the last few years is the discovery that smell loss is among the first, if not the first, signs of such common neurodegenerative diseases as Alzheimer’s

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disease (AD) and idiopathic Parkinson’s disease (PD), and that disorders sharing similar motor signs to PD, such as progressive supranuclear palsy (PSP) and MPTP-induced parkinsonism (MPTP-P), are largely unaccompanied by such loss (see Chapter 23). Such observations imply that olfactory testing can be of value not only in the detection of some neurodegenerative disorders early in their development, but in differential diagnosis. Indeed, odor identification testing accurately differentiates between patients with AD and those with major affective disorder (i.e., depression) (Solomon et al., 1999; McCaffrey et al., 2000). Interestingly, longitudinal studies have now appeared indicating that olfactory dysfunction can be predictive of AD in individuals who are at risk for this disorder, particularly when considered in relation to other risk factors (Bacon et al., 1998; Graves et al., 1999; Devanand et al., 2000). The only neurodegenerative disorder for which a definitive physiological basis has been found to date, however, is multiple sclerosis, where a –0.94 correlation has been observed between odor identification test scores and the number of plaques, as measured by MRI, in the subtemporal and subfrontal regions of the brain (Doty et al., 1997, 1998, 1999). While the olfactory bulbs of patients with schizophrenia are markedly reduced in size, the functional significance of this is yet to be elucidated (Turetsky et al., 2000). Diseases or disorders in addition to those noted above that have been found to be associated with smell loss include severe alcoholism, amyotrophic lateral sclerosis (ALS), chronic obstructive pulmonary disease, cystic fibrosis, epilepsy, the Guam ALS/PD complex, head trauma, Huntington’s disease, Kallmann’s syndrome, Korsakoff’s psychosis, pseudohypoparathyroidism, psychopathy, restless leg syndrome, schizophrenia, seasonal affective disorder, and Sjogren’s syndrome. Neurological disorders in which olfaction seems to be spared, in addition to PSP and MPTP-induced PD, are corticobasal degeneration, depression, panic disorder, essential tremor, and multiple chemical hypersensitivity (for review, see Doty, 2001). In addition to traditional medical means for treating or managing diseases responsible for decreased olfactory function, surgical intervention at the level of the olfactory neuroepithelium (e.g., by selectively ablating or stripping away the diseased tissue) or the olfactory bulb (e.g., by removal of one or both olfactory bulbs in an anterior cranial approach) has successfully eliminated or markedly reduced the symptoms of some forms of chronic dysosmia or phantosmia (see Chapter 22). Recent advances in understanding the deleterious influences of oxygen radicals on neural tissue, as well as changes that occur in olfactory tissue at menopause, have led to ongoing studies of the prophylactic potential of antioxidants, hormones, and other agents in mitigating toxin-induced damage to the olfactory mucosa (e.g., Dhong et al., 1999).

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VI.

CONCLUSIONS

In this introduction, a brief description of the significant role that tastes and odors have played throughout the course of human history has been presented. In addition, a number of key studies, events, and trends have been identified which form the backdrop of much of today’s chemosensory research enterprise, providing perspective for the chapters that follow. The chapters of this volume provide detailed contemporary information related to most of these trends and address the important role of chemosensory science in both basic and applied situations. Until recently, the chemical senses have engendered, relative to the other major senses, comparatively little attention on the part of the scientific and medical communities. This is due to a number of factors, including (1) the lack of a simple physical dimension analogous to wavelength that correlates with olfactory or taste quality, (2) the fact that chemosensory dysfunction rarely produces obvious influences on such everyday activities as locomotion and social interaction, and (3) the widespread belief that the chemical senses are of little importance to humans. As Boring (1942, p. 437) so aptly put it: If human culture could have been founded on a dog’s life, smell and not vision would be the great chapter of sensory psychology, and Helmholtz would have written three huge volumes of a Handbuch des Physiologischen Geruchs, as well as a Die Lehre von den Geschmacksempfindungen als Physiologische Grundlager für die Theorie der Geschmackslehre. In the not-too-distant future, the amount of knowledge within the chemical senses may well rival the level of knowledge of the visual sciences that was evidenced in Helmholtz’s classic 19th-century three-volume treatise (Helmholtz, 1851–1866/1924). When this occurs, the ramifications will be far-reaching, as suggested by Lewis Thomas (1983, p. 14): I should think that we might fairly gauge the future of biological science, centuries ahead, by estimating the time it will take to reach a complete, comprehensive understanding of odor. It may not seem a profound enough problem to dominate all the life sciences, but it contains, piece by piece, all the mysteries. ACKNOWLEDGMENTS I thank Drs. Marion Frank, Alan Mackay-Sim, Bert Menco, Igor Kratskin, David Smith, Gabriele Ronnett, Martin Witt, and Klaus Reutter for their constructive comments on this introduction. This paper was supported, in part, by Grants PO1 DC 00161, RO1 DC 04278, and RO1 DC 02974 from

Introduction and Historical Perspective

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1 Anatomy of the Human Nasal Passages Dean M. Clerico Valley ENT, Forty Fort, Pennsylvania, U.S.A.

Wyatt C. To and Donald C. Lanza The Cleveland Clinic Foundation, Cleveland, Ohio, U.S.A.

I. A.

1993). However, it was not until the work of Max Schultze, in 1856, that the first reasonably accurate description of human olfactory receptor and supporting cells was presented (Zippel, 1993). In 1892, von Brunn set out to determine the precise extent of olfactory mucosa within the nasal cavity. He studied four men ages 30–45 years, who succumbed by decapitation. According to von Brunn, the position of the olfactory neuroepithelium is restricted to the superior turbinate and nasal septum. In 1908, Read characterized, in a Ph.D. thesis, the olfactory neuroepithelium in a 1-year-old child and a “30–40 year old man”. Read suggested that the distribution of the olfactory mucosa was more extensive than that indicated by von Brunn but less than that depicted by Scarpa and others. Read also found that the olfactory nerve fibers originating in the neuroepithelium ascended vertically through the cribriform plate without anastomoses or formation of a plexus, as had been previously held. Variations in the distribution of olfactory neuroepithelium are now well documented and seem to be related to a variety of factors. In 1941, Smith recorded that atrophy of the olfactory nerves in adults was very common. He postulated that such atrophy might help prevent the entry of neurotropic viruses. In 1970, Naessen described a macroscopic staining technique to visualize the extent of olfactory neuroepithelium. He subsequently (1971) explored the effects of aging upon the morphology of the olfactory neuroepithelium. In 1984, Nakashima,

INTRODUCTION Historical Perspective

Scientific advancements leading to our current understanding of olfaction have evolved considerably since the midseventeenth century. Until that time, it was generally held that olfaction occurred via the direct access of odors to the brain. In 1655, Schneider suggested that the sense of smell did not occur as a result of such air passage. Instead, he reported that the superior aspects of the nasal mucosa were extremely sensitive, and he suspected this tissue was responsible for olfaction. Moreover, he proposed that the secretions that drained through the nose were not produced by the brain, as previously maintained. He believed that the nasal membranes themselves produced these secretions (Zippel, 1993). Subsequent study of olfaction in humans has focused upon the distribution of olfactory nerves and the nature of olfactory neuroepithelium. However, further advancements in delineating the anatomy of olfactory tissue have been limited by two key issues: its relative inaccessabilty in living humans and its vulnerability to rapid decomposition in the immediate post-mortem period. Thus, without suitable fixatives, histological evaluations prior to the twentieth century had to be performed quickly and immediately after death. In 1785, Antonio Scarpa described an extensive plexus of olfactory nerve fibers within the human nose (Zippel, 1

2

Clerico et al.

Kimmelman, and Snow evaluated 26 specimens from fetal age through the ninth decade of life. These researchers found olfactory neuroepithelial degeneration to be characteristic of adult human tissue. The age-related changes were seen in the cellular arrangement and topographic distribution of the olfactory mucosa. Whether or not age alone could be responsible for such changes remains unclear. Alterations in the olfactory epithelium seem to be subject to a wide variety of factors, which include exposures to viral and bacterial infections (see Chapters 3, 5, and 26), head injury (see Chapter 30), neurodegenerative disorders (see Chapters 23 and 24), and chemical exposures (see Chapters 25 and 27). Importantly, metabolic changes may predispose an individual’s olfactory system to greater susceptibility to damage from such environmental factors (Rehn et al., 1981). It should be emphasized that the ability to appreciate smells goes beyond the proper function of the olfactory neuroepithelium. It is contingent upon the correct functioning of all components in the olfactory system. Stimulation of this system begins when odorants are delivered through the nasal passages to the olfactory neuropithelium. Physiological or pathological alterations of the nasal passages can alter the perception of odors. Thus, understanding nasal anatomy and the dynamic nature of these passageways is essential to a complete understanding of human olfactory function. In this chapter, we review nasal anatomy as it relates to human olfaction. When appropriate, structures with synonyms are followed by their alternate names in parentheses. The terminology used to describe anatomical relationships of the nose is presented in Figure 1. It should be noted that the nasal anatomy has not been thoroughly investigated in all ethnic groups. Therefore, some of the anatomical description in this chapter may not generalize to non-Caucasian individuals. The overview that follows is a brief introduction to the major components that form the nasal passages. B.

Anatomical Overview

The nasal passages are complex and dynamic conduits through which respiration begins. They communicate with the external environment through the nose, a pyramidshaped, bony, cartilaginous, and soft tissue structure which rests upon an elliptical bony opening into the midface (Fig. 1). This elliptical opening is known as the pyriform aperture (anterior choanae) and is the anterior boundary of the nasal cavity (nasal fossa, cavum). The nose and nasal cavity are separated into two nasal passages by a central partition called the nasal septum. The lateral wall of each nasal cavity is shaped by three (or occasionally four) bony

Figure 1 Lateral view of external nose: (1) ascending process of maxilla; (2) accessory cartilages; (3 and 4) lateral and medial crura of lower lateral cartilages; (5) upper lateral cartilage; (6) nasal bone.

protuberances known as turbinates (conchae). A cleft (meatus) is present lateral to each of these turbinates. Four paired groups of paranasal sinuses are ventilated through these meati. Posteriorly, the nasal passages end at the posterior choanae. The posterior choanae are bounded by the posterior aspects of the nasal septum and inferior turbinate; they represent the anterior openings to the nasopharynx. The majority of the bony and cartilaginous structures that support the nasal cavity are covered with mucussecreting epithelia referred to as mucous membranes (mucosa, Schneiderian membranes, tunica mucosa nasi). Secretions from the nasal mucosa are regulated by the innervation and vascular supply of the nasal cavity. These regulators are in turn affected by many factors. The nasal structures that most influence the delivery of odorants are emphasized in the more detailed description of the airway that follows. II.

THE NOSE

Externally, the nose can be divided into three separate segments (Lanza et al., 1991). The superior one third is composed of the paired nasal bones fused with the ascending (frontal) processes of the maxilla. The middle third of

Anatomy of the Human Nasal Passages

the nose arises at the caudal end of the nasal bones, where they join the cephalic portions of the upper lateral cartilages (lateral cartilages). The lower third of the nose originates at the juncture of the upper and lower lateral cartilages (alar cartilages). The lower lateral cartilages are comprised of the medial and lateral crura (Fig. 1). These cartilaginous support structures help maintain the caudal one third of the nose. The medial crura of the lower lateral cartilages abut one another at the midline to form the most caudal partition known as the columella. The columella and nasal septum divide the nose into separate nasal passageways. The nasal septum, which lies directly posterior to the columella, supports the caudal one third of the nose. Minor changes in the three-dimensional structure of the caudal one third of the nose, secondary to either trauma or surgery, can have a dramatic affect upon nasal airflow, as well as cosmesis. The nasal musculature regulates nasal airflow by controlling the aperture of the nares and nasal valve regions (see Chapter 21 and below). A.

Nasal Septum

The nasal septum (Fig. 2) is composed of three anatomical regions: the membranous septum, the cartilaginous septum, and the bony septum. However, a small portion of this vertical midline partition is created by contributions from the maxillary and the palatine bones. Both of these bones send up vertical crests measuring 3–10 mm in height to which the cartilaginous and bony septum are attached (Lang, 1989). The membranous septum is the most anterior portion of the septum and is comprised of squamous epithelium overlying connective tissue. It extends from the cephalic border of the columella to the caudal end of the cartilaginous septum. The cartilaginous septum (quadrangular or quadrilateral cartilage) is situated just posterior to the membranous septum and traverses the nose and nasal cavity. Its hyaline cartilage is 2– 4 mm thick and is covered by mucoperichondrium (Lang, 1989). Its basal attachment to the maxillary crest is termed the footplate. The cartilaginous septum widens at several locations, which include its base, its junction with the upper lateral cartilages, and the anterior septal body (anterior tubercle, septal intumescence). The anterior septal body is a thickened area of mucosa that has characteristics resembling erectile tissue. This is situated on the septal cartilage just anterior to the middle turbinates. According to Lang (1989), Zuckerkandl noted in 1884 that this body marks the entrance to the olfactory cleft. Thin cartilaginous strips present at the base of the quadrilateral cartilage are known as the paraseptal cartilages (Jacobson’s cartilage). They are present in most adults and may ossify to form paraseptal ossicles (Lang, 1989). The

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Figure 2 Schematic diagram, representing a sagittal view of nasal septum depicting (a) artery, (aa) arteries, and (n) nerves. A star marks the perpendicular plate of ethmoid and the (*) indicates the quadrangular cartilage. (From Lanza et al., 1990.)

vomeronasal organ (Jacobson’s organ, Ruysch tube) may be identified as a blind pouch in the septal mucosa. The opening to the vomeronasal organ, first located by Ruysch in 1703 (Bahtnagar, 1996), is located near the base of the nasal septum approximately 10–17 mm posterior to the anterior nasal spine (Smith et al., 1998). The documented prevalence of this organ varies. Moran et al. (1991) used an operating microscope to identify bilateral vomeronasal organs in all 200 patients examined. Using anterior rhinoscopy, Garcia-Velasco and Moudragon (1991) was able to identify the vomeronasal organ in 808 of 1000 patients. Won et al. (2000) performed rigid nasal endoscopy to identify the organ in 22 of 78 patients. A more detailed discussion of the vomeronasal organ is found in Chapter 46. The bony septum lies directly posterior to the cartilaginous septum and is thus situated within the nasal cavity. It is formed by the vomer and perpendicular plate of the ethmoid. Both of these bones are covered by mucoperiosteum (Fig. 2). Deviations in the septum are extremely common and may result from a number of causes. Most are asymptomatic but, when severe, can lead to bilateral nasal obstruction and anosmia. The junction of the cartilaginous septum with the bony septum is a common site for septal spurs to occur (a form of septal deviation). Disruption of the septum in childhood by either trauma or surgery has been reported by some investigators to stunt nasal growth (Farrior and Connolly, 1970; Jugo, 1987).

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B.

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Nasal Vestibule and Nasal Valve

The nares open into the anterior nasal chambers, which are known as the vestibules. They are lined by keratinizing squamous epithelium, a hair-bearing epithelium containing sweat and sebaceous glands. The nasal hairs are known as vibrissae. Caudally, the vestibule is bounded by the free margin of the ala and nasal sill (Fig. 3). Posteriorly, the vestibule leads to the pyriform aperture and the nasal cavity. The line demarcating the junction between the skin of the nasal vestibule and the mucosa of the nasal fossa is called the limen vestibuli. Superiorly, this transition line roughly corresponds with the cephalic border of the lower lateral cartilages. Inferiorly, the limen vestibuli approximates the location of the pyriform aperture. The floor of the vestibule, at least in Caucasians, usually lies at a slightly lower level than the inferior rim of the pyriform aperture. The narrowest portion of the nasal passage is the functional segment known as the nasal valve area. It is situated within the nasal vestibule. The superolateral margin of the valve area is the caudal border of the upper lateral cartilage. The medial boundary is the quadrilateral cartilage. Inferiorly and posteriorly, its limits are the pyriform aperture and anterior portion of the inferior turbinate (Bridger and Proctor, 1970; Kern, 1978). The nasal valve area (Fig. 3) is distinguished from the nasal valve. The nasal valve is a specific slit-like structure situated between the caudal ends of the upper lateral cartilages and the septum. In Caucasians it is the major flowlimiting segment in the entire airway, accounting for about 50% of total resistance to respiratory airflow (Cole, 1993). Whether or not this is true for other ethnic groups is not well established. The velocity of airflow through the valve during normal breathing approaches gale-force speed (Cole, 1993). Thus, even small vestibular lesions, such as cysts and papillomas, can have a substantial impact upon airflow at the entrance of the nose. Although anatomical boundaries can be assigned to the nasal valve area and the nasal valve, these structures are best considered functional segments whose anatomical boundaries may vary from individual to individual. They are

Figure 3 Basal view of the external nose and nasal vestibule demonstrating the nasal valve area. (From Lanza et al., 1990).

positioned as described in the previous paragraph. Ethnic origin, nasal trauma, and gender are just a few of the important variables that can affect the components and positioning of the flow limiting segment to each nasal passage. Several theories exist concerning the function of the nasal valve. Inhalation against upper airway resistance produces increased intrathoracic pressure. This may aid alveolar gas exchange by prolonging the expiratory phase of breathing. With regard to olfaction, the nasal valve disrupts the laminar airflow entering the nares. The resultant turbulent stream in the nasal cavity can promote interaction of odorants with the olfactory neuroepithelium (Berglund and Lindvall, 1982). The vestibular counterpart to the nasal valve is the culde-sac (diverticulum, infundibulum). It represents a dilation of the lateral vestibule between the caudal border of the upper lateral cartilages and the cephalic border of the lower lateral cartilage. Cottle (1987) suggested that the cul-de-sac and the nasal valve together represent a series of baffles for temperature and humidity control of respired air. C.

Nasal Musculature

The muscles of the nose can be categorized into those that elevate, depress, dilate, and compress its structure (Tardy and Brown, 1990). While all nasal muscles may have an impact upon both appearance and function, some appear to play a greater role in affecting mimetic expression, whereas others play a greater role in affecting airflow. Typically, muscle groups function synergistically to achieve either effect. Muscles of the nose are compartmentalized by two aponeuroses: the superficial muscular aponeurotic system (SMAS) and the perichondrial aponeurosis. The muscles lie deep to the SMAS (Daniel and Letourneau, 1988; Tardy and Brown, 1990). The muscle group that elevates includes the procerus, levator muscle of the upper lip and ala, and the anomalous nasi. The depressor group includes the alar portion of the nasalis muscle and the depressor nasi septi labii. The depressor nasi septi labii depresses the membranous septum and draws the nasal tip downward, thereby narrowing and elongating the vestibule. It also contributes to expanding the nares during deep inspiration. The compressors of the nose include the transverse portion of the nasalis and the compressor narium minor. According to Tardy (1990), the dilator group includes the anterior dilator naris. However, Fomon et al. (1950) argue that the same effect is achieved with the alar portion of the nasalis (Fig. 4). All of the nasal muscles are innervated by the lower zygomatic and buccal branches of the facial nerve. The procerus receives additional neural input from the temporal branch of the facial nerve. In 1977, Sasaki et al. used electromyography

Anatomy of the Human Nasal Passages

Figure 4 Nasal musculature: (1) medial fascicle of procerus muscle; (2) levator of the upper lip; (3) levator of the upper lip and ala; (4) anterior dilator naris; (5) compressor narium minor muscle; (6) orbicularis oris muscle (7) depressor septi nasi labii; (8) transverse (a) and alar (b) portions of the nasalis muscle; (9) anomalous nasi muscle; (10) lateral fascicle of procerus muscle. (Adapted from Tardy and Brown, 1992.)

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The ethmoid bone may be conceptualized as a horizontal bony plate from which a series of parallel vertical plates emanates (Fig. 5). The center portion of the horizontal plate is known as the cribriform plate (lamina cribrosa). As its Latin root implies, this sieve-like structure is perforated with multiple openings known as foramina. Each side of the cribriform plate contains between 20 and 71 foramina (Lang, 1989). Through these foramina pass the fila olfactoria, which are the coalescence of unmyelinated axonal filaments arising from the sensory neuroepithelium in the olfactory cleft. The olfactory cleft is the space situated between the medial surface of the turbinates (middle and superior) and the bony septum (perpendicular plate of the ethmoid) (Figs. 5 and 6) (Douek et al., 1975; Lanza et al., 1993; Lovell et al., 1982). Axons from the olfactory cleft relay their messages centrally through synapses in the olfactory bulbs. The cribriform plate is divided at the midline into approximately equal halves. Superiorly it is divided by the crista galli and inferiorly by the perpendicular plate of the ethmoid. The crista galli is occasionally pneumatized and may even be involved with disease extending from the ethmoid sinus. The perpendicular plate of the ethmoid forms the superior bony portion of the nasal septum.

to demonstrate that the nasal dilator muscles functioned in direct correlation with ventilatory resistance. Cole et al. showed in 1985 that electrical activity recorded with alar electromyograms ceased with mouth breathing. III.

THE NASAL CAVITY

Each nasal cavity can be thought of as a modified box that is open at opposite ends, with a roof, a floor, and two side walls. The anterior limit of the nasal cavity is the pyriform aperture and its posterior limit the posterior choanae. These bony constituents and the soft tissue elements, including the vasculature, innervation, and epithelium, are discussed below.

A.

Osteology

1.

Paranasal Sinuses

a. Ethmoid Complex and Sinuses. Since both the olfactory bulbs and the olfactory neuroepithelium rest upon the ethmoid complex, the integrity of this intricate structure is essential for normal olfaction. The ethmoid is a bony complex that articulates with 13 bones and forms a central part of the nasal roof. Its articulations include the paired frontal, sphenoid, nasal, maxillary, lacrimal, palatine, and inferior turbinate bones and the unpaired vomer (Gray, 1973).

Figure 5 Schematic drawing of the ethmoid bone separated into anterior and posterior segments. (1) anterior ethmoid sinuses; (2) middle turbinate attached to cribriform plate above (ant. segment) and lateral attachment to the lamina papyracea (post. segment); (3) crista galli; (4) perpendicular plate; (5) lateral lamella of cribriform; (6) superior turbinate; (7) olfactory fossa; (8) lamina papyracea; (9) middle meatus.

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The superolateral edge of the cribriform plate gives rise to the lateral lamellae of the cribriform. These lamellae form the lateral border of the olfactory fossa intracranially. A portion of this lateral lamella frequently contains a structure referred to as the ethmoidal sulcus of the olfactory fossa. This sulcus is a groove through which the anterior ethmoidal artery courses once it has traversed from lateral to medial along the ethmoid roof (see below). The bone thickness of the sulcus has been recorded at 0.05 mm (Stammberger, 1991). The significance of these measurements lies in the propensity of this region to fracture during ethmoid surgery, resulting in cerebrospinal fluid (CSF) leaks (Kainz and Stammberger, 1989). The lateral lamellae vary widely in height and orientation. Keros described three types that define the variations of the olfactory fossa (Kainz and Stammberger, 1989). Type I denotes a flat olfactory fossa where the lateral lamella of the cribriform are short in height, between 1 and 3 mm. Type II olfactory fossa are those where the lateral lamella are a little taller (4–7 mm) and the roof of the ethmoid is steeper. A Keros type III olfactory fossa occurs

Figure 6 Coronal CT of paranasal sinuses. Bone is represented by white, soft tissues are depicted in gray, and black indicates aircontaining spaces. The (*) is situated in right maxillary sinus, below the right eye. The inferior portion of the left middle turbinate is marked by the arrowhead and inferior turbinate by the circle. Note the attachment of the middle turbinate to the cribriform above. A short white arrow in the left anterior ethmoid sinuses points to the anterior ethmoidal neurovascular bundle as it emerges from the left orbit and courses along the roof of the ethmoid. The open arrow located in the anterior cranial fossa is directly superior to the bony crista galli. The long thin arrow situated within the olfactory cleft points superiorly to the cribriform plate. The star in the right olfactory fossa is adjacent to the vertical lamella of the cribriform plate. This most closely resembles a Keros type II olfactory fossa.

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when the lateral lamellae are very long (8 mm plus) and the roof of the ethmoid is well above that of the cribriform plate. Under this circumstance the bone of the lateral lamellae is very thin and the olfactory fossa is very deep. This is the anatomical condition under which iatrogenic CSF leak is most likely to occur during sinus surgery (Fig. 6) (Kainz and Stammberger, 1989; Stammberger, 1991). The inferolateral border of the cribriform plate gives rise to the paired middle and superior turbinates. Lateral and inferior to each of these turbinates are clefts known as the middle and superior meati, respectively. The superior turbinate is often the most posterior of the lamellae within the ethmoid complex. Occasionally, a supreme turbinate is present. Posterior to the superior concha rests the anterior wall of the sphenoid sinus. Anatomical variations in the turbinates, such as their pneumatization (with concha bullosa or interlamellar cells), are not uncommon (Bolger et al., 1991) (Fig. 7). Development of such anatomical variations could theoretically compromise airflow to the

Figure 7 Coronal CT scan of the paranasal sinuses showing brain (black check), orbital contents (white check), olfactory fossa (white asterisk), nasal septum (black asterisk), right middle turbinate (black star), left middle turbinate with concha bullosa (white star), inferior turbinate (large black curved arrow), nasal cavity (small white curved arrow), ethmoid bulla (large arrow), uncinate process (small arrow), and drainage pathway of the maxillary sinus (arrowhead).

Anatomy of the Human Nasal Passages

olfactory cleft. However, to date no studies have documented such an effect. The most lateral vertical plates of the ethmoid bone are the paired laminae papyracea (orbital plate of ethmoid). The lamina papyracea form the majority of the medial orbital wall. Medial to these plates and lateral to the middle and superior turbinates are the ethmoid sinuses (Figs. 5 and 6). Anatomically, the development and pneumatization of the ethmoid sinuses varies from individual to individual. The anterior and posterior ethmoid sinuses are a maze of individual sinuses (cells), which collectively have earned the term “ethmoid labyrinth.” The ethmoid sinuses are divided into anterior and posterior sinus groups by the lateral attachment of the middle turbinate to the medial orbital wall. This attachment is known as the basal (grand) lamella of the middle turbinate. The anterior ethmoid sinuses are generally smaller and more numerous than the posterior ethmoid cells. They are ventilated through the middle meatus. However, the posterior ethmoid cells drain through the superior meatus and occasionally through a supreme meatus if a supreme turbinate is present. The roof of the ethmoid sinuses is not completely formed by ethmoid bone (McMinn and Hutchings, 1977; Stammberger, 1991). Instead, a significant portion of this roof is formed by the frontal bone (Stammberger, 1991). The anterior ethmoid sinuses, as well as the frontal and maxillary sinuses, drain through a narrow region located in the anterior middle meatus. This region is referred to as the ostiomeatal complex. The ostiomeatal complex is created by the ethmoid infundibulum, uncinate process of the ethmoid bone, hiatus semilunaris, ethmoid bulla, and middle turbinate (Lanza and Kennedy, 1993) (Fig. 7). The hook-shaped, uncinate process is the most anterior structure exposed to inspired air within the middle meatus. A few millimeters posterior to the uncinate process is the anterior face of ethmoid bulla (bulla ethmoidalis). The ethmoid bulla is generally the largest and most constant ethmoid air cell (sinus). The two-dimensional space between the uncinate process and the ethmoid bulla is the hiatus semilunaris inferioris (Messerklinger, 1978; Stammberger, 1991). It leads anteriorly to a three-dimensional, funnel-shaped space lateral to the uncinate process termed the ethmoid infundibulum. When inflammatory conditions afflict the nose and paranasal sinuses, the ostiomeatal complex is frequently involved. This is believed to be due to the narrowness of this region and the fact that only a small amount of mucosal swelling is required to occlude drainage through this area. Stagnation of drainage can promote regional inflammation and infection (Kennedy et al., 1987). Moreover, since nearly 66% of inspired air passes through the anterior middle meatus, environmental agents (viruses, pollutants, and

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allergens) are likely to have a significant impact upon the ostiomeatal complex (Wolfsdorf et al., 1969). Resultant ethmoid sinusitis, particularly in the case of polyposis, can produce hyposmia. Appropriate medical and or surgical therapy can sometimes reverse these conditions. b. Maxillary Bone and Sinus. The maxilla is the second-largest facial bone (after the mandible) and contributes to the structure of the oral, nasal, and orbital cavities. Each maxilla articulates with eight bones: the zygomatic, frontal, palatine, nasal, ethmoid, lacrimal, palatine, and inferior turbinate. Its intranasal surfaces form the pyriform aperture, anterior floor of the nasal cavity, inferior nasal septum, and the lateral nasal wall below the orbit. The maxillary sinus (antrum, maxillary antrum) develops within the maxilla and has a mean volume of about 14 mL (Maran and Lund, 1990). The medial maxillary bone is open at the maxillary hiatus. This hiatus is partially closed by bony contributions from the palatine, lacrimal, and inferior turbinate bones. Furthermore, a connective tissue sheet, covered by mucosa, spans the gap that remains. Zuckerkandl described the portions of this fibrous connective tissue sheet anterior and posterior to the uncinate process as “the anterior (inferior) and posterior fontanelles,” respectively (Fig. 8) (Messerklinger, 1978). Mucus generated within the maxillary sinus is propelled by cilia off the sinus floor in a star-shaped pattern (Messerklinger, 1978; Stammberger, 1991) (Fig. 9). It exits through the maxillary ostium, an opening along the anteriorsuperior aspect of the medial antral wall. Typically maxillary sinus secretions follow a path through the ethmoid infundibulum and cross the hiatus semilunaris into the middle meatus. From the middle meatus these secretions eventually drain into the nasal cavity beneath the eustachian tube orifice in the posterior nasal cavity (Kennedy et al., 1987). Smaller accessory ostia are commonly found within the posterior fontanelle. In most cases mucus from the maxillary sinus bypasses these openings in favor of exiting through the natural ostium (Kennedy et al., 1987). c. Frontal Bone and Sinus. The paired frontal bones articulate with the ethmoid, lacrimal, maxillary, nasal, parietal, sphenoid, and zygomatic bones (Gray, 1973). Anteriorly the frontal bones meet with one another at the midline. In most cases, the frontal bone contains a nasal spine, which abuts the perpendicular plate of the ethmoid and the undersurface of the nasal bones, helping to stabilize and support them. Posteriorly, at the midline, the frontal bone articulates with the cribriform plate. The foveolae ethmoidales of the frontal bone joins the lateral lamella of the cribriform plate to create the roof of the ethmoid sinuses (Stammberger, 1991). The foveolae

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Figure 8 Schematic sagittal view of lateral nasal wall: (1) frontal bone; (2) ethmoid bone; (2a) bulla ethmoidalis; (2b) uncinate process; (3) sphenoid bone; (4) perpendicular process of the palatine bone; (5) maxillary bone; (6) lacrimal bone; (7) nasal bone; (8) inferior turbinate bone; (9a and b) upper and lower lateral cartilage. Note the lacrimal bone (6) is depicted more prominently here than it would be seen in life from this sagittal view. In actual fact a greater portion of this bone rests more anterolaterally to the uncinate process. This diagram, however, highlights its relationships to the ethmoid, inferior turbinate, maxillary, and frontal bones.

ethmoidales region has a mean thickness of 0.5 mm, whereas the lateral lamellae of the cribriform plate averages 0.2 mm in thickness (Stammberger, 1991). The frontal sinus is the most variable of the paranasal sinuses, being completely absent in a small percentage of the population. It is formed by pneumatization, which originates in the ethmoid. An intersinus septum is usually present inferiorly at the midline but can deviate markedly as it courses upwards. Some investigators have documented continued growth and expansion of the frontal sinus well into adulthood, though this is thought to be more the exception than the rule (Lang, 1989). Mucus generated within the frontal sinus circulates prior to exiting through the frontal sinus ostium. This opening is located anteromedially in the floor of the frontal sinus (Fig. 9). The frontal sinus ostium drains into a channel within the anterior superior ethmoid complex known as the frontal recess. The term “nasofrontal duct” had been used to describe this area of drainage, but is falling out of favor because only in the minority of cases does a discrete bony canal exist. Thus, anterior ethmoid sinus disease may promote obstruction of the frontal recess and block mucociliary clearance from the frontal sinuses. A frontal sinusitis may develop in association with such an obstruction. d. Sphenoid Bone and Sinus. The sphenoid bone forms the most posterior extent of the nasal cavity. It is the

largest and most central bone of the skull base. It articulates with the ethmoid, frontal, vomer, occipital, parietal, temporal, zygomatic, and palatine bones (Gray, 1973). Its articulation with ethmoid appears at the cribriform plate, perpendicular plate, lamina papyracea, and the posterior aspect of the ethmoid labyrinth. The vomer meets the anterior wall of the sphenoid sinus in the midline at an area termed the sphenoid crest. The perpendicular process of the palatine bone

Figure 9 Schematic diagram of the mucociliary clearance patterns of the frontal (above) and maxillary (below) sinuses.

Anatomy of the Human Nasal Passages

articulates with the body of the sphenoid more laterally in the area of the sphenopalatine foramen. The medial pterygoid plate of the sphenoid meets the posterior surface of the perpendicular process of the palatine bone to form the most posterolateral recesses of the nasal cavity and nasopharynx. The sphenoid sinus has been classified into three types on the basis of size and degree of pneumatization (Moss-Salentijn, 1991): (1) the rare conchal type, with minimal posterior extension, (2) the presellar type, with extension to the anterior wall of the pituitary fossa, and (3) the common postsellar (sellar, postsphenoid) type, with posterior pneumatization beneath and sometimes even behind the pituitary fossa. The average volume of the sphenoid sinus is about 5–7 mL. A mid-sagittal intersinus septum usually is present but, as in the case of the frontal sinus septum, may be in an eccentric position. The lateral wall of the sinus may be indented by the optic nerve and internal carotid artery, forming recesses above and below these structures. Of note is that several investigators have found bony dehiscence overlying these structures, making surgical manipulation of the lateral sphenoid wall extremely dangerous (Kennedy et al., 1990). Microdehiscences in the lateral wall of the sphenoid sinuses are thought to serve as a route for the intracranial spread of infection. Mucus is actively transported out of the sphenoid sinus through the sphenoid ostia. The ostium is located just 2–3 mm lateral to the sphenoid crest in the sphenoethmoidal recess. Inflammatory disease in the sphenoethmoidal recess can cause blockage of the sphenoid os and subsequent sphenoid sinusitis. Drainage from the posterior ethmoidal sinuses eventually joins that of the sphenoid sinus in the sphenoethmoidal recess. Mucus clearance from these sinus groups is usually seen together draining above the eustachian tube orifice. 2.

Other Bones of Nasal Cavity

a. Inferior Turbinate Bone. The inferior turbinate bone articulates with the maxilla anteriorly, the perpendicular process of the palatine bone posteriorly, and the lacrimal bone superiorly (Clemente, 1981; Gray, 1973) (Fig. 8). Occasionally it also interfaces superiorly with the uncinate process of the ethmoid bone. The cleft lateral to the inferior turbinate is known as the inferior meatus. The nasolacrimal duct opens into the anterior segment of the inferior meatus. b. Lacrimal Bone. The lacrimal bone is the smallest bone of the lateral nasal wall and articulates with the frontal process of the maxilla, the inferior turbinate, the lamina papyracea, and the frontal bone (Fig. 8) (Zide and Jelks, 1985). The lacrimal bone together with the

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ascending process of the maxilla forms the lacrimal fossa, which houses the lacrimal sac in the anterior orbit. The nasolacrimal duct drains this sac through the medial floor of this fossa. The duct traverses inferiorly to drain into the inferior meatus. c. Palatine Bone. The palatine bone articulates with the inferior concha, maxilla, ethmoid, sphenoid, vomer, and opposing palatine bones (Clemente, 1981; Gray, 1973). Its L shape forms the posterior portions of the floor and lateral wall of the nasal cavity. Its perpendicular process has a conchal crest to which the posterior portion of the inferior turbinate bone attaches. This perpendicular process also forms part of the medial wall of the maxillary sinus and joins the sphenoid bone. The articulation with the sphenoid is located just beyond the posterior end of the middle turbinate and marks the region of the sphenopalatine foramen. This foramen is the opening through which the sphenopalatine neurovascular bundles emerge (see below) (Fig. 8). d. Vomer. This midline unpaired bone forms the inferior portion of the bony nasal septum. It articulates with the perpendicular plate of the ethmoid, the sphenoid, palatine, and maxillary bones, as well as with the septal cartilage (Clemente, 1981; Gray, 1973). B.

Soft Tissue Anatomy

1.

Vasculature of the Nasal Cavity

The description that follows represents a basis for understanding the predominant circulatory patterns of nasal passages. The arterial supply to the nasal passages is formed by a plexus of vessels derived from several sources. Thus, several texts report subtle differences in the origin of vessels. a. Arterial Supply. The nasal cavity receives its blood supply from both the internal and external carotid arteries. Intracranially the internal carotid artery gives rise to the ophthalmic artery. The ophthalmic artery, in turn, branches to form the anterior and posterior ethmoid arteries. The ethmoid arteries cross from the orbit into the ethmoid labyrinth through a foramina located near the frontoethmoidal suture. These arteries usually course within bony canals situated within the ethmoid labyrinth along the foveolae ethmoidales. However, the position of the vessels may vary considerably, from 2 mm below to 4 mm above the level of the cribriform plate (Lang, 1989; Stammberger, 1991) (Fig. 6). Once both ethmoid arteries cross the ethmoid roof, they enter the cranial vault to give

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rise to meningeal and nasal branches. The meningeal branches supply the dura matter, and the nasal branches descend through the cribriform plate to supply the nasal cavity. Specifically, the posterior ethmoidal arteries supply the superior turbinate and posterior septum. After the anterior ethmoid artery courses within the ethmoidal sulcus of the olfactory fossa (Stammberger, 1991), its terminal branches supply the lateral nasal wall, including the anterior middle turbinate and the septum. A small division courses to the external nose between the caudal border of the nasal bones and upper lateral cartilages (Gray, 1973). The external carotid artery delivers blood to the nasal cavity via two main branches, the facial and the internal maxillary arteries. The facial artery has two terminal branches which supply the nose and anterior nasal cavity: the superior labial and angular arteries. Significant portions of the nasal cavity derive their arterial distribution from the internal maxillary artery. Within the pterygopalatine fossa (pterygomaxillary space), the internal maxillary artery divides into many branches. Most notable for this discussion are the sphenopalatine and descending palatine arteries. Variations can occur in the manner in which these vessels arise, anastomose, and supply the nasal cavity. According to Gray (1973), the descending palatine is also known as the greater palatine artery; however, Lang (1989) distinguishes these vessels from one another. Lang asserts that the descending palatine artery gives rise to the greater palatine vessel. Regardless, the descending palatine artery, which arises in the medial aspect of the pterygopalatine fossa, contributes to the blood supply of the septum and lateral nasal wall (Clemente, 1981; Gray, 1973; Lang, 1989; Pansky, 1979). The portion known as the greater palatine artery courses through pterygopalatine canal and exits into the oral cavity through the greater palatine foramen. This vessel courses along the hard palate, where its terminal branches pass through the incisive canal to supply the nasal septum (Figs. 2, 10). The sphenopalatine artery emerges from the pterygopalatine fossa, along with some branches of the descending palatine artery via the sphenopalatine foramen. This foramen is located just superior to the posterior attachment of the middle turbinate. The sphenopalatine artery divides into posterior lateral and posterior septal branches. The posterior lateral branches contribute to the supply of the turbinates, their respective meati, and to the paranasal sinuses (Gray, 1973). The posterior septal branch gives rise to the nasopalatine artery, which runs in a groove along the vomer to reach the incisive foramen. Anteriorly on the nasal septum and just superior to the incisive foramen, anastomoses between the septal branches of the superior labial, anterior ethmoid, greater palatine, and sphenopalatine arteries form Kiesselbach’s plexus,

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Figure 10 Schematic diagram of sagittal view of lateral nasal wall depicting (a) artery, (aa) arteries, and (n) nerve. (From Lanza et al., 1990.)

located on the septum in a region known as Little’s area. This is by far the most common source of epistaxis within the nasal cavity. However, most cases of severe posterior nasal bleeding involve the sphenopalatine artery. b. Venous Drainage. Nasal veins arise from a rich network within the nasal mucosa and generally course along the reverse route of the arterial supply. Since a system of valveless veins constitutes the drainage from the nasal passages, the potential for spread of infection to the cavernous sinus is real. Venous drainage from the septum generally corresponds with the course of the sphenopalatine artery. Venous blood coursing in this direction eventually reaches the pterygopalatine and infratemporal fossa. The pterygoid plexus is located in the infratemporal fossa and eventually communicates with the cavernous sinus. The ethmoidal veins exit to the orbit and anterior cranial fossa. Orbital drainage via the ophthalmic vein is linked to the cavernous sinus. Ethmoidal drainage can also join the venous drainage from the dura mater and exit through the superior sagittal sinus (Lang, 1989). The area of the nares is drained by a small external nasal plexus and ultimately drains into the facial vein. c. Lymphatic Drainage. Lymph from the nasal cavity drains both anteriorly and posteriorly. The nasal vestibule drains into the facial vein and submandibular lymph nodes (Gray, 1973). The lymphatics of the septum run along the floor of the nose to join drainage from the lateral nasal wall. The lymphatic pathways of the lateral nasal wall are divided into anteroinferior and posterosuperior trunks

Anatomy of the Human Nasal Passages

(Lang, 1989). The anteroinferior trunk drains the inferior turbinate and anterior face of the middle turbinate. The posterosuperior trunk filters the olfactory cleft, superior turbinate, posterior middle turbinate, and sphenoethmoidal recess (Lang, 1989). These two trunks join posterior to the eustachian tube to drain into the lateral retropharyngeal nodes. Other lymph node chains, namely the jugulodigastric and the deep cervical, also receive lymphatic drainage.

d. Microcirculation and Cavernous Plexuses. Three different types of capillary vessels supply the nasal cavity. Capillaries that directly supply the epithelial cells are known as the subepithelial capillaries. These exhibit large fenestration in their endothelial lining and probably play a role in the humidifying the air (Cauna, 1982). Deeper within the mucosa, capillaries associated with glands are fenestrated to a lesser extent. Capillaries not associated with the epithelium or glands are not fenestrated (Cauna, 1982). Discrete regions within the nasal mucosa resemble erectile tissue and are known as cavernous plexuses. These are networks of tortuous valveless veins which can rapidly alter the dimensions of the nasal passages. The cavernous plexus is best developed over the septum and inferior and middle turbinates. Frequently, they may be developed adjacent to the openings of the paranasal sinuses (Cauna, 1982). The cavernous plexuses derive their blood supply from both arterial and venous sources. They consist of a superficial and deep layer. The superficial layer is formed by the union of veins which drain the subepithelial and glandular capillaries. The deep layer of the plexus runs along the periosteum and perichondrium. It is of interest that olfactory ability may improve when the nasal passageways are narrowed somewhat—i.e., when the mucosa is moderately congested, wet, and red (Schneider and Wolf, 1960). Factors such as hypoxia, hypercapnia, exercise, and increased sympathetic tone cause constriction, thereby increasing nasal patency, whereas cold air, irritants, and hypocapnia can cause dilation (Cole, 1993). Emotional states, posture, and allergic and inflammatory conditions can also affect cavernous plexuses (Cole, 1993). Interestingly, normal noses undergo an alternating pattern of leftright congestion and decongestion, a phenomenon termed the nasal cycle (see Chapter 21). In the nonpathological nose, this fluctuating resistance to inspiratory airflow is seldomly appreciated. Importantly, greater airflow through the right nostril relative to the left is associated with sympathetic predominance, greater left hemispheric integrated EEG activity, and heightened olfactory sensitivity (Frye and Doty, 1992).

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2.

Innervation of the Nasal Cavity

Besides the special sensory function associated with cranial nerve (CN) I, the nasal cavity contains general sensory and autonomic fibers. The general sensory innervation is derived from the ophthalmic (V1) and maxillary (V2) divisions of the trigeminal nerve. The autonomic input originates from the cervical sympathetic chain and superior salivary nucleus in the midbrain (parasympathetic). There are two areas of neural supply within the nose which are poorly understood in humans: the nervus terminalis and vomeronasal organ (Jacobson’s organ). a. General Sensory Supply. The trigeminal nerve (fifth cranial nerve, on CN V) (see Chapter 47) is the largest of the cranial nerves and relays both sensory and motor information (e.g., muscles of mastication). The sensory root of this nerve has its ganglionic cell bodies situated within the semilunar (Gasserian, trigeminal) ganglion. The trigeminal nerve trifurcates as it emerges from the semilunar ganglion within the anterior middle cranial fossa. The ophthalmic division of the trigeminal nerve enters the posterior orbit through the superior orbital fissure and gives rise to the nasociliary nerve. The nasociliary nerve divides into the anterior and posterior ethmoid nerves. These join the anterior and posterior ethmoid arteries as they course through their respective foramina at the level of the frontoethmoid suture. The anterior ethmoid nerve divides into an internal and external branch before it descends upon the anterior septum. The internal branch innervates the anterior lateral nasal wall and the external division supplies a small dorsal area of the external nose. The posterior ethmoid nerve supplies the posterior and superior regions of the septum and lateral nasal wall (Figs. 2, 10). The maxillary division leaves the middle cranial fossa via the foramen rotundum. It crosses the roof of the pterygopalatine fossa and traverses the floor of the orbit within the infraorbital canal. Within this canal a division known as the anterior superior alveolar nerve sends fibers to the upper incisors and canine teeth. This division also supplies the anterior portions of the inferior meatus and floor of the nasal cavity (Gray, 1973). The infraorbital nerve emerges distally and supplies sensory fibers to the middle third of the face (including the lower lateral nose). Divisions of V2 that supply the nasal cavity diverge from the maxillary nerve in the pterygopalatine fossa. Along with autonomic input from the sphenopalatine ganglion and the sphenopalatine vessels, these fibers traverse the sphenopalatine foramen. They enter the nasal cavity as several branches. The posterolateral nasal branches from V2 supply sensation to the mucosa over the turbinates and

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lateral nasal wall. Medial branches cross the posterior nasal roof and descend upon the nasal septum as the nasopalatine (Scarpa’s) nerve (Figs. 2, 10). The nasopalatine nerve courses anteriorly and eventually traverses the incisive foramen. Its terminal branches form anastomoses with those from the greater palatine nerve. They may innervate the anterior and superior gingiva as far laterally as the canine teeth (Lang, 1989). Of clinical interest is the fact that the nasal mucosa has a limited ability to localize tactile and painful stimuli (Cauna, 1982). This may contribute to the phenomenon of referred head and facial pain seen in some sinonasal disorders (including sinusitis). Though no temperature receptors have been found histologically, clinical and animal experiments suggest that the nasal mucosa does indeed have a thermal sense (Jones et al., 1989). Two other nerves of the nasal septum warrant mention. The nervus terminalis (terminal nerve) is a nerve of unknown function lying in the anterior superior aspect of the septum (see Chapter 48). Olfactory, general sensory, and autonomic functions have been attributed to it, but its actual role in humans remains uncertain (Gray, 1973; Lang, 1989). The cell bodies for this nerve lie in a nerve plexus in the olfactory region of the septum. Preganglionic fibers course through the cribriform plate into the anterior cranial fossa where multiple central connections are made. The vomeronasal nerve and organ (Jacobson’s organ) are believed by many to be vestigial in humans. However, it is highly developed in some animal species. b. Autonomic Supply. The vascular bed and glandular structures within the nasal mucosa are under sympathetic and parasympathetic control. The sympathetic pathway originates in the thoracolumbar spinal cord as preganglionic fibers. From there, the fibers join the vagosympathetic trunk and then terminate in the cervical sympathetic ganglion. The postganglionic fibers then course along the internal carotid artery and form the deep petrosal nerve. This nerve then unites with the greater superficial petrosal nerve to form the Vidian nerve (nerve of the pterygoid or Vidian canal). The Vidian nerve emerges from its canal to arrive within the pterygopalatine fossa, where it contributes to the sphenopalatine ganglion. Sympathetic fibers do not synapse there, but proceed to join the nasopalatine and posterior lateral branches of V2 and are dispersed to the nasal mucosa. The ophthalmic division of the trigeminal nerve also conveys sympathetic fibers from the carotid plexus via the ethmoidal nerves. The parasympathetic pathway to the nose begins in the superior salivary nucleus of the midbrain. Fibers run

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in the nervus intermedius portion of the facial nerve (cranial nerve VII) to the geniculate ganglion. They exit this ganglion, without synapsing, as the greater superficial petrosal nerve. This nerve merges with the deep petrosal nerve (sympathetic) to form the nerve of the Vidian canal. The nerve of the Vidian canal enters the pterygopalatine fossa, where its parasympathetic preganglionic fibers synapse in the sphenopalatine (pterygopalatine or Meckel’s) ganglion. Postganglionic fibers course along with the sensory and sympathetic fibers of the nasopalatine and posterior lateral nasal nerves to innervate the mucous membranes of the nose and hard palate. Other postganglionic fibers from sphenopalatine ganglion innervate the lacrimal gland. 3.

Epithelia of the Nasal Passages

Epithelia of different types are topographically distributed within the nasal passages. Anteriorly, the nasal vestibule is lined by stratified squamous epithelium. Posterior to the limen vestibuli the epithelium gradually changes from squamous to respiratory in nature. Small areas of squamous epithelium which persist over the anterior ends of the inferior and middle turbinate probably represent the influence of unmodified inspired air contacting these areas (Mygind et al., 1982). Posteriorly, ciliated cells are found in increasing numbers as the nasal mucosa transforms into a true respiratory epithelium (pseudostratified, ciliated, columnar epithelium) (Mygind et al., 1982). A discrete region of the nasal roof is covered by a yet different tissue, the olfactory neuroepithelium (see below). a. Respiratory Epithelium. The respiratory epithelium of the nasal cavity is composed of four basic cell types: basal cells, goblet cells, and ciliated and nonciliated columnar cells. The basal cells lie on the basement membrane of the mucosa and do not extend to the mucosal surface. They are no longer believed to be progenitor cells; they are now thought to support the columnar cell by assisting in their adherence to the basement membrane (Baroody and Naclerio, 1990). Goblet cells are found in their greatest concentrations on the inferior turbinate. These concentrations are considerable but diminished on middle turbinate and septum. Columnar cells have microvilli upon their apical surfaces, which may help to prevent dehydration through increases in surface area. The ciliated columnar cells found throughout the airway are essential to the proper function of the respiratory tree. These cells are responsible for mucus transport through a mechanism known as mucociliary clearance (Deitmer, 1989; Messerklinger, 1978).

Anatomy of the Human Nasal Passages

13

Cilia, which project from the columnar cells, possess intrinsic motility. Each ciliary stroke has a biphasic nature, with a rapid active stroke that is followed by a slower recovery beat (Deitmer, 1989). During the active stroke, cilia make contact with the thicker, more superficial gel layer of nasal mucus. During the slower recovery beat, the cilia only pass through the thinner sol layer of mucus, which is closer to the cell surface. The overlying mucus blanket is propelled by this coordinated and synchronized mucociliary clearance mechanism. Inhaled particulate matter trapped by the viscous mucus is thus swept back out of the nasal cavity and ultimately swallowed. The glands associated with the nasal passages produce between 1 and 2 L of mucus daily. Mucus is about 96% water and 4% glycoprotein (Widdicombe and Wells, 1982). This essential fluid serves several functions: (1) protection—mucus contains proteins that defend the underlying epithelium against various harmful particles, pathogens, and noxious substances inhaled from the environment; (2) humidification—the nasal passages lined with mucus warm and humidify inspired air, making it more suitable for the lower respiratory tract; (3) olfaction—mucus affects the ability of odorant molecules to reach and react with the olfactory epithelium.

There are three main types of glands associated with the nasal respiratory epithelium: (1) the serous (anterior nasal) glands, located within the vestibular epithelium, (2) the seromucous glands, and the (3) intraepithelial glands. Most nasal secretions are produced by seromucous glands, with a lesser contribution from the epithelial goblet cells. Seromucous glands, numbering in the tens of thousands, are situated submucosally, and their fluid production is primarily responsible for keeping the nasal mucosa moist. The serous glands outnumber the mucin-producing ones by about 8:1. Intraepithelial glands are thought by some to be pathological and found by others to be present in normal noses. Regardless, they appear to be few in number, be irregularly distributed, and produce only a small amount of mucus. There is a lower density of glandular elements in the paranasal sinuses than in the nasal cavity. The glands

Figure 11 Comparison of the work of von Brunn in 1892 and Read in 1908 in mapping the extent of the olfactory neuroepithelium. Note both depict the olfactory neuroepithelium ranging onto the superior aspect of the middle turbinate (one half natural size). (From Read, 1908.)

Figure 12 Percentage of olfactory tissue in 71 biopsy specimens collected from 23 healthy individuals. The number of samples containing olfactory epithelium was divided by the number of samples collected in each area (n). (From Feron et al., 1998).

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Figure 13 Maps of the nasal lining illustrating the location of the biopsy specimens and their histological composition. The plus sign (+) indicates that the biopsy specimen taken from that area contains olfactory epithelium and/or fasicles of the olfactory nerve (and thus was olfactory mucosa originally). The minus sign (-) indicates that no olfactory epithelium or nerve was seen. For convenience of illustration, biopsy specimens of the right-side septal mucosa and of the left-side turbinate mucosa were translated onto representations of the left-side septum and right-side turbinates, respectively. (From Leopold et al., 2000).

within each sinus appear to have a higher concentration around the ostial regions. Furthermore, the anterior ethmoid sinuses contain more glands than the posterior ethmoid (Tos, 1982). b. Olfactory Neuroepithelium. Traditional teaching places the olfactory neuroepithelum at the cribiform plate extending a short distance inferiorly and onto the superior turbinate (see Sec. I). Several classic diagrams have suggested that the middle turbinate may also have olfactory tissue (Bucher, 1973; Lang, 1989; Read, 1908; von Brunn, 1892) (Fig. 11). Recent studies have demonstrated a more extensive distribution of olfactory neuroepithelium, extending as anteriorly as the anterior middle turbinate insertion (Leopold, 2000) and as inferiorly as the body of the middle turbinate itself (Feron et al., 1998). Feron et al., (1998) took 97 biopsies from six different regions in 33 subjects. Olfactory neuroepithelium was found in 30–76% of specimens, depending on the region of biopsy (Fig. 12). Leopold et al. (2000) performed electro-olfactographic studies demonstrating responses to olfactory stimuli from leads placed at the anterior insertion of the middle turbinate. Biopsies of

this region confirm the presence of olfactory neuroepithelium (Fig. 13). The dimensions and distribution of the olfactory neuroepithelium are known to vary with between individuals (Paik et al., 1992). Paik et al., (1992) performed biopsy of the uppermost portion of the septal mucosa and noted that the ability to obtain a positive biopsy positive for olfactory neuroepithelium decreases with age. Other factors such as chemical exposure, bacterial or viral infection, and head trauma are also thought to affect the distribution of olfactory neuroepithelium. Macroscopically, the neuroepithelium has been described by some authors to have a yellow appearance, which distinguishes it from the surrounding respiratory epithelium (Read, 1908; Zippel, 1993). Further discussion on the microscopic anatomy of the olfactory epithelium is presented in Chapters 2, 3, 5, and 6.

REFERENCES Baroody, F., and Naclerio, R. M. (1990). Review of Anatomy and Physiology of the Nose. American Academy of OtolaryngologyHead and Neck Surgery Foundation, Inc., Alexandria, VA, p. 13.

Anatomy of the Human Nasal Passages Berglund, B., and Lindvall, T. (1982). Olfaction. In The Nose: Upper Airway Physiology and the Atmospheric Environment, D. F. Proctor and I. Andersen (Eds.). Elsevier Biomedical Press, Amsterdam pp. 279–285. Bhatnagar, K. P., and Reid, K. H. (1996). The human vomeronasal organ, I: Historical perspectives. A study of Ruysch’s (1703) and Jacobson’s (1811) reports on the vomeronasal organ with comparative comments and English translations. Biomed Res. 7:219–229. Bolger, W. E., Butzin, C. A., and Parsons, D. S. (1991). Paranasal sinus bony anatomic variations and mucosal abnormalities: CT analysis for endoscopic sinus surgery. Laryngoscope 101:56–64. Bridger, G. P., and Proctor, D. F. (1970). Maximum nasal inspiratory flow and nasal resistance. Ann. Otol. 79:481–488. Bucher, O. (1973). Cytologie, Histologie und Mikroskopische Anatomie des Menschen, 8th ed. Huber, Bern, p. 330. Cauna, N. (1982). Blood and nerve supply of the nasal lining. In The Nose: Upper Airway Physiology and the Atmospheric Environment, D. F. Proctor and I. Andersen (Eds.). Elsevier Biomedical Press, Amsterdam p. 51. Cauna, N., Cauna, D., and Hinderer, K. H. (1972). Innervation of the human nasal glands. J. Neurocytol. 1:54. Clemente, D. D. (1981). Anatomy: A Regional Atlas of the Human Body. Urban and Schwarzenberg, Baltimore-Munich, pp. 600–615. Cole, P. (1993). Respiratory Role of the Upper Airways. MosbyYear Book, St. Louis, pp. 8, 93. Cole, P., Haight, J. S. J., Love, L., and Oprysk, D. (1985). Dynamic components of nasal resistance. Am. Rev. Respir. Dis. 132:1229–1232. Cottle, M. H. (1987). The anatomy of the nasal septum and external nasal pyramid. In Rhinology, the Collected Writings of Maurice H. Cottle, MD, P. A. Barelli, W. E. E. Loch, and E. B. Kern (Eds.). American Rhinologic Society, Chicago, IL, pp. 89–94. Daniel, R. K., and Letourneau, A. M. (1988). Rhinoplasty: nasal anatomy. Ann. Plastic Surg. 20:5–13. Deitmer, T. (1989). Physiology and Pathology of the Mucociliary System: Special Regards to Mucociliary Transport in Malignant Lesions of the Human Larynx. Karger, New York. Doty, R. L. (1979). A review of olfactory dysfunctions in Man. Am. J. Otolaryngol. 1:1. Douek, E., Bannister, L. H., and Dodson, H. C. (1975). Recent advances in the pathology of olfaction. Proc. R. Soc. Med. 68: 467–470. Farrior, R. T., and Connolly, M. E. (1970). Septorhinoplasty in children. Otolaryngol. Clin. North Am. 3:345. Feron, F., Perry, C., McGrath, J. J., and Mackay-Sim, A. (1998). New techniques for biopsy and culture of human olfactory epithelial neurons. Arch. Otolaryngol. Head Neck Surg. 24:861–866. Fomon, S., Gilbert, J. G., Caron, A. L., and Segal, S. Jr. (1950). Collapsed ala: pathologic physiology and management. Arch. Otolaryng. 51:465–484. Frye, R. E., and Doty, R. L. (1992). The influences of ultradian anatomic rhythms, as indexed by the nasal cycle, on unilateral

15 olfactory thresholds. In Chemical Signals in Vertebrates 6, R. L. Doty and D. Moller-Schwarze (Eds.). Plenum Press, New York, pp. 595–598. Garcia-Velasco, J. G., and Mondragon M. (1991). The incidence of the vomeronasal organ in 1000 human subjects and its possible clinical significance. J. Steroid Biochem. Mol. Biol. 39:561–563. Gray, H. (1973). Anatomy of the Human Body. Lea and Febiger, Philadelphia, pp. 164–196, 577–593, 683–690, 740–747, 911–930. Jafek, B. W., Eller, P. M., Esses, B. A., and Moran, D. T. (1989). Post-traumatic anosmia: ultrastructural correlates. Arch. Neurol. 46:300–304. Jones, A. S., Wight, R. G., and Durham, L. H. (1989). The distribution of thermoreceptors within the nasal cavity. Clin. Otolaryngol. 14:235–239. Jugo, S. B. (1987). Total septal reconstruction through decortication (external) approach in children. Arch. Otolaryngol. Head Neck Surg. 113:173. Kainz, J., and Stammberger, H. (1989). The roof of the anterior ethmoid: a place of least resistance in the skull base. Am. J. Rhinol. 4:191–199. Kennedy, D. W., Zinreich, S. J., Shaalan, H., Kuhn, F., and Naclerio, R. (1987). Endoscopic middle meatal antrostomy: theory, technique, and patency. Laryngoscope 97(suppl. 43): 81. Kennedy, D. W., Zinreich, S. J., and Hassab, M. H. (1990). Internal carotid artery as it relates to endonasal sphenoethmoidectomy. Am. J. Rhinol. 4:7–12. Kern, E. B. (1978). Surgical approaches to abnormalities of the nasal valve. Rhinology 16:165–189. Lang, J. (1989). Clinical Anatomy of the Nose, Nasal Cavity and Paranasal Sinuses. Thieme Medical Publishers, New York, pp. 16, 30–40, 49–98, 100–121. Lanza, D. C., and Kennedy, D. W. (1993). Endoscopic sinus surgery. In Head and Neck Surgery-Otolaryngology, B. J. Bailey (Ed.). J. B. Lippincott Company, Philadelphia, pp. 389–401. Lanza, D. C., Kennedy, D. W., and Koltai, P. J. (1991). Applied nasal anatomy and embryology. Ear Nose Throat J. 70(7):416–422. Lanza, D. C., Moran, D. T., Doty, R. L., Trojanowski, J. Q., Lee, J. H., Rowley, J. C. III, Crawford, D., and Kennedy, D. W. (1993). Endoscopic human olfactory biopsy technique: a preliminary report. Laryngoscope 103(7):815–819. Leopold, D. (1986). Physiology of olfaction. In OtolaryngologyHead and Neck Surgery, C. W. Cummings (Ed.). C. V. Mosby Company, St. Louis, p. 528. Leopold, D. A., Hummel, T., Schwob J. E., Hong, S. C., Knecht, M., and Kobal G. (2000). Anterior distribution of human olfactory epithelium. Laryngoscope 110(3):417–421. Lovell, M. A., Jafek, B. W., Moran, D. T., and Rowley, J. C. III (1982). Biopsy of human olfactory mucosa. Arch. Otolaryngol. 108:247–249. Maran, A. G. D., and Lund, V. J. (1990). Clinical Rhinology. Thieme Medical Publishers, Inc., New York, pp. 17–23. McMinn, R. M. H., and Hutchings, R. T. (1977). A Colour Atlas of Human Anatomy. Wolfe Medical Publications Ltd., London, pp. 25–32.

16 Messerklinger, W. (1978). Endoscopy of the Nose. Urban and Schwarzenberg, Baltimore, pp. 11–14. Moran, D. T., Jafek, B. W., and Rowley, J. C., III (1991). The vomeronasal (Jacobson’s) organ in man: ultrastructure and frequency of occurrence. J. Steroid Biochem. Mol. Biol. 39:545–552. Moss-Salentijn, L. (1991). Anatomy and embryology. In Surgery of the Paranasal Sinuses, A. Blitzer, W. Lawson, and W. H. Friedman (Eds.). W. B. Saunders, Philadelphia, p. 18. Mygind, N., Pedersen, M., and Nielsen, M. H. (1982). Morphology of the upper airway epithelium. In The Nose: Upper Airway Physiology and the Atmospheric Environment, D. F. Proctor and I. B. Andersen (Eds.). Elsevier Biomedical Press, New York, pp. 70–97. Naessen, R. (1970). The identification and topographical localization of the olfactory epithelium in man and other mammals. Acta Otolaryngol. 70:51–57. Naessen, R. (1971). An enquiry on the Morphological characteristics and possible changes with age in the olfactory region of man. Acta Otolaryngol. 71:49–62. Nakashima, T. Kimmelman, C. P., and Snow, J. B. (1984). Structure of human fetal and adult olfactory neuroepithelium. Arch. Otolaryngol. 110:641–646. Paik, S. I., Lehman, M. N., Seiden, A. M., Duncan, H. J., and Smith, D. V. (1992). Human olfactory biopsy: the influence of age and receptor distribution. Arch. Otolaryngol. Head Neck Surg. 118:731–738. Pansky, B. (1979). Review of Gross Anatomy, 4th ed MacMillan Publishing Co., Inc. New York, p. 35. Read, E. A. (1908). A contribution to the knowledge of the olfactory apparatus in dog, cat and man. Am. J. Anatomy 8:17–47. Rehn, B., Breipohl, W., Schmidt, C., Schmidt, U., and Effenberger, F. (1981). Chemical blockade of olfactory perception by N-methyl-formimino-methylester in albino mice. II. Light microscopical investigations. Chem. Sens. 7:317–328. Sasaki, C. T., Suzuki, M., Fukuda, H., and Mann, D. G. (1977). Dilator naris muscle. Ann. Otol. Rhinol. Laryngol. 86:362–370. Schneider, R. A. and Wolf, S. (1960). Relation of olfactory activity to nasal membrane function. J. Appl. Physiol. 15:914–920. Schultz, E. W., and Gebhardt, L. P. (1934). Olfactory tract and poliomyelitis. Proc. Soc. Exp. Biol. Med. 31:728–730. Smith, C. G. (1941). Incidence of atrophy of the olfactory nerves in man. Arch. Otolaryngol. 34:533–539. Smith, T. D., Siegel, M. I., Burrows, A. M., Mooney, M. P., Burdi, A. R., Fabrizio, P. A., and Clemente, F. R. (1998). Searching for the vomeronasal organ of adult humans: preliminary

Clerico et al. findings on location, structure, and size. Microsc. Res. Tech. 41:483–491. Stammberger, H. (1991). Functional Endoscopic Sinus Surgery. B. C. Decker, Philadelphia, pp. 17–47, 49–87. Talamo, B. R., Feng, W-H., Perez-Cruet, M., Adelman, L., Kosik, K., Lee, V. MY., Cork, L. C., and Kauer, J. S. (1991). Pathologic changes in olfactory neurons in Alzheimer’s disease. Ann. NY Acad. Sci. 640:1–7. Tardy, E., and Brown, R. J. (1990). Anatomy of the Nose. Raven Press, New York. Tomlinson, A. H., and Esiri, M. M. (1983). Herpes simplex encephalitis. Immunohistological demonstration of spread of virus via olfactory pathways in mice. J. Neurol. Sci. 60:474–484. Tos, M. (1982). Goblet cells and glands in the nose and paranasal sinuses. In The Nose: Upper Airway Physiology and the Atmospheric Environment, D. F. Proctor and I. B. Andersen (Eds.). Elsevier Biomedical Press, New York, pp. 99–144. Trojanowski, J. Q., Newman, P. D., Hill, W. D., and Lee, V. M-Y. (1991). Human olfactory epithelium in normal aging, Alzheimer’s disease, and other neurodegenerative disorders. J. Comp. Neurol. 310:365–376. von Brunn, A. (1892). Beiträge zur Mikroskopischen Anatomie der Menschlichen Nasenhöhle. Arch. Mikr. Anat. 39:632–651. Widdicombe, W. G. and Wells, U. M. (1982). Airway secretions. In The Nose: Upper Airway Physiology and the Atmospheric Environment, D. F. Proctor and I. B. Andersen (Eds.). Elsevier Biomedical Press, New York, pp. 215–244. Wolfsdorf, J., Swift, D. L., and Avery, M. E. (1969). Mist therapy reconsidered: an evaluation of the respiratory deposition of labelled water aerosols produced by jet and ultrasonic nebulizers. Pediatrics 43:79–808. Won, J., Mair, E. A., Bolger, W. E., and Conran, R. M. (2000). The vomeronasal organ: an objective anatomic analysis of its prevalence. Ear Nose Throat J. 79:600–605. Yamagishi, M., and Nakano, Y. (1992). A re-evaluation of the classification of olfactory epithelia in patients with olfactory disorders. Eur. Arch. Oto-Rhino-Laryngol. 249: 393–399. Yamagishi, M., Hasegawa, S., and Nakano, Y. (1988). Examination and classification of human olfactory mucosa in patients with clinical olfactory disturbances. Arch. Otorhinolaryngol. 245:316–320. Zide, B. M., and Jelks, G. W. (1985). Surgical Anatomy of the Orbit. Raven Press, New York, p. 5. Zippel, H. P. (1993). Historical aspects of research on the vertebrate olfactory system. Naturwissenschaften Aufsätze 80: 65–76.

2 Morphology of the Mammalian Olfactory Epithelium: Form, Fine Structure, Function, and Pathology Bert Ph. M. Menco Northwestern University, Evanston, Illinois, U.S.A.

Edward E. Morrison Auburn University, Auburn, Alabama, U.S.A.

I.

INTRODUCTION

largest gene family in multicellular organisms, including humans (Glusman et al., 2001). This chapter reviews the morphology and structure of the main olfactory epithelium of mammals, including humans, in health and disease. The relationship between morphology and biochemistry and physiology is addressed when possible, and, when appropriate, contrasts are made with some of the other chemosensory systems, such as the vomeronasal organ. The reader is referred elsewhere for reviews on the structure of the chemosensory systems of a wide range of species, including higher-order projection centers (Menco, 1992a; Smith, 1998; Tolbert, 1993).

In most mammals, chemicals, particularly volatile ones, are sensed by several intranasal systems, the main olfactory organ (tuned to odors in general), the vomeronasal or Jacobson’s organ (tuned to chemicals employed in social and sexual activities) (Evans, 2002) (see Chapter 46), the septal olfactory organ (a patch of olfactory tissue on the anterior septum of some vertebrates that likely responds to the same agents as the main olfactory system, and perhaps serves an alerting role) (Adams, 1992), and the trigeminal intranasal somatosensory system (responsive to pungent and irritative odors) (see Chapter 47). Humans likely do not possess a septal organ, and their vomeronasal organ is rudimentary and nonfunctional (Giorgi et al., 2000; Smith et al., 2001; Trotier et al., 2000). The chemosensory neurons of main, septal, and vomeronasal olfactory organs have several characteristics that set them apart from neurons of the central nervous system. First, because of their peripheral location they are exposed to the external environment. Second, their axons project directly to the forebrain without synapsing in the thalamus (see Chapters 7 and 8). Third, they have a remarkable capacity for continued postnatal neurogenesis, even into old age (see Chapters 5 and 6). Fourth, reflecting a need to distinguish between many compounds, the subgenome of the main olfactory system comprises the

II.

EARLY OBSERVATIONS

Massa (1536) and Scarpa (1789) first described the course of human olfactory nerves (see Graziadei, 1971; Seifert, 1969). These early investigators chronicled the presence of fine delicate bundles of nerves that originate from the nasal cavity mucosa and extend into the cranial vault, attaching directly to the brain. The early histological investigations were limited by the difficulty in obtaining fresh olfactory tissue, unsuitable fixation and staining methods, and the overall poor quality of available microscopes. For example, Ecker (1855) thought that the olfactory region lacked cilia, and others reported that only the sustentacular cells have 17

18

Figure 1 Light microscopy of rodent olfactory neuroepithelium illustrates an apical row of supporting cell (s) nuclei, olfactory receptor cell nuclei (o) that occupy the basal two thirds of the epithelium compartment, and globose (g) and horizontal basal cells (h). Bar
25 m.

Figure 2 High-voltage transmission electron micrograph of a section (1 m) parallel to the olfactory epithelial surface through an olfactory mucus layer (calf, septum). Cilia (C) radiate from the dendritic endings (De) of olfactory receptor cells. The tissue was conventionally fixed and embedded. Bar
10 m.

Menco and Morrison

Figure 3 Human olfactory dendritic ending (scanning electron micrograph) with proximal parts of olfactory cilia surrounded by, in this case short, microvilli of nearby supporting cells. Bar
1 m. (After Morrison and Costanzo, 1990.). Figure 4 As in Figure 3, but in an 18-day-old rat embryo. Cilia, radiating from the dendritic ending, have 1–2 m long thick proximal parts (about 0.3 m across, fat arrow) that taper to 0.1 m (large arrow). Developing dendritic endings nearby may just have one (primary) cilium (small arrow) in embryos. Microvilli (asterisk) of olfactory epithelial supporting cells intermingle with receptor cell cilia in a course perpendicular to these. Bar
1 m.

flagellum-like processes. Improved optical lenses, new chemical dyes that yielded superior staining techniques, and methods for obtaining tissue quickly (thereby allowing better fixation and tissue preparation) developed in the late nineteenth century provided opportunities for more accurate and detailed descriptions of the olfactory mucosa. Early histologists (Ecker, 1855; Krause, 1876; Schultze, 1856) showed that the vertebrate olfactory mucosa was primarily composed of three cellular components: olfactory receptor, supporting cells, and basal cells (for reviews, see Seifert, 1969; Zippel, 1993). Schultze (1862) provided what is thought to be the first accurate description of the vertebrate olfactory mucosa. He suggested that olfactory cilia were the endings of olfactory nerves. Further studies on a variety of animals over the course of the following 140 years supported Schultze’s astute observations. He also emphasized the uniform structural pattern of the olfactory mucosa in vertebrates (Fig. 1) (Graziadei, 1973; Seifert, 1969). Parker (1922) was apparently the first to propose that olfactory cilia contain the receptive elements for olfactory receptor neurons. Such cilia, located on the exposed dendritic tips of olfactory receptor neurons (Figs. 2–15), were hypothesized to increase the sensory surface area available for contact with odors. Todd and Bowman (1847) were the first to describe glands in the lamina propria. These glands,

Morphology of the Mammalian Olfactory Epithelium

Figure 5 Surface of human olfactory epithelium (scanning electron micrograph) where thin parts of olfactory cilia form a blanket covering the epithelial surface. The opening of a Bowman’s gland duct containing secretory product (asterisk) can be seen. Bar
10 m.

later named after Bowman by von Kölliker (1858), produce mucous secretions that reach the epithelial surface; olfactory cilia are embedded within this mucus (Fig. 2). Milne-Edwards (1844) proposed that the specialized secretions form a microenvironment that enhances odorant absorption, a hypothesis that is still posed today (see Chapter 3). Thus, odorant-binding proteins within the nasal mucus are thought to enhance odorant-receptor functioning (Pelosi, 1994). Such proteins may be especially active in VNO signaling (Pelosi, 2001; Tegoni et al., 2000). After World War II, the development of the electron microscope ushered in a new era of the study of cell structure. Engström and Bloom (1953) provided the first electron microscopic observations of the human olfactory epithelium. They determined that olfactory cilia have a 9(2)

19

Figure 6 As in Figure 5, but in a rat. Whereas in the adult human of Figure 5 the thin tapering parts of the cilia are no longer aligned, this is not true in a healthy young (2 months old) rat. Thin parts of olfactory cilia of many dendritic endings run parallel to each other. The cilia contain many expansions. Some of these may be a fixation artifact, whereas others are genuine. Tips of supporting cell microvilli “peep” through the parallel array of cilia (asterisks). Bar
1 m.

 2 microtubule arrangement, typical of almost all cilia and flagella (Bloodgood, 1990; Burton, 1992), that there were over 1000 cilia per receptor cell, and that these cilia were 1–2 m in length and 0.1 m in diameter. Later studies showed that the number of cilia was a significant overestimate that may have been due to respiratory metaplasia within the neuroepithelium. Also, we now know that olfactory cilia are actually much longer and that only the distal parts of these cilia have the smaller diameter (Figs. 4–15) (Menco, 1977, 1983; Seifert, 1970, 1972). Engström and Bloom also showed a presence of mitochondria within the receptor cell dendrites and a system of intracellular membranes, later identified as endoplasmic reticulum.

Figure 7 Freeze-fracture platinum/ carbon (Pt/C) replica transmission electron micrograph of an olfactory receptor cell dendritic ending with radially oriented cilia (rat, fixed). Membranes of the cilia have an array of spiraling particles at their base, the ciliary necklace (arrow). Several cilia are seen cleaved just below these necklaces (arrowhead). The base of the dendritic ending displays a tightjunctional belt. The area of this belt marked with an asterisk is the region where two other cells joined this cell. The dendritic ending and its cilia display many globular membrane particles, whereas those in the apices of the surrounding supporting cells are mostly dumbbell-shaped (curved arrow) (Menco, 1980a, 1984, 1988a). As these particles represent transmembrane proteinaceous or lipidic entities (Menco, 1986), the distinctions give some indication of molecular differences between cells and also between subcellular regions. Bar
1 m.

Figure 8 Freeze-substituted, unfixed rat olfactory epithelial surface (see Menco, 1995a, b, for techniques). Cilia that originate from the dendritic endings have thick proximal parts with a complete 9(2)  2 axonemal configuration (large arrow, see Fig. 10), that taper to thin long distal parts that have most commonly two microtubules (small arrows, Fig. 11) and that align parallel to the epithelial surface (see Figs. 6 and 9). Dendrites are packed with microtubules and associated proteins, basal bodies (small asterisks), and mitochondria. Supporting cell microvilli (large asterisk) have a course perpendicular to that of the cilia (see Figs. 3, 4, 6, and 27), their tips reaching the mucus surface. Bar
1 m.

Morphology of the Mammalian Olfactory Epithelium

21

Figure 10 Cross section through the proximal part of a rat olfactory cilium with a complete 9(2)  2 microtubule axonemal configuration (see also Burton, 1992). Bar
0.1 m.

Figure 9 Freeze-fracture replica (Pt/C) transmission electron micrograph of an olfactory epithelial mucus layer (rat, fixed) depicting parallel distal parts of olfactory cilia (small arrow), that emerge from the thicker proximal parts (large arrow; see Figs. 4 and 8). Ciliary membranes are studded with particles reflecting a heterogeneous population of proteins (see also Figs. 7 and 12–14). Bar
1 m.

III.

turbinates in other vertebrates, such as dog and fox (Morrison et al., 1983; Negus, 1958), which can expand over many centimeters. The nonsensory respiratory region is covered by a stratified columnar epithelium consisting of ciliated/microvillous cells interspersed with goblet cells. This epithelium predominantly lines the inferior, middle, and a portion of the superior turbinate. The mesenchyme below the basement membrane contains diffuse lymphoid tissue and blood vessels and mucous and serous glands. During development, this layer plays a major role in the formation of the olfactory pathway by way of inductive signals (LaMantia et al., 2000). Mucous secretions pass through glandular ducts that extend to the mucosal surface. The nonsensory respiratory portion of the nasal cavity warms, cleans, and humidifies the inspired air.

ANATOMY OF THE OLFACTORY MUCOSA

The human nasal cavity typically has three structures extending from each lateral wall of the ethmoid, termed the inferior, middle, and superior “turbinates” or “conchae.” Other animals can have more (Negus, 1958), e.g., the rat has four (Menco and Jackson, 1997a). The turbinates and septum, the latter being a cartilaginous structure that separates both halves of the nose, are covered with an epithelium that, depending on its location, is either nonsensory (respiratory) or sensory (olfactory). The human olfactory neuroepithelium is located high in the superior region of the nasal vault (Chapter 1). From cadaver measurements, this region appears to be approximately 1–2 cm2, varying among individuals (Moran et al., 1982a). This area is modest relative to the nasal

Figure 11 Cross section through the distal parts of rat olfactory cilia that here have two to four microtubules. Bar
0.1 m.

22

Menco and Morrison

Figure 12 Parts of distal segments of rat (fixed) olfactory cilia, rotary-replicated with Pt/C from a 45° angle. The cilia have smaller (small arrows) and larger (large arrows) membrane particles, that may reflect different transmembrane proteins. Bar
0.1 m. Figure 13 Part of the distal segment of a rat olfactory cilium (unfixed), rotary-replicated with tantalum/tungsten (Ta/W) from a 20° angle. Some larger membrane particles in this fine grain replica have pores (large arrows), that may reflect ion channels. Smaller particles lack pores (small arrows). Bar
0.1 m. Figure 14 Part of the distal segment of a rat olfactory cilium (large arrow), fixed and labeled with the lectin wheat germ agglutinin (WGA, binding to N-acetylglucosamine residues) conjugated to 5 nm gold grains, rapidly frozen, etched, and rotary-replicated with Pt/C from a 45° angle (see Fig. 12). WGA bound to several surface particles, reflected as dark dots inside the particles (small arrows) (Menco, 1992b). Surrounding mucus is virtually free of labeling, suggesting that the label bound to molecules specific to the ciliary surface. Bar
0.1 m.

Trapped dust and other particulate matter is transported to the nasopharynx by ciliary movements. The vasculature of the nasal cavity forms an erectable plexiform network beneath the mucous membrane (Proctor and Anderson, 1982). An adaptation, possibly peculiar to nasal septum and turbinates of primates, including humans, are small invaginations termed “olfactory pits” that may serve to better protect some sectors of olfactory epithelium from external damage or, though minimally, enhance the receptive surface area in these species (Feng et al., 1997). Otherwise, the human olfactory epithelium has a structure similar to that of other vertebrates. It is pseudostratified columnar, composed of olfactory receptor neurons, nonsensory supporting cells, and two types of basal cells, horizontal (HBC) and globose (GBC) (Fig. 1). It also contains leukocytes (Suzuki et al., 1995) and other cells besides supporting cells that have microvilli (Menco and

Jackson, 1997b; Moran et al., 1982b; Pixley et al., 1997). A discussion of the structure and, to some degree, function of most of these cell types is presented below. A.

Olfactory Receptor Neurons

Vertebrate olfactory receptor neurons are slender and bipolar, have 5- to 7-m-wide cell bodies that are generally located within the lower two thirds of the neuroepithelium, ciliated dendrites, and occur in densities of 106–107 per cm2 (Güntherschulze, 1979; Menco, 1983). The single dendrites of olfactory receptor cells can take a rather tortuous path, winding around adjacent receptor cell bodies, other dendrites, and supporting cells as they extend toward the mucosal surface. Each dendrite is slightly thicker near the soma and contains a Golgi body, smooth and rough endoplasmic reticulum, mitochondria, microtubules, and

Morphology of the Mammalian Olfactory Epithelium

23

Figure 15 Diagram of mammalian olfactory (top) and respiratory (bottom) cilia, and of lengths of olfactory cilia (inset center) (after Menco, 1977, 1983). Features in the three diagrams have been drawn to scale. The olfactory cilium is interrupted at two places, indicating that the cilia are actually much longer (inset). A–E represent basal body cross sections; F–H: cross sections through proximal regions of olfactory cilia (top) and homologous regions of respiratory cilia (bottom); I–K: cross sections through distal parts of olfactory cilia. The section of Figure 10 is similar to cross section G. Figure 11 shows the cross sections J and K. Other structures: R: striated rootlet of respiratory cilium; 1: fibrogranular microtubule pool (cilium precursor pool); 2: microtubules inside dendritic endings; 3: microvilli of dendritic endings (sparse) and of ciliated respiratory cells; 4: coated vesicles (Bannister and Dodson, 1992); 5: ciliary necklaces (see Fig. 7; 7 strands for olfactory cilia, 5 strands for respiratory cilia) (Menco, 1980c); 6: ciliary membranes studded with membrane particles, that reflect proteins. Olfactory cilia have many more of these than respiratory cilia (see Figs. 7, 9, and 12–14) (Menco, 1977, 1983, 1992a. 1997); 7: nearby glycocalix; 8: bundle of tapers of other, nearby, cilia; 9: vesiculated expansion along distal part of cilium (see Figs. 6 and 9); 10: ciliary tips; olfactory cilia terminate in a small vesicle. The inset demonstrates that the cilia of one receptor cell dendrite can extend over about 15 other endings. Olfactory cilia are drawn over about 60 m, that is, 120 m from the tip of one cilium to the tip of an other cilium across (Seifert, 1970). Bar top and bottom
1 m; center: 10 m.

vesicles. Some of these features can be seen in Figure 8 (Andres, 1969; Bannister and Dodson, 1992; Burton, 1992; Naguro and Iwashita, 1992). Dendrites have variable lengths, mostly extending nearly the total depth of the epithelium, from the surface deep into the epithelium, but some are extremely short, only a brief distance from cell bodies lying close to the epithelial surface (Moran et al., 1982a: Morrison and Costanzo, 1990, 1992). Dendrites end in a swelling at the epithelial surface, called the olfactory dendritic ending, knob, or vesicle. Olfactory dendritic endings extend usually, but not always, above the epithelial surface, are spherical or cylindrical,

and measure 1–2 m in diameter (Figs. 2–4, 7, 8). At the apical surface, just below the dendritic ending, a belt-like tight-junctional complex—a transmembrane barrier, characteristic of most epithelial tissues — attaches the dendrite to adjacent supporting cells, as well as supporting cells to other supporting cells (Fig. 7) (Kerjaschki and Hörandner, 1976; Menco, 1980b, 1988c). Receptor cells are closely associated with olfactory supporting cells at other levels as well, where desmosomes are found between them. Olfactory dendritic endings contain basal bodies (Burton, 1992). Many of these give rise to sensory cilia that project perpendicularly from the dendritic ending into the

24

overlying mucus layer. Each cilium consists of a short proximal part that tapers to a longer and thinner distal part, which aligns parallel to the epithelial surface. Thus, olfactory cilia are much longer than the nonsensory cilia of the respiratory epithelium. The distal, aligned, parts of the olfactory cilia form the interface between the external odorous environment and the luminal surface of the olfactory epithelium (Figs. 2 – 9, 15, 27). In most mammals, including humans, lengths of olfactory cilia are around 50 m (Figs. 5, 6) (Seifert, 1970). In nonmammalian vertebrates, such as the frog, they can be as long as 200 m (Reese, 1965). Individual olfactory receptor cells possess 1–50 sensory cilia (Figs. 2 – 4) (Chuah and Zheng, 1992; Menco, 1983; Menco and Farbman, 1985b; Morrison and Costanzo, 1990, 1992; Ohno et al., 1981). Thus, sensory cilia number and special morphology result in an increased surface area, as much as 40 times (inset Fig. 15) (Menco, 1983, 1992b), for interaction with odors. As noted earlier, the proximal parts of the olfactory cilia have a 9(2)  2 axonemal configuration (Figs. 10, 15). At their very base they have a ciliary necklace (Figs. 7, 15) that consists of spiraling arrays of membrane particles, presumably special proteins. Both features are typical of almost all forms of cilia. However, olfactory cilia have more of such spiraling strands than nonsensory respiratory cilia (Menco, 1980c, 1988b; Naguro and Iwashita, 1992). The exact function of the necklaces is still unclear, but it has been suggested that they may serve as anchors, molecular barriers, and calcium-binding entities (see references in Menco, 1980c, 1988b; Plattner and Klauke, 2001). The thin membrane-lined distal parts of mammalian olfactory cilia have only one to four, but most commonly two, microtubules inside (Figs. 11, 15). Mammalian olfactory cilia, including those of humans, are not intrinsically motile, unlike the case in some other vertebrates (Lidow and Menco, 1984). The nine doublets in the proximal parts lack dynein arms (Fig. 10). Dynein is a Mg2-ATPase protein necessary to generate the force for cilium motility (Stephens, 1974). The overall fine structure of the receptor cell cilia of the septal olfactory organ resembles that of those of the main olfactory organ (Adams, 1992; Miragall et al., 1984). The blanket of sensory cilia covering the olfactory region varies with location. Some regions of the septum and superior turbinates can have a dense, matted sensory ciliary surface (e.g., Figs. 5, 6), whereas adjacent regions can have only a few scattered olfactory receptor cells. This topographic distinction is discussed in Sec. IV of this chapter. Since the aforementioned early investigations by Schultze (1856, 1862), cilia have been suspected of harboring the odorant receptors (Menco, 1977; Parker, 1922; Rhein and Cagan, 1981). The plasma membranes of

Menco and Morrison

olfactory cilia have a special morphology, as they are studded with numerous (1000–2000/m2), intramembranous particles (Figs. 7, 9, 12 – 15). The density of these particles in olfactory cilia is about twice that of such particles of motile cilia of respiratory ciliated cells (Fig. 15) (Menco, 1977, 1980a, 1983, 1992b, 1997). Particles of olfactory cilia may reflect odorant receptors (Buck, 1996, 2000; Buck and Axel, 1991; Mombaerts, 1999), but also the transmembrane signaling proteins Type III adenylyl cyclase (AC) (Bakalyar and Reed, 1990; Krupinski et al., 1989) and olfactory cyclic-nucleotide gated (CNG) channels (Dhallan et al., 1990). Ultrastructural research (reviewed in Menco, 1997) supports physiological and biochemical evidence (Ache and Restrepo, 2000; Paysan and Breer, 2001; McClintock, 2000; Nakamura, 2000) (see Chapters 4 and 11) that the cilia contain the biochemical mechanisms of olfactory signal-transduction. In somewhat more detail, olfactory signal transduction begins when odorants interact with members of the GTPbinding protein (or G-protein)–linked odorant-receptor superfamily that characteristically traverse the membrane seven times (Buck, 1996; Buck and Axel, 1991; Mombaerts, 1999; Sullivan and Dryer, 1996). This stimulus receptor interaction leads to activation of a G-protein, probably Golf , but perhaps Gs (especially in embryos) as well (Belluscio et al., 1998; Menco et al., 1994). The G-protein  subunits, Golf and Gs, activate calcium(Ca2)/ calmodulin-sensitive Type III AC, making cyclic AMP (cAMP). The cAMP opens CNG channels. This results in an electrical signal (Belluscio et al., 1998; Brunet et al., 1996; Gold and Nakamura, 1987; Jones and Reed, 1989; Kleene, 1994; Wong et al., 2000; reviewed by Nakamura, 2000; Schild and Restrepo, 1998) (see Chapter 4). Finestructural studies have shown that all proteins involved in the onset of the AC/cAMP cascade are highly concentrated in the olfactory cilia, particularly the distal parts. This includes odorant receptors (Figs. 16, 17) (Menco et al., 1997), Gs and Golf , Type III AC (Asanuma and Nomura, 1991; Mania-Farnell and Farbman, 1990; Menco et al., 1992, 1994), and CNG channels (Fig. 18) (Matsuzaki et al., 1999a) (Fig. 27 and Table 1). Regulators of G-protein–signaling (RGS) proteins are a group of GTPase-activating proteins (GAPs) (Kehrl, 1998). These RGS proteins have recently also been identified in rodent (Norlin and Berghard, 2001) and canine olfactory (unpublished) and vomeronasal systems, with some showing spatial restrictions correlating with other olfactory signaling molecules (Ressler et al., 1993; Vassar et al., 1993). RGS proteins were first discovered in the yeast Saccharomyces cerevisiae and the nematode Caenorhabditis elegans. To date, 19 mammalian genes are known to encode RGS-cognate sequences. In the

Morphology of the Mammalian Olfactory Epithelium

25

Figures 16 and 17 Two nearby sections through the same mouse olfactory epithelial surface labeled with polyclonal antibodies to putative odorant receptor M4 (dilution: 1:100, arrowhead). Proximal (large arrow) and distal segments (thin arrow) of cilia of one receptor cell dendritic show binding, while those of nearby receptor cells do not (asterisk). Freeze-substituted tissue was fixed with paraformaldehyde (Menco et al., 1997). Gold particles, conjugated to secondary goat-anti-rabbit antibodies, are 10 nm across. Bar
1 m.

26

Menco and Morrison

Figure 18 Distal parts of mouse olfactory cilia (thin arrow) are immunopositive for polyclonal antibodies to -subunits of CNG channels (dilution: 1:25), unlike dendritic endings, proximal cilium parts (large arrow), and supporting cell microvilli (asterisk). Unfixed tissue was rapidly frozen and freeze-substituted (Matsuzaki et al., 1999a). Gold particles, conjugated to goat-anti-rabbit antibodies, are 10 nm across. Bar
1 m.

main olfactory system RGS2 probably contributes to the ability of olfactory neurons to discern odors by controlling AC activity (Sinnarajah et al., 2001). Alternative routes, particularly in invertebrates (Hatt and Ache, 1994), may work through activation of a phospholipase C (PLC)/trisphosoinositide (IP3) cascade. G-proteins are thought to be the catalysts for these routes as well (Nakamura, 2000; Schild and Restrepo, 1998). However, in vertebrates evidence for a role of the PLC/IP3 cascade in olfactory signaling, though present (Cadiou et al., 2000; Vogl et al., 2000), is ambiguous: knockout mice studies favor the cAMP/AC cascade (Belluscio et al., 1998; Brunet et al., 1996; Wong et al., 2000), and two proteins thought to be involved in the PLC/IP3 cascade, Gq (DellaCorte et al., 1996) and IP3 receptors (Cunningham et al., 1993; Kalinoski et al., 1994), have been localized to supporting cell microvilli besides receptor cell cilia. For some other proteins conceivably involved in olfactory signal onset and most proteins putatively involved in signal termination and signal modulation, the exact subcellular location is less clear (Table 1). The former includes several proteins implemented in the multiple roles potentially played by Ca2 in olfactory signaling (Lindemann, 2001; Nakamura, 2000; Schild and Restrepo, 1998; Zufall and Leinders-Zufall, 2000), such as Ca2-exchanger (Noë et al., 1997) and -binding proteins (Kishimoto et al., 1993; Yamagishi et al., 1993). However, fine structural energydispersive x-ray microanalysis suggests that Ca2-gated Cl channels in olfactory cilia conduct inward currents carried by Cl efflux into the mucus (Reuter et al., 1998). In motile cilia there is evidence that the ciliary necklace (Fig. 7) (Menco,

1980c, 1988b) contains regulatory components as well as target structures involved in Ca2 signaling (Plattner and Klauke, 2001). Other fine structural evidence suggests that olfactory cilia possess at least some signal-terminating and -modulating proteins, phosphodiesterases (PDEs; Asanuma and Nomura, 1993) and Na, K-ATPase (Table 1 and Fig. 27) (Kern et al., 1991; Menco et al., 1998). The latter may play a role in the restoration of the receptor potential. CO and NO conceivably also help to regulate and modulate olfactory signaling. However, heme-oxygenase-2 immunoreactivity is found in perinuclear regions of receptor cells rather than in their cilia (Wenisch et al., 2001), and cytochemical activities of proteins involved in CO and NO metabolism are not restricted to the receptor cells, but also involve supporting cells (Wenisch et al., 2000). In vertebrates, fine structural localization is lacking for odorant-binding proteins (Bastianelli et al., 1995), but in insects binding proteins for pheromonal compounds occur in different antennal hemolymph compartments than those for more general odorants (Steinbrecht, 1999). Antibodies to putative odorant receptors label in particular cilia and only those of a few receptor cells (Figs. 16, 17) (Menco et al., 1997), the latter as would be predicted from in situ hybridization studies (Buck, 1996; Buck and Axel, 1991). Interestingly, several other proteins are also present in only a select group of olfactory receptor cells. One of these is the heat shock protein 70 (HSP70) (Carr et al., 1994), which is confined to a subset that is much smaller than that of cells expressing specific odorant receptors (Buck and Axel, 1991). Immunoreactivity for HSP70 is present throughout the

Morphology of the Mammalian Olfactory Epithelium

27

Table 1 Olfactory Signal-Onset Molecules and the Signal-Modulating and -Terminating Molecules Targeting Them Signal modulation Signal onset

Odorant receptors

Signal termination

GRK3, PKA, PKC (?)

Golf ␣

RGS2

PKC γ, δ, and λ (?)

Type III AC (minor cell population: GC)

CNG channel

PDE (PDE1C2, PDE4A?), CaMKII, -arrestin-2, (PDE2 for GC)

CaM

Horizontal arrows give the sequentially activated onset molecules, from odorant receptor to current generating channel. Exact locations of these molecules have been established (bold italics; see also Fig. 27). For all molecules marked in plain italics, especially those of signal modulation and signal termination, exact such knowledge is still absent. The vertical arrows near signal-terminating and -modulating molecules point to the part of the signal-onset cascade targeted by these molecules. Signal-onset molecules: Golf
olfactory GTP-binding protein; AC
adenylyl cyclase; CNG channels
cyclic nucleotide-gated channels. Signal-termination molecules: Protein kinases A and possibly C (PKA and PKC) may act in concert with GRK3 (formerly called -adrenergic receptor kinase-2 or ARK2) (Borisy et al., 1982; Dawson et al., 1993; Peppel et al., 1997; Schleicher et al., 1993) or other GRKs at the level of odorant receptors (Boekoff and Breer, 1992; Breer, 1994). CaM interacts with CNG channels (Chen et al., 1994; Kurahashi and Menini, 1997), while phosphodiesterases (PDEs) (Firestein and Shepherd, 1991), possibly CaM-activated PDE (PDE1C2, formerly called CaM-PDE) (Borisy et al., 1982; Juilfs et al., 1997; Yan et al., 1995), -arrestin-2 (Dawson et al., 1993), and CaM kinase II (CaMKII) (Wei et al., 1998) act at the level of AC. Some PDEs other than PDE1C2, PDE4A (formerly called PDE2) (Cherry and Davis, 1995) and PDE2 (not to be confused with PDE4A, formerly called PDE2), may also be involved in signal termination. PDE2 is expressed in a minor population of receptor cells that use guanylyl cyclase (GC) instead of AC (Gibson and Garbers, 2000; Juilfs et al., 1997) and, likely, cGMP-selective CNG channels instead of cAMP-selective CNG channels (Meyer et al., 2000). Ultrastructurally GC has also been localized to olfactory dendritic knobs and cilia, but to supporting cell apices and microvilli as well (Spreca and Rambotti, 1994). Signal-modulation molecules: A regulator of G-protein signaling proteins [RGS2; RSGs act as GTPase-activating proteins (GAPs)] attenuates odorant-elicited cAMP production (Sinnarajah et al., 2001). Besides a possible role in signal termination, PKCs (, , and ) may modulate signals by increasing CNG channel sensitivity. For reasons of comprehension, not all molecules that may be involved in olfactory signaling, such as several Ca2-binding proteins, are included in this table, but see text (Müller et al., 1998; also Nakamura, 2000; Schild and Restrepo, 1998) (see also Chapter 4).

cytoplasm but seems to exclude the cilia (Fig. 19) whereas odorant receptors are primarily located in membranes of receptor cell cilia (Figs. 16, 17) (Menco et al., 1997). The implication of the fact that morphologically similar receptor cells can have different, membranous as well as cytoplasmic, proteins is unclear. There are at least two other, larger, subsets of olfactory epithelial receptor cells. Both of these may have specific roles. One of these is a subset of receptor cells that displays carbonic anhydrase activity (Brown et al., 1984; Coates 2001; Okamura et al., 1996). Carbonic anhydrase is a zincdependent metalloenzyme that catalyzes the reversible hydration of CO2 to produce HCO3 and H. This enzyme is thought to play a role in CO2 chemoreception (Coates, 2001). The other subset is one that uses guanylyl cyclase and cGMP-gated CNG channels instead of ACIII and cAMPgated CNG channels. These cells of this subset terminate in a special region of the olfactory bulb, the so-called necklace region.* These cells may, like vomeronasal receptor cells, be involved in certain aspects of conspecific recognition (Gibson and Garbers, 2000; Meyer et al., 2000) (see also *The

bulbar necklace region, a glomerular cellular assembly, should not be confused with the ciliary necklaces mentioned earlier. The latter are special subcellular structures at the base of cilia.

Table 1). The two cellular subsets considered here are likely not the same, as carbonic anhydrase positive cells also occur in the nasal respiratory epithelium. In the VNO microvilli of receptor cells are thought to be the subcellular sites that interact with incoming VNO-targeted odors (see Chapter 46). Indeed, analogous to the cilia of main olfactory epithelium receptor cells, these microvilli are selectively enriched in proteins putatively involved in VNO signal transduction (Matsuoka et al., 2001; Menco et al., 2001). Olfactory marker protein (OMP) is a low molecular weight soluble protein. It may, either directly or indirectly, modulate part of the olfactory signaling cascade (Buiakova et al., 1996) and olfactory neurogenesis (Carr et al., 1998). Labeling for OMP is rather evenly distributed throughout receptor cells that have sprouted cilia. The labeling includes the cilia (Johnson et al., 1993; Margolis, 1988; Menco, 1989). Thus, the labeling pattern for OMP differs from that of the antibodies to the signaling proteins that label most receptor cells, such as those to CNG channels (Fig. 18). The latter label the cilia much more prominently than other cellular compartments (Menco et al., 1992, 1994). However, antibodies to OMP and signaling proteins have in common the fact that they label many receptor cells. This contrasts with the labeling patterns of antibodies to odorant receptors (Figs. 16, 17), HSP70 (Fig. 19), and

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Menco and Morrison

Figure 19 An olfactory receptor cell dendrite (large asterisk) shows binding for monoclonal antibodies to HSP70 (undiluted) (Carr et al., 1994) throughout its cytoplasm apart from that inside the cilia (large arrow). This receptor cell is the only one out of thousands that displays labeling; an unlabeled dendrite can be seen nearby (small asterisk). Surrounding supporting cells, including their microvilli, are also devoid of label. The tissue was fixed with paraformaldehyde and glutaraldehyde before cryofixation and freeze-substitution (Griffith 1993; Menco, 1995b). Gold particles, conjugated to secondary goat-anti-rabbit antibodies, are 15 nm across. Bar
1 m.

cGMP cascade proteins and of carbonic anhydrase cytochemistry. Olfactory axons arise from the basal region of the receptor cell bodies and transmit information about odor intensity and quality to the brain. Olfactory axons are always unmyelinated and unbranched and are among the smallest fibers (0.1–0.7 m) in the nervous system (Fig. 20). Olfactory axons form small intraepithelial bundles, pass through the basal lamina, and then combine in larger fascicles, the fila olfactoria. The latter are surrounded by ensheathing or Schwann cells (Doucette, 1992). It is noteworthy that odorant receptors are also expressed in axons and axon terminals (Harrington et al., 1997; Ressler et al., 1994; Vassar et al., 1994; reviewed by Buck, 1996; Mombaerts, 1999) in addition to olfactory cilia (Menco et al., 1997). This intriguing finding suggests that these receptor cells use the same odorant receptors for odor recognition and for axonal targeting to appropriate secondary mitral and tufted cells in the olfactory bulb (Mombaerts, 1999; Mori et al., 2000). Olfactory ensheathing cells have several unique characteristics. For example, they do not surround individual

axon fibers but extend tongues of cytoplasm that wrap bundles of 50 – 200 olfactory axons (see, e.g., Figs. 2D and 8A in Doucette, 1992). This unique packaging of axons, in direct contact with one another, provides the opportunity for interaction of fibers in terms of metabolism, ionic flux, and electrical currents during transduction (Eng and Kocsis, 1987; Gesteland, 1986; Zhang et al., 2000). Olfactory ensheathing cells lack a surrounding basement membrane and contain GFAP and S-100 protein, two biochemical markers characteristic of central nervous system (CNS) astrocytes (Barber and Lindsay, 1982; Takahashi et al., 1984). However, not all olfactory ensheathing cells are immunopositive for both proteins (Pixley, 1993). Some resemble astrocytes, while others resemble Schwann cells (Franklin and Barnett, 2000). They are derived from the olfactory placode, accompany the axons they surround, and cross the peripheral nervous system (PNS)-CNS boundary (Doucette, 1992). Thus, along with their unique morphological characteristics, olfactory ensheathing Schwann cells are more similar to central glial cells than to peripheral Schwann cells.

Morphology of the Mammalian Olfactory Epithelium

29

some present deep within the bulb relative to the olfactory nerve layer. B.

Supporting Cells

Supporting cells can be distinguished from receptor cells, which occasionally are found in the upper epithelium, by their width and their oval and elongated nuclei. Supporting cells are columnar; they span the neuroepithelium throughout and taper basally where they attach by foot-like processes to the basal lamina (Fig. 20). Like receptor cells, supporting cells exhibit a cellular polarity, also cytochemically (Figs. 24, 25) (Menco et al., 1998 and unpublished). Olfactory receptor cell bodies, dendrites, and axons are often surrounded by supporting cell sleeve-like extensions. Scanning microscopy has shown many fine cellular exten-

Figure 20 Olfactory axons (human, transmission electron micrograph) form small intraepithelial fascicles that exit through the basal lamina (arrow) into the underlying lamina propria. O, olfactory receptor cells; S, supporting cells; B, horizontal basal cells. The tissue was conventionally fixed and embedded. Bar
5 m.

Because of their axon growth–promoting properties, they may be an important therapeutic asset in nerve reconstitution following nerve injury (Franklin and Barnett, 2000; Imaizumi et al., 2000; Ramón-Cueto and Avila, 1998). After projecting, unbranched, centrally through small foramina in the cribriform plate of the ethmoid bone (Fig. 21), the olfactory axons terminate in the olfactory bulb in characteristic spherical neuropils called glomeruli (Fig. 23) (see Chapter 7). Within these glomeruli, olfactory receptor axons form asymmetrical synapses with secondorder mitral and tufted neuronal cells (Fig. 22). Axons of these second-order neurons project to subcortical and cortical regions where higher-level processing of olfactory information and discrimination occurs (Chapters 8, 9). The human glomerular layer appears not to exhibit the continuity observed in other species (Smith et al., 1991). Glomeruli tend to be somewhat smaller (25–100 m) and fewer and are more widely dispersed than that seen in other mammals,

Figure 21 Intracranial view of the human anterior cranial fossa, cribriform plate ethmoid bone region. Olfactory axon fascicles (arrows) project from the nasal epithelium through the foramen of the cribriform plate to reach the olfactory bulb (OB). It is within this region that the delicate olfactory axon fascicles are susceptible to injury, i.e., head trauma. Asterisk: crista galli, d: dura mater. Bar
1 mm.

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Menco and Morrison

material from the mucus. Mammalian olfactory supporting cells, however, do not contain glycoconjugates characteristic of mucus-producing cells (Foster et al., 1991). This role is mainly played by Bowman’s gland cells (see Sec. III. E). Also, supporting cell apical glycoproteins differ distinctly from those of surrounding ciliated receptor and other microvillous cells (Ferrari et al., 1999; Foster et al., 1992; Menco, 1992c). The membrane appearance of supporting cell apices and microvilli is quite different from that of receptor cell dendritic endings and cilia. Densities of membrane-associated particles are considerably higher in the supporting cell apical structures. Also, membranes of supporting cell apices contain a special type of rod- or dumbbell-shaped particle (Fig. 7) (Menco, 1980a, 1988a). These have often been associated with transport processes within epithelia (Menco et al., 1998). Indeed, several lines of evidence suggest that one of the putative functions of the supporting cells is to maintain a water and salt balance by way of

Figure 22 Plastic section, 1 m thick, toluidine blue stained, of human olfactory bulb. Olfactory axons (arrows) from the outer fiber layer, enter the bulb, and terminate in characteristic neuropile structures, called glomeruli (G). Bar
100 m.

sions, forming multiple contacts with olfactory receptor cells throughout the epithelium (Breipohl et al., 1974; Morrison and Costanzo, 1990). The apical part of the supporting cell is covered with long microvilli. These microvilli extend into the mucus and terminate at the mucous surface, where they intermingle with the thin parts of the olfactory cilia (Figs. 6, 8, 27) (Andres, 1969; Bannister and Dodson, 1992; Naessen, 1971b; Okano et al., 1967; Seifert, 1970, 1972). Ultrastructural observations of supporting cells have shown differences between them and the columnar mucus secretory goblet cells of the respiratory epithelium. Unlike that of the latter cells, supporting cell apical cytoplasm contains a rich supply of organelles that become scarce basally (Carr et al., 2001; Moran et al., 1982a; Naguro and Iwashita, 1992). Cytoplasmic vesicles have been observed fusing with apical supporting cell surface membranes (Bannister and Dodson, 1992), suggesting that the supporting cells release materials in and/or absorb

Figure 23 Transmission electron micrograph of an olfactory bulbar glomerulus showing receptor cell axon terminals with synaptic vesicles (arrows) and dendrites (D) of second-order neurons. The tissue was conventionally fixed and embedded. Bar
1 m.

Morphology of the Mammalian Olfactory Epithelium

31

Figure 24 Olfactory supporting cell microvilli (large asterisk) bind polyclonal antibodies to amiloride-sensitive Na-channels (dilution: 1:5); apical regions of the supporting cells from which the microvilli sprout (arrow), and olfactory receptor cell dendritic knobs (small asterisk) and cilia (curved arrow) do much less so, if at all. Unfixed tissue was rapidly frozen and freeze-substituted. Protein G, conjugated to 5 nm colloidal gold, was used as secondary probe (Menco et al., 1998). Bar
1 m.

transporting channels. For example, supporting cell microvilli have amiloride-sensitive sodium channels (Fig. 24) (Menco et al., 1998), and at least one water channel, aquaporin Type 3, is present in the lateral membranes of these cells (Fig. 25) (Verkman and Mitra, 2000). The latter is in line with findings that aquaporin 3 is present in basolateral membranes of some ciliated cells (Matsuzaki et al., 1999b). Aquaporin 3 does not appear to be present elsewhere in the olfactory epithelium. While aquaporins 1 and 2 have been found to be immunopositive in VNO tissues, this was not the case in main olfactory epithelial tissues. This includes receptor as well as supporting cells (unpublished). Besides playing a role in ion and water regulation (Kern and Pitovski, 1997; Menco et al., 1998), supporting cells are, together with those of Bowman’s glands, involved in metabolism of xenobiotic compounds. This includes odorant metabolism (Chapters 3, 27). The specific expression of an ubiquitin-positive membrane array in supporting cell supranuclear regions, following excessive odor exposure, is one sign of supporting cell involvement in metabolism of xenobiotic compounds (Fig. 26) (Carr et al., 2001).

Vomeronasal epithelial supporting cells lack this array. Ubiquitination serves to modify proteasomes, multiprotein complexes involved in the regulated breakdown of proteins. Chains of added ubiquitin enable these proteasomes to participate in protein degradation (Bonifacino and Weissman, 1998). Supporting cells may also be involved in removing debris of dying cells and act as phagocytes (Suzuki et al., 1996). Their apical surfaces undergo remarkable morphological transformations paralleling endocrine activity during the ovarian cycle (Da Pos and Arimondi, 1983; Saini and Breipohl, 1976). The close association between supporting cells and receptor neurons (Breipohl et al., 1974) has led to the belief that supporting cells have glial characteristics. They are thought to electrically isolate adjacent olfactory receptor neurons and to regulate the potassium concentration in the extracellular fluid compartment (Graziadei, 1971; Morrison and Costanzo, 1989; Rafols and Getchell, 1983). However, neither supporting cells nor any of the other olfactory epithelial cells express glial fibrillary acidic protein (GFAP). This suggests that these cells do not resemble glial ensheathing cells (Ophir and Lancet, 1988;

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Figure 25 Lateral membranes of olfactory supporting cells are immunopositive for polyclonal antibodies to aquaporin 3 (small arrows; dilution: 1:100). No other structure is seen labeled, including supporting cell microvilli (large asterisk), olfactory receptor cell dendritic knobs (small asterisk), and cilia (large arrow). Freeze-substituted tissue was fixed with paraformaldehyde. Gold particles, conjugated to secondary goat-anti-rabbit antibodies, were 15 nm across. Bar
1 m.

Figure 26 Supranuclear region of olfactory epithelial supporting cells of a rat exposed to 1.0 mL lavender essential oil extract for 6 hours prior to sacrifice. -Ubiquitin (dilution: 1:100) immunoreactivity outlines a conical, somewhat electron-dense array, in this region (asterisk) (Carr et al., 2001). The array consists of a heterogeneous assembly of fragmented membranes of organelles normally present in the supranuclear regions, such as those of endoplasmic reticulum, Golgi body, and mitochondria. Tissue treatment was as in Figure 26. n: nucleus of labeled supporting cell. Bar
1 m.

Volrath et al., 1985). Nevertheless, in some mammalian species, supporting cells (their apical structures), as well as receptor cells and Schwann cells, may contain S-100 (S100) proteins, a biochemical marker for glial cells. In the receptor cells this protein may be involved in microtubule assembly. The presence of S-100 in supporting cells suggests that these cells may share at least some properties with glial (Schwann) cells (Rambotti et al., 1989). A heterogeneous population of supporting cells has been observed in humans and other vertebrates (Costanzo and Morrison, 1989; Rafols and Getchell, 1983; Yamada, 1983). Some areas of the nasal cavity may even consist

exclusively of supporting cells and horizontal basal cells (Suzuki et al., 2000). Such variations may be due, in part, to altered physiological conditions (Saini and Breipohl, 1976). Other supporting cell heterogeneity may be a specific topographic protein expression (Miyawaki et al., 1996), paralleling the topographic expression of odorant receptors in olfactory receptor cells (Mori et al., 2000; Ressler et al., 1993; Strotmann et al., 1994; Vassar et al., 1993) and topographic physiological odor responsivity (Scott and Brierley, 1999). Indeed, scanning electron microscopic observations suggest that, at least in part, the expression of the odorant-receptor zones is determined by

Morphology of the Mammalian Olfactory Epithelium

33

a distinct morphological appearance of supporting cell apices as well as of receptor cell apices in each zone (Menco and Jackson, 1997a). Thus, supporting cell heterogeneity may play a role in the formation of the odorant receptor-specific epithelial zones (Fig. 27). C.

Basal Cells

There are two types of basal cells—horizontal (HBC) and globose (GBC) (Figs. 1, 20). Both are roughly 4–7 m in diameter and have a round, centrally located nucleus. HBCs are found near the basal lamina and contain keratins, intermediate filaments, or tonofilaments characteristic of proliferating epithelial cells (Holbrook et al., 1995; Suzuki and Takeda, 1991a, b). They also contain ecto-5-nucleotidase, a marker for neural development (Braun and Zimmerman, 1998). Several of the histochemical characteristics of the HBCs are shared with basal cells of the nasal respiratory epithelium (Holbrook et al., 1995). GBCs are possibly a heterogeneous population of cells in themselves (Goldstein and Schwob, 1996). They are located above the HBCs. Their cytoplasm is more electronlucent and contains basal bodies. GBCs are not immunoreactive for keratin. Animal studies show basal cells to be stem cells capable of postnatal neurogenesis; mitotic figures are evident in the lower epithelial region. In vitro and in vivo evidence suggests that at least some GBCs give rise to new olfactory neurons. In rodents, HBCs are more slowly dividing and replenish the GBCs (Figs. 1, 20) (Caggiano et al., 1994; Goldstein and Schwob, 1996; Huard et al., 1998; Ohta and Ichimura, 2001; Suzuki and Takeda, 1993; Suzuki et al., 1998). Supporting cell progenitors may be multipotent basal cells (Caggiano et al., 1994; Goldstein and Schwob, 1996; Schwob et al., 1994) and/or reside in Bowman’s gland ducts (Huard et al., 1998; Weiler and Farbman, 1998) (see Chapters 5, 6). D.

Figure 27 Summarizing diagram of the fine structural localization of important olfactory epithelial signal-transduction proteins (see also Table 1 and Menco, 1997) and of several proteins that may play supportive roles in this transduction process. Antibodies to all signaltransduction proteins mainly label cilia, in most cases especially their distal parts (Cunningham et al., 1994; DellaCorte et al., 1996; Kern et al., 1991; Matsuzaki et al., 1991a; Menco et al., 1992, 1994, 1997, 1998). Immunoreactivity for OMP includes cytoplasmic compartments of knobs and dendrites (Menco, 1989), whereas, when present, HSP70-immunoreactivity (Carr et al., 1994) is localized in all cytoplasmic compartments of the cells apart from cilia (unpublished preliminary observations). Supporting cell apices seem to be involved in ion (Menco et al., 1998) and water transport (Matsuzaki et al., 1999b), and in detoxification (Carr et al., 2001) (see Chapters 3 and 27). The thin parts of the cilia align near the interface mucus/external odorous environment where they intermingle with the tips of supporting cell microvilli (see Chapter 4). Bar
1 m.

Microvillous Cells

Besides the major populations of olfactory epithelial cells—the ciliated receptor cells, microvillous supporting, and basal cells—there are at least five other much less abundant cell types that line the nasal cavity with microvilli. The term microvillous is used here generically for all cell types that have microvilli to prevent confusion with the term “microvillar,” which has been used to describe specific microvillous cell types in the nose (Moran et al., 1982a,b; Rowley et al., 1989) (see below under cell Types 2 and 4). First, there are brush cells, which occur in olfactory and respiratory epithelia and which have microvilli with a more rigid appearance than those of supporting cells. Collectively, the microvilli of these cells resemble a brush (Andres, 1969; Jeffery and Reid, 1975;

Jourdan, 1975; Menco, 1977). A second type of infrequent microvillous cell has its microvilli aligned in parallel, and these microvilli have a more uniform diameter and length than those of supporting cells. Depending on the fixation method used, the cytoplasm of this cell is either more electron-opaque (Agasandyan, 1990; Carr et al., 1991; Erhardt and Meinel, 1979; Johnson et al., 1993; Jourdan, 1975; Pyatkina and Agasandyan, 1991; Rowley et al., 1989) or more electron-lucent than that of surrounding supporting cells (Menco, 1992c, 1994; Pixley et al., 1997). A third type of infrequent microvillous cell is more electron-lucent in conventionally fixed tissues than surrounding supporting cells, while its microvilli are more compacted than those of cell Type 2 above (Miller et al., 1995). A fourth

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microvillous cell, also electron-lucent in conventionally fixed tissues and found in humans (Moran et al., 1982a, b), is flask-shaped and has short microvilli (Fig. 28) and a subnuclear pole-like process (Morrison and Costanzo, 1990, 1992). These cells are present at a level of approximately 10% of the neuronal population. A fifth cell resembles in its apex hair cells of the ear and has, so far, only been shown to be present during development. This cell is very sparse and is zonally distributed (Menco and Jackson, 1997b). There is no evidence that any of these cells resemble microvillous receptor cells of the vomeronasal organ. For example, the latter have ample basal bodies in their apices (e.g., Vaccarezza et al., 1981), unlike the microvillous cells that may have, at most, two (Bannister and Dodson, 1992; Menco and Jackson, 1997b). Also, none of them resemble fish olfactory epithelial microvillous receptor cells (Moran et al., 1992d; Rhein et al., 1981; Zielinski and Hara, 1992). The latter are more similar to vomeronasal receptor cells of higher vertebrates (Anderson et al., 1999; Eisthen, 1992). Microvillous cell Type 4 above is immunopositive for the calcium-binding proteins Spot-35 and calbindin

(Yamagishi et al., 1993). Membranes of its microvilli have a specific lectin-labeling pattern distinct from that of cilia of surrounding receptor cells and microvilli of supporting cells (Fig. 29) (Menco, 1992c). Membranes of microvilli of Type 2 cells immunolabel in their apical membranes with an antibody named 1A6 (Carr et al., 1991) and display ecto-5-nucleotidase activity (Braun and Zimmerman, 1998). Also, despite the different appearances, Type 2 and Type 4 cells may be a same polymorphous cell. The shape of the apex of this cell is conceivably affected by fixation; in fixed tissues we saw more Type 2 cells and in unfixed tissues we saw more Type 4 cells. The functional implications of these labeling patterns are still unclear, and the exact role of any of the microvillous cells is unknown. The brush cell, Type 1, may help to regulate concentrations of electrolytes, probably NaHCO3 (Ogata, 2001). Speculatively, because of their resemblance to inner ear hair cells, Type 5 cells with stiff microvilli could be mechanoreceptors (Menco and Jackson, 1997b). Type 2 and/or 4 were thought to be bipolar neurons (Rowley et al., 1989), but the evidence for this is controversial (Carr et al., 1991).

Figure 28 Transmission electron micrograph of a flask-shaped microvillous cell (M; human), probably Type 4, surrounded by apices of supporting cells (S) and by olfactory epithelial sensory receptor cell dendrites (D). The tissue was conventionally fixed and embedded. Bar
1 m.

Figure 29 Microvilli (small arrows) of microvillous cell Type 4 bind the lectin peanut agglutinin (specific for -galactose residues; lectin conjugated to colloidal gold, 5 nm) in a neuraminidase-treated section (removes sialic acid). Nearby supporting cell microvilli (large asterisk) and receptor cell cilia (small asterisk) are devoid of label (Menco, 1992c). Unfixed tissue was rapidly frozen and freeze-substituted. Bar
1 m.

Morphology of the Mammalian Olfactory Epithelium

E.

Lamina Propria, Bowman’s Glands, and Mucus

The olfactory mucosa resides on a lamina propria that contains axon fascicles, blood vessels, connective tissue, and Bowman’s glands. Axons of olfactory receptor cells fasciculate, form small intraepithelial bundles, and enter the lamina propria, where they form larger bundles (20–100 m) that project centrally to the olfactory bulb. Bowman’s glands are present in the olfactory region of all vertebrates except for fish. Human Bowman’s glands are spherical (20–40 m diameter) and are composed of serous and stem cells (Breipohl, 1972; Getchell and Getchell, 1992; Huard et al., 1998; Seifert, 1971, 1972). The serous cells are pyramidal, with a spherical nucleus and short stubby microvilli, and surround a central lumen. Myoepithelial cells surround the acini and contain actin filaments. They squeeze secretory cells and aid in moving secretory products toward a simple duct, which extends through the epithelium to deliver the products to the mucous surface. Thus, Bowman’s gland cells, together with supporting cells (especially in lower vertebrates), produce the microenvironment in which sensory transduction occurs (Getchell and Getchell, 1990, 1992; Getchell and Mellert, 1991; Pelosi, 2001; Seifert, 1971; 1972). There are at least two types of Bowman’s gland serous cells, one with electron-lucent droplets and one with opaque droplets, suggesting that these glands secrete multiple mucous products (Seifert, 1971). Such heterogeneity is reflected in the mucus, which can consist of several distinct domains (Foster et al., 1992; Menco and Farbman, 1992). The exact implications of this heterogeneity, although still unclear, may relate to odorant binding, clearance, and/or maintenance of a mucus consistency, all allowing the process of olfaction to properly take place (Getchell and Getchell, 1990; Pelosi, 1994). IV. TRANSIENT ASPECTS OF THE OLFACTORY MUCOSA A.

Structural Aspects of Embryonic Development and of Neuronal Plasticity

Since other chapters in this book deal with the development of the olfactory mucosa and its special plasticity (see Chapters 5, 6, and 29), only a few aspects of these processes, notably those touching on fine structure, are discussed here. The developmental aspects of the olfactory epithelium have been staged in mice (Cuschieri and Bannister, 1975a,b), as well as in humans (Bossy, 1980; Pyatkina, 1982). The latter authors noted differentiated olfactory

35

receptor cells by week 11. During development and throughout the receptor cell’s life, centrioles migrate through the dendrites to the receptor cell dendritic knobs. This process is thought to be important in cilium formation, cilium replacement, and possibly cell renewal (Heist and Mulvaney, 1968; Mulvaney and Heist, 1971). In this context it is noteworthy that vomeronasal receptor cell dendrites and dendritic endings are stacked with centrioles that do not give rise to cilia, but to microvilli instead (Vaccarezza et al., 1981). That abundance of centrioles is still enigmatic. In general, olfactory receptor cell dendritic endings sprout primary cilia before the full complement of olfactory cilia arises (Fig. 3) (Menco, 1988a; Menco and Farbman, 1985a,b).* There is a distinct topographical transition period, probably within hours, when the olfactory epithelial surface becomes characteristically olfactory. This includes the appearance of tight junctions (Menco, 1988c). In rats this occurs by day 14 following conception (total time of pregnancy 22 days). As part of olfactory ciliogenesis, densities of membrane particles (Menco, 1988a) and strands of ciliary necklaces (Menco, 1988b) increase, and most of the signaling proteins begin to become apparent (Matsuzaki et al., 1999; Menco et al., 1994; reviewed in Menco, 1997, especially Fig. 15, and Tarozzo et al., 1995, Table 2). Immunohistochemical (Menco et al., 1994) and knockout studies (Belluscio et al., 1998) suggest that a Gs signaling cascade precedes the one involving Golf. Initial receptor cell formation does not need odorant receptors, but such receptors are needed for their proper projections to secondary cells (Lin and Ngai, 1999). Neurotrophic factors influence the formation of the neuroepithelium (Mackay-Sim and Chuah, 2000). Special pioneer cells may precede this process (Whitlock and Westerfield, 1998). All of this parallels onset of physiological responsivity (Gesteland et al., 1982).† *A

case can be made for parallel evolution in some form. Whereas in many invertebrates single modified primary cilia seem to form the odorant-receptive sensory cellular structures, in most vertebrates these structures involve modified secondary cilia. In again other instances such structures involve modified microvilli, such as in vomeronasal chemoreception (Eisthen, 1992; Menco, 1992b; Steinbrecht, 1999; Vaccarezza et al., 1981). † Interestingly, in the invertebrate nematode C. elegans, there is distinct evidence that special transcription factors are involved in sensory neuron cilium formation (Swoboda er al., 2000). So far none of these factors has been directly implicated in vertebrate olfactory cilium formation, but receptor cells that do not yet have cilia lack the immunocytochemical expression of OMP (Menco, 1989).

36

The olfactory epithelium has a zonal topography during fetal development (Menco and Jackson, 1997a) that roughly parallels zones in which odorant receptors (Ressler et al., 1993; Strotmann et al., 1994; Vassar et al., 1993; review Mori et al., 2000) and perhaps also RGS proteins (Norlin and Berghard, 2001), as well as zones of odorinduced functional responsivity (Scott and Brierley, 1999) are expressed. Supporting cells, too, show the zonal patterning (Menco and Jackson, 1997a; Miyawaki et al., 1996). Also, like receptor cells, they undergo developmental transformations; their microvilli become longer and the number of supranuclear organelles increases (Cuschieri and Bannister, 1975b; Menco and Farbman, 1985a,b; Mendoza and Kühnel 1991). Olfactory neurons have a number of unique characteristics that set them apart from most other neurons within the nervous system. First, their peripheral location, which exposes them to the external environment, renders them especially vulnerable; most other sensory receptor neurons are located internally, protecting them from the environment. Second, olfactory neurons are among the few neurons that can replace themselves (postnatal neurogenesis), normally and when injured. This remarkable capacity for regeneration, which involves trophic (Mackay-Sim and Chuah, 2000; Newman et al., 2000; Plendl et al., 1999) as well as adhesive factors (Plendl and Sinowatz, 1998) (see Chapter 5), allows them to keep functioning in an often hostile environment. In vitro studies suggest that the human olfactory epithelium also retains the capacity for neurogenesis (Murrell et al., 1996). In primates, olfactory axotomy results in immediate retrograde degeneration of the olfactory receptor neurons. Third, the receptor cells literally form a conduit from the environment to the central nervous system, providing a pathway for movement of exogenous agents into the brain (see Chapter 26). At a structural level, experimental animal studies have shown that injury to olfactory axons (axotomy) results in profound changes in the neuroepithelium. Olfactory receptor neurons degenerate after their axons are severed, leaving an epithelium mostly populated by supporting cells and basal cells, besides degenerating neurons. Following degeneration, GBCs become mitotically active. These then give rise to new neurons that mature, grow axons to the olfactory bulb, where they reestablish anatomical and functional connections, eventually reconstituting the neuroepithelium (Costanzo, 1991; Farbman, 1992; Graziadei and Monti Graziadei, 1979; Monti Graziadei et al., 1980; Morrison and Costanzo, 1989, 1995). Olfactory receptor cells display a remarkable degree of target independence with regard to this regeneration, and this independence involves odorant receptors (Conzelmann et al., 1998; Lin et al., 2000; Wang et al., 1998).

Menco and Morrison

Another developmental process important for correct tissue formation is that of programmed cell death or apoptosis. In the olfactory mucosa apoptosis involves programmed developmental elimination of neurons and of mesenchymal cells (Pellier et al., 1996). B.

Aging

It is difficult to determine the life span of human olfactory neurons. However, in nonhuman vertebrates, olfactory receptor neurons appear to have a life span of at least one year, depending on factors such as the environment, health of the animal, and ability to form synapses (Hinds et al., 1984; Mackay-Sim and Kittel, 1990; Weiler and Farbman, 1997). In human adult epithelium it is not uncommon to find olfactory receptor neurons in all regions, even close to the epithelial surface. Based on their morphology receptor neurons observed near the epithelial surface may be “long-lived” or “old.” Typically, they have short, thick (2–3 m) dendrites and often a more irregular “bumpy” cell surface than neurons found in lower parts of the epithelium (Morrison and Costanzo, 1990). However, Strotmann et al. (1996) offer a distinct alternative. They showed that, based on odorant-receptor expression, a definite vertical zonal organization of olfactory neurons exists besides the horizontal zonal organization (Ressler et al., 1993; Strotmann et al., 1994; Vassar et al., 1993). Each laminar zone contains receptor cells that express a distinct group of odorant receptors. Conceivably, then, the distribution of cells could reflect intrinsic differences in cell populations, age-related processes, or both. The number of receptor cells decreases with age (Breckenridge et al., 1997; Naessen, 1971a; Rosli et al., 1999). In epithelial surfaces, the numbers of cilia and supporting cell microvilli are reduced (Hirai et al., 1996). Areas containing sparse numbers of receptor cells may have been subject to local insults, e.g., from airborne toxic agents, bacteria, or viruses, and/or may reflect gradual epithelial changes inherent in the aging process (Lenz, 1977; Morrison and Costanzo, 1990; Naessen, 1971b; Seifert, 1969) (see next section). Age-related accumulation of several types of electron-dense granules in supporting cell apices (Naessen 1971a) and basal feet (Naguro and Iwashita, 1992) may reflect processes that ultimately compromise function. The basis for these accumulations, including their chemical nature, is not clear, although the basal feet granules are thought to contain lipofuscin. The various transformations and the decrease in amount of sensory epithelium, overall or in cell numbers and cilia, likely contribute to the decreased olfactory ability experienced by many elderly (Spielman, 1998) (see Chapters 23 and 24).

Morphology of the Mammalian Olfactory Epithelium

C.

Ultrastructural Correlates of Olfactory Pathologies and Biopsies

37

A thorough understanding of the distribution of olfactory epithelia is relevant to studies of olfactory biopsy material in clinical cases. Obtaining a reliable tissue sample from the nearly inaccessible olfactory region has presented a challenge to the clinician. The procedure is also dangerous, since part of the olfactory mucosa is located on a thin, bony shelf (cribriform plate of the ethmoid bone) that separates the nasal and anterior cranial cavities. In 1982 Lovell and colleagues developed an instrument and technique to obtain small biopsies. Together with direct endoscopic observation (Lanza et al., 1993), this and similar instruments have been used for safe removal of human olfactory epithelium (e.g., Lehman et al., 2000; Leopold et al., 2000; Paik et al., 1992; Yamagishi et al., 1988) (see Chapter 1). This has led to an increased use of biopsy samples in ultrastructural, immunocytochemical, and pathologi-

cal evaluation of human olfactory tissues (Getchell et al., 1991). An irregular and patchy distribution of olfactory epithelium mixed with respiratory epithelium (Figs. 30 – 33) (Morrison and Costanzo, 1990; Naessen, 1970, 1971a; Nakashima et al., 1991; Rossli et al., 1999; Schultze, 1862; Talamo et al., 1994; von Brunn, 1892; Yamada et al., 1980) must be taken into account when sampling for biopsies or for studies on the physiological responsivity of the human olfactory epithelium (Leopold et al., 2000; Rawson, 2000). Several attempts may be needed to obtain samples that contain olfactory neuroepithelium. Olfactory dysfunction may have a genetic component (Belluscio et al., 1998; Brunet et al., 1996; Wong et al., 2000). Here we restrict ourselves to some structural aspects of olfactory dysfunction. However, emerging methods of functional imaging that take the whole olfactory system into account (see Chapters 12 and 28) may be very helpful in the diagnosis of olfactory deficits (e.g., Yousem et al., 1996). (Ultra)structural abnormalities of the

Figure 30 Scanning electron micrograph of transition region between sensory (darker areas, labeled O) and respiratory epithelial regions (lighter areas, R) in human nasal septum. The border between the two epithelia is irregular. Bar
1 mm.

Figure 31 A transition region between the two epithelia (human) at higher magnification. The bottom half displays olfactory epithelium, the top half respiratory epithelium. Arrows identify olfactory receptor cell dendritic endings with cilia. Bar
5 m.

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Figures 32 and 33 Schematic sagittal sections through the human nose corresponding to Figure 30. The dashed lines outline the olfactory epithelial areas (O; dark), patches of respiratory epithelium within them (R; light). OB: olfactory bulb. The somewhat erratic distributions of both epithelia has to be taken into account for biopsies (see also Fig. 4 in Naessen, 1970).

olfactory mucosa accompany pathological states that lead to total or partial loss of olfactory function (Rawson, 2000; Spielman, 1998). These states include traumatic anosmia (Hasegawa et al., 1986; Jafek et al., 1989; Moran et al., 1985, 1992a, c; Yamagishi et al., 1988) (see Chapters 29 and 30), postviral olfactory dysfunction (Douek et al., 1975; Jafek et al., 1990; Moran et al., 1992a; Yamagishi et al., 1988), Alzheimer’s and Parkinson’s diseases (Brouillard et al., 1994; Moran et al., 1992b) (see Chapters 23–26), Kallmann’s syndrome (Schwob et al., 1993; Truwitt et al., 1993), olfactory epithelial tumors (Reznik-Schüller, 1983; Takahashi et al., 1986; Taxy et al., 1986), rhinosinusitis (Kern, 2000), and exposure to xenobiotic toxic compounds (Hurtt et al., 1988; Mancuso et al.,

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1997; Schwob et al., 1995; Sunderman, 2001) (see Chapters 3, 25–28). Dysfunction can be caused by damaged receptor, supporting, basal, and Bowman’s gland cells (Mancuso et al., 1997; Nakashima et al., 1991; Schwob et al., 1995) or combinations of the above, but special protective mechanisms (Carr et al., 2001) may make supporting and gland cells more resistant to damage than receptor cells (Nakashima et al., 1991). Significant olfactory deficits often occur as a result of head injury (Chapter 30). This may lead to transection of olfactory receptor axons, either by fracture of the cribriform plate or through rapid displacement of the brain. The limited data available suggest that a number of so-called “traumatic anosmics” show marked changes in the ultrastructure of their olfactory epithelia (Hasegawa et al., 1986; Jafek et al., 1989). The olfactory epithelium looks disorganized, lacking the normal “layered” appearance seen in normal individuals. Many pyknotic and metabolically active neurons cause the epithelium to resemble a regenerative one. Numerous olfactory axon fascicles are displaced within the epithelium and lamina propria, indicating axon proliferation. The number of olfactory receptor cells is greatly reduced. Few dendrites reach the surface; those that do usually are devoid of cilia. New receptor cells that develop following trauma-induced axotomy may try to send their axons centrally, but most are unable to penetrate the fibrotic healing of the cribriform plate. Though, in rare instances, slight recovery of olfactory function occurs, suggesting some potential for regeneration and reconnection (Doucette et al., 1983). In Kallmann’s syndrome the symptoms are somewhat similar, but possibly even more extreme. As the olfactory bulb is hypoplastic or aplastic (Truwitt et al., 1993), developing olfactory neurons cannot reach their targets. Consequently the olfactory epithelium is severely degenerated with vastly reduced numbers of receptor and supporting cells. The few axons present also reflect this degeneration (Schwob et al., 1993). Olfactory function is also commonly lost following intranasal viral infections; the condition is known as postviral olfactory dysfunction (see Chapter 26). Patients who have experienced this problem often also show ultrastructural changes in their olfactory epithelia that are quite similar to those in traumatic anosmics, that is, the number of ciliated olfactory receptor cells is reduced, and those that are present have few olfactory cilia. Postviral hyposmics, who have partial loss of their sense of smell, have more ciliated olfactory receptor cells than do postviral anosmics, emphasizing that there is a correlation between function and the number of olfactory neurons. (Jafek et al., 1990).

Morphology of the Mammalian Olfactory Epithelium

39

Various degrees of loss of olfactory function as a consequence Alzheimer’s and Parkinson’s disease (Chapters 23 and 24) are accompanied by olfactory epithelial ultrastructural alterations (Brouillard et al., 1994; Moran et al., 1992b). Compared to other brain areas, these degenerative changes preferentially extend to the olfactory cortex (Reyes et al., 1993). Olfactory epithelia of patients having Alzheimer’s and Parkinson’s disease exhibit a greatly reduced number of ciliated receptor cells. When present, these cells have reduced numbers of cilia (Figs. 34 – 37). In Alzheimer’s disease some of the bipolar neurons have thickened dendrites; many dying cells are evident. Near the basement membrane, the olfactory epithelium of Alzheimer’s patients contains increased numbers of axons, many of them swollen (Fig. 36). Supporting cells also show signs of degeneration (Brouillard et al., 1994). In patients with Parkinson’s disease, the “layering” of nuclei normally seen in healthy olfactory epithelia is disrupted in places. Receptor cell supranuclear regions are often swollen. Numbers of axon profiles near the basement membrane

Figure 35 Transmission electron micrograph of the same biopsy material as used for Figure 34. Olfactory dendritic knobs lack cilia (arrows) and axon bundles have invaded the base of the epithelium (arrowheads). O, olfactory receptor neurons. Bar
5 m.

are greatly increased, and these axons are often enlarged and of variable diameter. Large axon bundles “invade” the epithelium (Figs. 34, 35).

V.

Figure 34 Light micrograph (1 m thick section) of an olfactory epithelial biopsy of a patient with Parkinson’s disease. The epithelium has a disorganized appearance (arrows). Olfactory axons (Ax) and Bowman’s glands are present in the underlying lamina propria. Bar
20 m.

SUMMARY AND CONCLUSIONS

In this chapter we have reviewed the overall and functional morphology of the mammalian olfactory system, including that of the human, as well as structural aspects of normal and regenerative development, aging, and some important pathologies. The evidence is compelling that the olfactory receptor cell cilia (and also VNO receptor cell microvilli) possess all the properties necessary to transform odorant-receptor interactions into an electrical signal. Thus, these cilia and microvilli are highly specialized organelles that resemble in many respects the modified cilia that form vertebrate retinal photoreceptor cell outer segments (Müller and Kaupp, 1998). The olfactory epithelial supporting cells appear to play a large number of roles. These include insulation of receptor cells, transport and regulation of ions and other

40

Figure 36 Transmission electron micrograph of olfactory tissue from an Alzheimer’s patient. The epithelium is disorganized with several degenerating neurons (arrows). Olfactory knobs generally lack cilia and there is an increased number of axon fibers invading epithelium near the basal lamina (arrowheads). Bar
5 m.

substances in surrounding receptor cells and extracellular fluid, metabolism of xenobiotic compounds, protection against aging, phagocytosis, response to hormonal variations, structural support, maintaining of a transmembrane permeability boundary, and guiding of developing receptor neurons. There are two types of basal cells, horizontal and globose, the latter being the precursors for the receptor cells. Bowman’s glands make a heterogeneous mucus and may contain the stem cells for the supporting cells. Though some insights as to the nature of changes in the olfactory epithelia associated with aging and a number of diseases have been observed, the data are still sparse. New biopsy techniques offer the opportunity for a more systematic study of the histopathology of the olfactory epithelium in a variety of disease states. ACKNOWLEDGMENTS The authors thank the editorial staff of Marcel Dekker Inc., the editor of this book, Dr. Richard Doty, Dr. Virginia Carr (the HSP70 and ubiquitin project), Maya Yankova and

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Figure 37 As in Figure 36, but showing an absence of normal ciliated olfactory receptor cells: swollen dendrites (arrows), Supporting cells (S). Bar
5 m.

Gene Minner (BPhMM), Debbie Allgood and Karen Wolfe (EEM) for their help, and Dr. D.T. Moran, whose collaboration on the earlier version of this chapter made our work here so much easier. The work was supported by NIHNIDCD (DC02491, BPhMM and DC01532, EEM), NSF (IBN-0094709, BPhMM), ONDCP, and FAA (EEM). REFERENCES Ache, B. W., and Restrepo, D. (2000). Olfactory transduction. In The Neurobiology of Taste and Smell, 2nd ed. T. E. Finger, W. L. Silver, and D. Restrepo (Eds.). Wiley-Liss, Inc., New York, pp. 167–177. Adams, D. R. (1992). Fine structure of the vomeronasal and septal olfactory epithelia and of glandular glands. Microsc. Res. Techn. 23:86–97. Agasandyan, Kh. V. (1990). Microvillar cells in swine olfactory epithelium. J. Evol. Biochem. Physiol. 26:194–198. Anderson, K. T., Hansen, A., and Finger, T. E. (1999). Localization of olfactory-type (Ors) and vomeronasal type (V2Rs) receptors in different olfactory receptor neurons of goldfish. Chem. Senses 24:593, abstr. 265. Andres, K.-H. (1969). Der olfaktorische Saum der Katze. Z. Zellforsch. mikrosk. Anat. 96:250–274.

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3 Olfactory Mucosa: Composition, Enzymatic Localization, and Metabolism Xinxin Ding New York State Department of Health and State University of New York at Albany, Albany, New York, U.S.A.

Alan R. Dahl Battelle Memorial Institute, Columbus, Ohio, U.S.A.

I.

their metabolic capacities are compared across several species. Finally, the potential for these enzymes to modulate the toxicity of inhalants and to influence odor signal detection is discussed. This chapter is not a review of all specific isozymes detected in nasal tissues, their localization, or the specific toxic effects they are thought to mediate. For such a detailed review of nasal enzymes, the reader is referred to Dahl and Hadley (1991), Ding and Coon (1993), and Thornton-Manning and Dahl (1997). This chapter describes the complexity of the composition and regulation of the major known biotransformation enzymes. Additionally, it discusses various physiological and pathological processes in which nasal metabolic activity is thought to play a role, providing the reader with a framework in which to incorporate enzyme-specific information and relevant sources for a more detailed examination of specific questions. In many cases, data on nasal metabolism have been obtained from whole tissue homogenates, making it impossible to determine the relative contributions of epithelial or subepithelial enzymes, or olfactory versus respiratory mucosa. Because the olfactory mucosa occupies the most caudal region of the nose, metabolism in other nasal regions can affect olfactory function as well. Therefore, data from nasal homogenates are also discussed and noted as such.

INTRODUCTION

The olfactory mucosa, as well as the nasal respiratory mucosa, has a very high metabolic capacity for endogenous and exogenous, or xenobiotic, substrates. Olfactory tissue also has a high degree of inflammatory and immune responsiveness stimulated by contact with foreign substances, exfoliates in response to toxic insult, and regenerates to varying degrees following this exfoliation. The olfactory epithelium is unique in containing the only recognized mammalian neurons that regenerate from precursor basal cells. In addition, these neurons are unique in contacting the external environment with their dendritic processes while the axonal processes of the same cells synapse within the central nervous system (CNS) in the olfactory bulbs. The olfactory mucosa, therefore, represents a tissue where interactions are continually occurring between secretory processes, immune responses, neural signaling, and cell death and development. This chapter examines the basic structure and cell types of the olfactory mucosa and then focuses primarily on the enzymatic capacity of this tissue. The anatomical characteristics generally common to all species are outlined and followed by a brief discussion of interspecies variability in the magnitude, localization, or occurrence of these characteristics. The localization of nasal enzymes and 51

52

II.

Ding and Dahl

ANATOMY OF THE NASAL CAVITY

To understand the contribution of metabolism in the olfactory mucosa, it is necessary to understand its relationship to other nasal tissues illustrated in Figure 1. Before reaching the olfactory mucosa, inhaled air comes in contact with three other epithelial types in the nasal cavity: squamous, transitional, and respiratory. These epithelial regions differ in their metabolic capacities, but metabolism in these tissues can alter the chemical composition of inhaled toxicants before they reach the olfactory mucosa. The anterior vestibule of the nasal cavity is lined with a stratified squamous epithelium. Although enzymes primarily involved in metabolism of endogenous substrates such as alkaline phosphatase and gamma-glutamyl peptidase have been localized to squamous epithelium (Randall et al., 1987), this epithelium has not been reported to have xenobiotic-metabolizing capacity. The squamous epithelium has neither secretory capacity nor cilia. The squamous epithelium gives way, in some species, to a narrow region of transitional epithelium, which is a cuboidal, nonciliated epithelium that has a high metabolic capacity for substrates of specific cytochrome P450 (P450 or CYP) enzymes (Bond et al., 1988). This transitional epithelium often

displays metaplastic changes in response to toxicants. For example, chronic exposure to ozone results in a metaplastic change of the transitional epithelium to secretory, respiratory epithelium, and cigarette smoke exposure produces squamous metaplasia in this region (Harkema, 1990). Continuing in a caudal direction, the nasal cavity is lined by a respiratory mucosa consisting of an epithelium made up of ciliated cells and mucus-secreting goblet cells. The ciliated cells are responsible for movement of the mucous layer through the nasal cavity. Underlying this epithelium are subepithelial glands that produce the majority of serous secretions in the nose. The subepithelial glands also secrete mucus to the mucous layer. The respiratory mucosa plays the major role in both production of nasal secretions and clearance of inhaled materials. High metabolic capacity is found in the respiratory mucosa as well. The most dorsal and caudal region of the nasal cavity is lined by the olfactory mucosa. The surface area of this mucosa is greatly enhanced by a convoluted turbinate structure, which varies greatly across species. The olfactory mucosa is composed of the olfactory epithelium lining the nasal cavity and separated from the underlying lamina propria by the basal lamina (Fig. 2). III. COMPOSITION OF THE OLFACTORY MUCOSA The following is a brief, general description of the olfactory mucosa discussing primarily those elements common to most species. For more detail, the reader is referred to the following reviews: for general nasal and olfactory tissue anatomy, Sorokin (1988) and Uraih and Maronpot (1990); for comparative anatomy, Reznik (1990); and for human olfactory anatomy, Chapters 1 and 2 in this volume. A.

Figure 1 Epithelia of the human nasal cavity. Olfactory epithelium (OE); Bowman’s gland (BG); olfactory nerve (ON); olfactory receptor cell (R); sustentacular cell (S); respiratory epithelium (RE); squamous epithelium (SE); transitional epithelium (TE); nasopharynx (NP); hard palate (HP); inferior turbinate (IT); middle turbinate (MT); superior turbinate (ST).

Epithelial Cell Types

The olfactory epithelium is made up of four primary cell types: the olfactory receptor cells, the sustentacular or supporting cells, the basal cells, and the duct cells of Bowman’s glands (Fig. 2). Cilia containing the olfactory receptors project from the receptor cells into the mucous layer lining the nasal cavity. These cells are unique in two respects: (1) they project directly into the brain before their first synapse, which makes them the only cells directly contacting both the CNS and the external environment; and (2) in contrast to almost all other neuronal cells, olfactory receptor cells regenerate from basal cells after damage (Graziadei and Monti-Graziadei, 1983; Huard et al., 1998).

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Figure 2 Cellular anatomy of the olfactory mucosa. The left panel shows a transverse section in the region of the ethmoturbinates of an adult rat. Tissues on the lumenal side of the basal lamina compose the olfactory epithelium (OE), and tissue inferior to the basal lamina forms the lamina propria (LP). The two layers are included in the olfactory mucosa (OM). Structures identified include sustentacular cell nuclei (sn), olfactory neuronal cells (n), basal cells (b), olfactory nerve bundles (on), Bowman’s glands (bg), Bowman’s gland ducts (d), blood vessels (bv), and nasal airway (NA). 5 m paraffin-embedded sections stained with hematoxylin and eosin; approximately x250 (Modified from Gu et al., 1997). The right panel shows a transmission electron micrograph of the olfactory epithelial surface of an adult rat (approximately x3500). The structures identified are olfactory receptor cells (RC); olfactory dendritic knobs (OK); cilia of receptor neurons (C); sustentacular (or supporting) cells (SC), and microvilli (MV) at the lumenal surface of supporting cells.

The cilia on the receptor cells are nonmotile. These receptor cells generally have very little xenobiotic-metabolizing capacity. The majority of xenobiotic-metabolizing enzymes in the olfactory epithelium have been localized to the sustentacular cells, the duct cells of Bowman’s glands, and the progenitor basal cells. Sustentacular cells have secretory functions in some species (Getchell et al., 1988; Zielinski et al., 1988), but generally are not the primary source of the seromucous secretions covering the olfactory epithelium.

B.

Subepithelial Structure

The lamina propria consists of the acinar cells of Bowman’s glands, olfactory nerves and their associated Schwann cells, blood vessels, and connective tissue. Because this tissue is so often exposed to inhaled foreign substances, cells associated with inflammation and immunity—including neutrophils, plasma cells, monocytes, and macrophages—often are present within the submucosa and epithelium.

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1. Bowman’s Glands These subepithelial glands are the primary source of mucous and serous secretions in the olfactory mucosa. The acinar and duct cells of Bowman’s glands contain many xenobiotic-metabolizing enzymes, although this localization is species dependent. In some cases there is evidence that these enzymes are secreted, again depending on the species (Bogdanffy et al., 1987; Chen et al., 1992; Lewis et al., 1992a). However, whether the secretion is from Bowman’s glands or from sustentacular cells has not been determined. 2. Blood Vessels The highly vascularized lamina propria in the olfactory mucosa is supplied by the ethmoidal artery, a source distinct from the sphenopalatine supply of the respiratory mucosa. In mice, the blood flow through the total nasal mucosa has been estimated at 0.87% of cardiac output (Stott et al., 1983), and in rats, 0.32 mL/min (Morris and Cavanagh, 1986), or 0.53% of cardiac output (Stott et al., 1983). The high perfusion rates in nasal tissues allow for rapid absorption and systemic distribution of substances that penetrate the olfactory epithelium. Conversely, this high perfusion can also allow toxicants in the bloodstream to come in contact with xenobiotic-metabolizing enzymes in the olfactory mucosa. Therefore, because of the high metabolic capacity for xenobiotics, the olfactory mucosa can show significant tissue damage following even systemic administration of toxicants that require metabolic activation. Examples of this are discussed in Sec. V.E. C.

Secretions of the Olfactory Mucosa

Acinar cells of Bowman’s glands and, in some species, sustentacular cells secrete acidic, sulfated, or neutral mucopolysaccharides, the percentage of each depending on the species and specific physiological, neuronal, and environmental conditions. The distribution of different carbohydrate residues in the mucociliary complex is not homogeneous (Getchell et al., 1993b). Human nasal secretions contain immune factors including IgA, IgM, and IgG (Kaliner, 1991). Secretory component and J chain have been localized to acinar and duct cells of Bowman’s glands, as well as the mucociliary apparatus in the human olfactory mucosa (Mellert et al., 1992). Other components of mucus thought to play a defensive role include the antimicrobial proteins lysozyme and lactoferrin (Mellert et al., 1992; Mullol et al., 1992), enzymatic constituents, including aminopeptidases, endopeptidases, carboxypeptidases, angiotensin-converting enzyme, peroxidase, and kallikrein (Kaliner, 1991; Ohkubo et al., 1998), and

a number of antioxidants (Cross et al., 1994), such as reduced glutathione (GSH), mucin, and an abundant, thiol-specific antioxidant protein belonging to the monocysteine subfamily of peroxiredoxins (Novoselov et al., 1999). As mentioned previously, preliminary evidence indicates that some xenobiotic-metabolizing enzymes may be secreted to nasal mucus as well. Nasal mucus also contains regulatory proteins and peptides, including secretory leukoprotease inhibitor (Lee et al., 1993), substance P, vasoactive intestinal peptide (Chaen et al., 1993), and insulin-like growth factor I and its binding proteins (Federico et al., 1999), as well as transport proteins such as odorant-binding proteins (Pelosi, 1996) and vomeromodulin (Krishna et al., 1995b). The proteins in the mucus are thought to result from either serum transudation (e.g., albumin, transferrin, and carboxypeptidase) or local synthesis and secretion (e.g., the metal-binding protein lactoferrin, lysozyme, neutral endopeptidase, and antiproteases) (Ohkubo et al., 1994, 1995). Nasal secretion may be controlled by nerve stimulation (Revington et al., 1997) and by corticosteroids (Fong et al., 1999). The latter, via mineralocorticoid receptors in supporting cells and Bowman’s glands (Robinson et al., 1999), may modulate olfactory Na,K-ATPase and active ion transport, which results in hyperosmolarity of mucus with respect to serum as well as secretion of water. The viscoelastic properties and fluidity of mucus are determined by interactions between mucous components, ion content, and pH. The composition of nasal secretions can change dramatically with inflammation, disease, or toxicant exposure. For example, inflammation, with the transient influx of neutrophils into the mucosa, results in large increases in the secretion of stored mucosubstances from the respiratory mucosa (Harkema et al., 1988). Chronic bronchitis and asthma also increase the quantity of nasal mucus; cigarette smoke and formaldehyde alter the surface viscoelasticity of the mucous layer, and cigarette smoke, antigens, and diethyl ether cause leakage of macromolecules and small ions into the mucous layer (Morgan et al., 1986). The levels of growth factors and neuropeptides in the mucus also change in pathological conditions. For instance, the levels of substance P and vasoactive intestinal peptide in nasal secretions of patients with nasal allergy are significantly higher than in normal subjects (Chaen et al., 1993), and the levels of insulin-like growth factor I and its binding proteins in the mucus of olfactory epithelium are decreased in patients with certain neurodegenerative diseases (Federico et al., 1999). The cilia on respiratory epithelial cells move mucus over the surface of the epithelium to produce mucociliary clearance of environmental airway contaminants.

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However, the olfactory epithelium does not contain beating cilia and therefore must rely on the movement and flow created by the cilia in the respiratory epithelium for clearance. As is the case with mucous composition, the efficacy of ciliary beating can be altered by toxicant exposure or disease. For example, beat frequency is reduced by cadmium salts and acetaldehyde, and the amplitude of the beat is reduced by dimethylamine (Morgan et al., 1986). The beat frequency and proportion of epithelial area with normal ciliary beat frequency are also decreased by oxygen radicals (Min et al., 1999). Cigarette smoke can cause loss of cilia, uncoordinated beating, and even reversal in the direction of beat (Iravani and Melville, 1974). The significance of these alterations in clearance for given individuals can vary widely. In humans, clearance rates have been described as characteristic of a given individual, which may vary from 1 to 20 mm/min (Proctor et al., 1978), and nasal ciliary beat frequency appears to be age independent (Jorissen et al., 1998). The presence in nasal secretions of macromolecules suggests that the protective function of secretions is not simply related to clearance [which decreases with increased secretion (Proctor et al., 1978)], but includes reactions such as bacterial destruction by lysozyme, proinflammatory peptide degradation by peptidases, viral inactivation by IgA interaction, and possibly metabolism of toxicants prior to tissue contact or absorption into systemic circulation. The precise activity and in vivo function of these macromolecules in mucous secretions have not been well studied to date. However, transport, binding, and clearance of xenobiotics occurring in the mucociliary apparatus will influence deposition of these substrates in nasal tissues. Therefore, alterations in mucous constituents and ciliary function will alter metabolism as well. D.

Comparative Aspects of Mucosal Composition

The primary interspecies anatomical differences in olfactory mucosa result from differences in turbinate structure and related proportion of the nasal cavity lined with olfactory mucosa. In general, the surface area to nasal cavity volume ratio reflects the reliance on olfaction of a given species (Fig. 3). For example, the rat has a surface area to lumenal volume ratio of 3350 mm2/cm3; macaque monkey, 775 mm2/cm3; and human, 820 mm2/cm3 with comparative lumenal volumes of 0.4 cm3 for rat and 25 cm3 for human (Harkema, 1991). Increased surface area results from an increase in the complexity of turbinates in the nasal cavity; generally the greatest difference occurs in the number of olfactory turbinates. For example, in the rat, the percentage of the nasal surface area covered by olfactory epithelium is nearly 50%, a much greater percentage

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Figure 3 Comparative anatomy of the nasal cavity. Shading represents the portion of the nasal lumen lined with olfactory epithelium. Note the decrease in proportional surface area of olfactory tissue progressing phylogenetically from rat to monkey to human. Also note the parallel proportional decrease in relative size of the olfactory bulbs. Olfactory bulb (OB); superior turbinate (ST); middle turbinate (MT); inferior turbinate (IT); hard palate (HP); nasopharynx (NP); nares (N); ethmoturbinate (ET); maxilloturbinate (MX); nasal turbinate (NT).

than in the human, as can be seen in Figure 3 (Harkema, 1991). Increased infolding in the turbinates also results in alteration in airflow patterns, and therefore in intranasal deposition patterns. However, studies on airflow indicate that the percentage of inspired air reaching the olfactory mucosa is roughly 15% in rat, monkey, and human (Hahn et al., 1993; Jaillardon et al., 1992; Kimbell et al., 1993). Because of the differences in relative proportion of olfactory tissue, however, the percentage of inhaled dose deposited in olfactory tissue may still be quite different across these species. These differences are therefore important considerations in extrapolating data derived from laboratory animal research to the human population. Although the cytoarchitecture of the olfactory epithelium is remarkably similar across mammalian species, the capacity and localization of xenobiotic metabolism can be markedly different. Thus, the relative activity of specific P450 enzymes varies between rat and human with some isoforms that show high activity in one species being apparently absent in the other (Dahl and Lewis, 1993; Gervasi et al., 1989; Hadley and Dahl, 1983; Morris, 1997; Sheng et al., 2000). Epoxide hydrolase and glutathione Stransferase (GST) both show greater activity in human respiratory tissue than in rat tissue; however, NADPHcytochrome c reductase activity in human respiratory tissue is only 25% that observed in rat tissue (Gervasi et al., 1989). Other interspecies differences in metabolic activity will be discussed in more detail in Sec. IV. Notably,

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although activities of specific enzymes may show large differences between species, no consistent differences across specific enzyme families or even isozymes within a given family allow for reliable generalizations regarding the relative overall enzymatic activity across species. Because biopsy samples of human olfactory mucosa are difficult to obtain, most human nasal enzyme activity to date has been studied in respiratory mucosa. Generally, the olfactory mucosa has a higher or equal level of activity for xenobiotic substrates than does the respiratory mucosa. An exception to this rule is aldehyde dehydrogenase. In rats, nasal respiratory mucosa shows higher aldehyde dehydrogenase activity than does olfactory tissue (Bogdanffy et al., 1998; Casanova-Schmitz et al., 1984), and olfactory tissue has very low immunoreactivity for this enzyme (Bogdanffy et al., 1986). The cells containing xenobiotic metabolic activity in the olfactory mucosa are relatively consistent across species. The primary localization for xenobiotic metabolizing enzymes is within the sustentacular, basal, and duct cells of the epithelium and within the acinar and duct cells of Bowman’s glands in the lamina propria. Much like squamous epithelial cells, olfactory receptor cells contain enzymes having primarily a homeostatic function, such as alkaline phosphatase (Bourne, 1948) and carbonic anhydrase (Brown et al, 1984); however, xenobiotic-metabolizing enzymes have generally not been localized to these cells. Although the cell types identified in the previous paragraph are consistent sites of enzyme localization across species, the specific distribution of a given enzyme within these cell types can vary across species. For example, the cyanide-metabolizing enzyme rhodanese is found in the acinar cells and duct cells of Bowman’s glands in bovine olfactory mucosa (Lewis et al., 1991). However, in the rat, rhodanese is localized to the sustentacular and basal cells rather than to Bowman’s glands. As will be discussed later, this localization can be an important determinant of toxicant-induced damage in different species and must be kept in mind when generalizing from one species to another. Conversely, some enzymes show remarkable similarity in distribution across species. For example, carboxylesterase localization by immunostaining is highly similar in the rat, dog, and human. However, the presence of inflammation in human respiratory tissue correlates with a marked reduction in immunoreactivity for the enzyme, whereas metaplastic lesions in the tissue are associated with a total loss of staining (Lewis et al., 1994b). Such findings indicate that caution must be used in extrapolating to the human population from clean laboratory studies because this extremely plastic tissue is vulnerable to toxicant and irritant-induced damage that can dramatically alter its enzymatic capacity.

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The distribution of specific enzymes within the nasal cavity can also be different. For example, zonal distribution has been observed for the expression of microsomal epoxide hydrolase in rat olfactory mucosa. The enzyme is absent from most of the dorsal medial meatus where immunoreactivity of a GST has been found to be abundant (Genter et al., 1995a). In contrast, expression of a sulfotransferase in mouse olfactory mucosa is localized to the most dorsal and medial zone (Miyawaki et al., 1996).

IV. IDENTITY, TISSUE-AND CELL-SELECTIVE EXPRESSION, AND DEVELOPMENTAL REGULATION OF NASAL BIOTRANSFORMATION ENZYMES The dramatic capacity of mammalian nasal mucosa to metabolize inhaled substances has only been recognized in the last two decades. Reports of alkaline phosphatase localization in olfactory tissue date back to 1948 (Bourne, 1948), and the possibility that esterases present in the olfactory apparatus of moths might play a role in metabolizing olfactory signals was suggested in 1981 (Vogt and Riddiford, 1981). Since the first reports that P450 activity in rat nasal mucosa sometimes exceeded activity in liver when normalized to tissue protein content (Hadley and Dahl, 1982), numerous laboratories have reported activity in the nasal mucosa for families of xenobiotic-metabolizing enzymes, including flavin-containing monooxygenases, aldehyde dehydrogenases, alcohol dehydrogenase, carboxylesterases, epoxide hydrolases, UDP glucuronosyltransferase, GST, and rhodanese (Dahl and Hadley, 1991). In addition, xenobiotic-metabolizing capacity has been demonstrated in olfactory and other nasal tissues from a broad range of species, including Drosophila melanogaster (Wang et al., 1999), lobsters (TrapidoRosenthal et al., 1990), rainbow trout (Starcevic and Zielinski, 1995), rabbits (Ding and Coon, 1988, 1990a; Shehin-Johnson et al., 1995), rodents (Genter et al., 1995b; Hadley and Dahl, 1982), dogs (Dahl et al., 1982), pigs (Marini et al., 1998), sheep (Larsson et al., 1994), cows (Longo et al., 1997), and humans (Gervasi et al., 1989; Getchell et al., 1993a; Gu et al., 2000; Lewis et al, 1991, 1994b). Rapid progress has been made in the identification and characterization of nasal biotransformation enzymes. Many new enzymes have been identified since this subject was recently reviewed (Dahl and Hadley, 1991; Ding and Coon, 1993). The majority of work was focused on members of the P450 gene superfamily, but significant progress has also been made in the molecular identification of other biotransformation enzymes. The biological model systems

Olfactory Mucosa

ranged from insects to fish and to humans. Although most of the xenobiotic-metabolizing enzymes localized in the nose are also found in other tissues, several enzymes have been found to be uniquely or preferentially expressed in the olfactory mucosa in a number of species. A.

Cytochrome P450

The P450 gene superfamily encodes over 500 structurally similar monooxygenases (Nelson et al., 1996). All P450s contain a heme prosthetic group ligated to a highly conserved cysteine residue in the carboxyl terminal portion of the proteins. In a single species, e.g., the humans, the total number of P450 genes can be more than 50, and individual genes are expressed more or less in tissue- and cell-selective fashions. Within a cell, the majority of P450s are located in the endoplasmic reticulum (microsomal fraction), while some are specifically located in the mitochondria. The substrates for microsomal P450s include physiologically important substances such as steroid hormones, eicosanoids, and retinoids, and xenobiotics such as drugs, procarcinogens, antibiotics, organic solvents, anesthetics, pesticides, and odorants. P450-catalyzed biotransformations lead to the formation of more polar compounds that are more readily excreted directly or after conjugation with water-soluble agents such as glucuronic acid and GSH (Porter and Coon, 1991). P450 and NADPH-cytochrome P450 reductase (CPR), a flavoprotein required for microsomal P450-catalyzed monooxygenase reactions, have been found in relatively high concentration in the olfactory mucosa of rodents, rabbits, cows, dogs, pigs, monkeys (Dahl and Hadley, 1991; Ding and Coon, 1993; Hua et al., 1997; Longo et al., 1997; Marini et al., 1998), and humans (Getchell et al., 1993a; Su et al., 1996). Both P450 and CPR have also been identified in the olfactory organ of D. melanogaster (Hovemann et al., 1997; Wang et al., 1999). On a per mg microsomal protein basis, the level of total microsomal P450 in olfactory mucosa is second only to liver among all tissues examined in rodents and rabbits; the level of CPR in olfactory mucosa microsomes is even higher than in liver (Ding et al., 1986; Reed et al., 1986). The evolutionarily conserved presence of the P450 enzymes supports their functional importance in olfaction. More than 10 different P450s have been identified in mammalian olfactory mucosa, including members of the CYP1A, 2A, 2B, 2C, 2E, 2G, 2J, 3A, 4A, and 4B subfamilies (Dahl and Hadley, 1991; Deshpande et al., 1999; Ding and Coon, 1993; Gu et al., 1998; Zhang et al., 1997). Additional forms are expected to be found since several subfamilies have not been examined, such as CYP2D, 2F, and 4F. Of these, CYP1A2, CYP2A, and CYP2G1 are the

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major forms (Ding and Coon, 1990a; Genter et al., 1998; Gu et al., 1998). Multiple genes are present in the CYP2A subfamily, which were named sequentially according to the time of discovery. The CYP2A genes expressed in the olfactory mucosa include CYP2A3 in rats, CYP2A5 in mice, CYP2A6 and CYP2A13 in humans, and CYP2A10 and CYP2A11 in rabbits (Koskela et al., 1999; Peng et al., 1993; Su et al., 1996, 2000). There appears to be only a single CYP2G gene in all mammalian animals studied; thus they are all called CYP2G1 (Ding et al., 1991; Hua et al., 1997; Nef et al., 1990). Originally, the rabbit CYP2As were called P450 NMa, which included both CYP2A10 and 2A11 when purified from nasal microsomes (Ding and Coon, 1988; Peng et al., 1993); similarly, the rabbit CYP2G1 was called P450 NMb (Ding and Coon, 1988), and the rat CYP2G1 was called P450 olf1 (Nef et al., 1989). In humans there may be two copies of the CYP2G gene, but both contain loss-of-function mutations in the majority of individuals, and a functional cDNA has not been identified to date (Sheng et al., 2000). In addition to the P450 forms in gene families 1–4, which are often referred to as the xenobiotic-metabolizing P450s, there are also several microsomal P450 gene families specifically involved in steroid biosynthetic pathways in the endocrine and reproductive organs or bile acid metabolism in liver, such as CYP7, 17, 19, 21, and 51 (Nelson et al., 1996). The expression of these genes in the olfactory mucosa has not been examined. Several olfactory mucosal P450s are specifically or preferentially expressed in this tissue. For example, CYP2G1 is only expressed in the olfactory mucosa (Ding and Coon, 1990a; Hua et al., 1997; Nef et al., 1989) and, at much lower levels, in the vomeronasal organ (Gu et al., 1999). Several CYP2As are expressed in olfactory mucosa at much higher levels than in other tissues (Ding and Coon, 1990a; Su et al., 1996). Preferential expression of a P450 in the olfactory organ was also found in Drosophila (Wang et al., 1999). Although the specific roles of these tissueselective P450s have not been identified, their unique or preferential presence in the olfactory mucosa strongly suggests functional importance in the chemosensory organ. Immunohistochemical studies of several olfactory mucosa microsomal P450s, including CYP1A, 2A, 2B, 2G, and 4B, indicated that they are expressed in nonneuronal cells, particularly in the sustentacular cells in the epithelium and in the Bowman’s glands in the submucosa (Adam et al., 1991; Chen et al., 1992; Getchell et al., 1993a; Thornton-Manning et al., 1997; Voigt et al., 1985, 1993; Zupko et al., 1991). Distribution of CPR was found to resemble that of the P450s (Adam et al., 1991; Baron et al., 1986; Voigt et al., 1985); however, CPR expression in olfactory receptor neurons (ORNs) has also been reported

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(Verma et al., 1993; Voigt et al., 1985). The lack of known microsomal P450 expression in the neuronal cells is also supported by toxicological studies implicating the Bowman’s glands and the supporting cells as the initial targets following chemical treatment (Brittebo, 1997). The localization of P450s to the mucus-producing cells in the Bowman’s glands and the detection of P450 immunoreactivity in the mucociliary complex at the epithelial surface led to suggestions that they may be secreted to the mucous layer where they may directly act on inhaled chemicals (Adam et al., 1991; Chen et al., 1992). Little is known about the molecular mechanisms that regulate the tissue- and cell-selective expression of P450s and other biotransformation enzymes in the olfactory mucosa. Two recent in vitro studies identified nuclear factor I–like cis-acting elements in the proximal promoter region of both CYP2A3 and CYP1A2 genes (Zhang and Ding, 1998; Zhang et al., 2000). These highly conserved DNA sequences, which are critical for transcriptional activity of the cognate P450 promoters in vitro, appear to interact with olfactory mucosa–restricted nuclear proteins. Identification of these potentially novel tissue-selective transcription factors will be important for understanding the regulation of these and other genes preferentially expressed in the olfactory mucosa. The developmental expression of P450s and CPR in olfactory mucosa has also been examined. In rabbits, CYP2G1 was detected at 2 days before birth (Ding et al., 1992). In rats, CYP2G1 expression was detected at E20, which was suggested to coincide with the appearance of Bowman’s glands (Margalit and Lancet, 1993). Prenatal expression of several P450s and CPR has also been found in humans (Gu et al., 2000). The earlier onset of P450 expression in olfactory mucosa than in other tissues may indicate a functional significance in the perinatal period when olfactory ability is important for the survival of the newborn. B. Other Enzymes GSTs catalyze the conjugation of GSH with numerous electrophilic substrates, including reactive intermediates formed in P450-catalyzed reactions, which decrease their reactivity with proteins and other cellular macromolecules (Armstrong, 1997; Eaton and Bammler, 1999), as well as unaltered odorants (Ben-Arie et al., 1993). Most GSTs are located in the cytosol, although some have also been found in microsomes and mitochondria (Eaton and Bammler, 1999). At least five cytosolic GST gene families are known in humans. Multiple GSTs have been detected in the olfactory mucosa in a number of species (Aceto et al., 1993; Banger et al., 1993; Ben-Arie et al., 1993; Krishna et al.,

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1995a; Rogers et al., 1999; Starcevic and Zielinski, 1995). In rats, these include rGSTA3, rGSTA4, rGSTM1, rGSTM2, rGSTM6, and rGSTP1 (Banger et al., 1993, 1996; Ben-Arie et al., 1993). In humans, GSTA and GSTP, but not GSTM, were detected in the olfactory mucosa by immunohistochemistry (Krishna et al., 1995a). In rats and cows, high GST activity was found in olfactory mucosa toward model substrates and odorants (Aceto et al., 1993; Ben-Arie et al., 1993). An olfactory tissue-specific GST has not been found in mammals, although one has been found in the sphinx moth Manduca sexta (Rogers et al., 1999). In rats, GSTA and GSTM immunoreactivity was detected in sustentacular cells and Bowman’s glands in the olfactory mucosa. In humans, GSTA immunoreactivity was detected mainly in the acinar cells of the Bowman’s glands, as well as in the supranuclear region of supporting cells, but GSTP immunoreactivity was detected only in the supporting cells in the olfactory mucosa (Krishna et al., 1994, 1995a). Olfactory mucosal GSTA and GSTM immunoreactivity was detectable at E16 in rats and increased postnatally, with peak expression around P11 (Krishna et al., 1994). The postnatal increases in the levels of GSTA and GSTM isoforms were confirmed by immunoblot analysis of olfactory S9 fractions (Banger et al., 1996). However, cytosolic GST activity measured with 1-chloro-2,4-dinitrobenzene as a substrate was constant in rat olfactory mucosa between P3 and P84, while microsomal GST activity remained low until P21 and then increased to reach adult levels at about P60 (Banger et al., 1996). In humans, both P450 2A and GST immunoreactivities were decreased in older adults (Getchell et al., 1993a; Krishna et al., 1995a). UDP glucuronosyltransferases (UDPGTs), also named UDP glycosyltransferases (UGTs), catalyze the conjugation of UDP glucuronic acid with a variety of substrates (Mackenzie et al., 1997). In mammals, the UDPGTs are found in microsomal fractions and belong to two different gene families, each having multiple genes (Mackenzie et al., 1997). An olfactory mucosa-specific UDPGT has been identified in rats, cows, and humans, named UGT2A1, which is active toward numerous compounds, including many odorants (Jedlitschky et al., 1999; Lazard et al., 1990, 1991; Mackenzie et al., 1997). A tissue-specific UGT (DmeUgt35a) has also been identified in the olfactory organ of D. melanogaster (Wang et al., 1999). Multiple UDPGTs are believed to be expressed in mammalian olfactory mucosa (Marini et al., 1998), but the specific enzymes have not been characterized, except for UGT2A1. Sulfotransferases (ST), which include phenol ST (PST), hydroxysteroid ST (HSST), and, in plants, flavonol ST (FST) gene families, catalyze the transfer of a sulfonate group from 3-phosphoadenosine-5-phosphosulfate to both endogenous and xenobiotic compounds

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(Weinshilboum et al., 1997). Mouse nasal cytosol had high activity for a number of phenolic aromatic odorants (Miyawaki et al., 1996; Tamura et al., 1997). A PST cDNA has been isolated from a mouse olfactory cDNA library (Matsui et al., 1998). PST proteins were also detected in the cytosol of rat and mouse nasal tissues using an antibody to rat liver PSTg (Miyawaki et al., 1996). Mouse PSTG immunoreactivity, which is detectable prenatally, is localized mainly in the sustentacular cells (Miyawaki et al., 1996). V. FUNCTIONS OF NASAL BIOTRANSFORMATION ENZYMES Nasal xenobiotic metabolism likely serves multiple functions. Four possibilities, discussed in more detail below, are (1) detoxication of inhaled and systemically derived xenobiotics, (2) protection of other tissues, such as the lung and CNS, from inhaled toxicants, (3) modification of inhaled odorants, including the special case of steroids as reproductive stimuli, and (4) modulation of endogenous signaling molecules. In addition, the roles of nasal biotransformation enzymes in the metabolic activation and toxicity of inhaled or systemically derived xenobiotics are also considered. A.

Detoxication of Inhaled Toxicants

The nose is the portal of entry for inhaled chemicals and, as such, is continually exposed to toxic insults. Therefore, one function of xenobiotic metabolism could be detoxication of inhaled toxicants. Because of the small mass of nasal mucosa, even though the activity of nasal enzymes is high, the total capacity to metabolize inhaled substrates is probably not high enough in most cases to provide systemic protection from inhaled toxicants. (Exceptions are discussed in the next section.) The protective function of nasal metabolism, therefore, is probably more a form of local tissue protection than protection of downstream tissues such as lung. It can be said for the vast majority of lipophilic compounds that would normally build up in the nasal tissue that combined P450 and phase II metabolism, or metabolism by other routes, decreases toxicity either by increasing solubility and subsequent clearance or by other chemical modification to less toxic forms. Examples of substrates for which this is the case are given in Table 1. In some instances, nasal metabolic systems may work in tandem to provide local protection. For example, a wide range of inhaled substrates, including methamphetamine, cocaine, nicotine, diesel soot extracts, and pyrilamine, are metabolized to formaldehyde by P450 isozymes in the

59 Table 1 Instances in Which Nasal Metabolism Probably Results in Detoxication Substrates Nitropyrenes 2,6-dichlorobenzonitrile Coumarin Cocaine Alkoxycoumarins Lactones Styrene oxide Naphthol Chlorodinitrobenzene Cyanide Nicotine Formaldehyde

Enzyme activities Oxidases and hydroxylases Hydroxylase 7-hydroxylase Demethylation Dealkylation Carboxylesterases Epoxide hydrolases Transferases Transferases S-transferases (rhodanese) Demethylases Aldehyde dehydrogenases

Source: Modified from Dahl and Hadley, 1991.

olfactory mucosa (Dahl and Hadley, 1983). Although inhalation of 15 ppm formaldehyde produces squamous metaplasia and squamous cell carcinomas of the respiratory mucosa of the nasal cavity (Morgan and Monticello, 1990), the presence of formaldehyde dehydrogenase can increase clearance and therefore protect tissues from metabolically produced formaldehyde. The activity of formaldehyde dehydrogenase in the olfactory mucosa is approximately double that of the respiratory mucosa (Bogdanffy, 1990). One would therefore predict that the olfactory mucosa will be less sensitive to formaldehyde toxicity. Indeed, olfactory mucosal lesions resulting from formaldehyde exposure are less common than respiratory mucosal damage. Nevertheless, the regional distribution of damage will be influenced by the relationships among airflow, deposition, and chemical solubility and reactivity, as well as enzyme localization. The enzyme rhodanese metabolizes cyanide to the less toxic metabolite thiocyanate. The activity of rhodanese in human nasal respiratory mucosa is high and probably serves to protect against toxic effects of inhaled cyanide (Lewis et al., 1991). There are many diverse environmental sources for inhaled cyanide such as combustion products from synthetic materials and cooking of cyanogenic fruits such as apricots and cherries. However, it is possible that rhodanese serves an additional protective function in secondarily metabolizing cyanide produced from the P450 metabolism of inhaled organonitrile compounds such as benzylnitrile and acetonitrile (Dahl and Waruszewski, 1990). With respect to the secondary detoxication of toxic metabolites, the cellular localization of specific enzymes may be important when generalizing across species. The organonitrile ,-iminodipropionitrile (IDPN) is toxic to the acinar cells of Bowman’s glands following systemic

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administration in rats (Genter et al., 1992). P450 metabolism of IDPN would yield cyanide. The cells of Bowman’s glands in the rat contain several isoforms of P450 (Dahl and Hadley, 1991); however, they do not contain rhodanese (Lewis et al., 1992b). It is therefore likely that the toxicity of IDPN in these cells is caused by a buildup of the metabolite cyanide. Because the cellular distribution of rhodanese differs across species, the target cells for IDPN toxicity may also differ. It should be remembered that nasal xenobiotic enzymes act not only on inhaled substrates, but on substrates in the systemic circulation as well, as evidenced by metaboliteinduced toxicity to the olfactory mucosa seen following intravenous administration of toxicants such as 4-(methylnitrosamino)-1-(3-pyridyl)-1-butanone (NNK) (Belinsky et al., 1990), 3-methylindole (Turk et al., 1986), and acetaminophen (Jeffery and Haschek, 1988). Teleologically, this metabolism of systemic compounds could serve to reduce stimulation of olfactory receptors by circulating odorants, thereby eliminating possible interference with or masking of inhaled odorants. In addition, such metabolism could serve to protect this important sensory tissue from damage induced by circulating toxicants. While, in the cases noted above, toxic metabolites are formed and the metabolic activity of the nasal tissue results in damage rather than protection, this is almost certainly not the general case, as discussed below.

B.

Protection of Other Tissues from Inhaled Toxicants

1. Lung Based on activity, most nasal enzymes probably have little effect on reducing concentrations of toxicants entering systemic circulation unless inhaled concentrations are very low. Two exceptions to this might be toxic substrates of carboxylesterases and cyanogenic compounds detoxified by the cyanide-metabolizing enzyme rhodanese (Dahl, 1988; Lewis et al., 1991). Across several species, the capacity of nasal carboxylesterases is sufficient to detoxify inhaled concentrations of esters such as ethyleneglycol monomethyl ether acetate in the 1000–3000 ppm range (Dahl, 1988), a concentration in excess of occupational exposure limits. However, in many cases, such as with dibasic esters, the metabolites of esters are themselves toxic to olfactory tissues (Bogdanffy, 1990). Likewise, nasal rhodanese activity is sufficient to detoxify inhaled concentrations of hydrogen cyanide as high as 2800 ppm in the rat (Lewis et al., 1991). This capacity to significantly alter systemic toxicant exposure through nasal metabolism does not hold for all

enzyme families. The capacity for nasal metabolism of some P450 substrates is considerably lower: 0.1–5 ppm for p-nitroanisole and 0.1–3 ppm for aniline. At these levels of activity, significant systemic protection from inhaled toxic substrates would probably not result from nasal metabolism. 2.

Central Nervous System

Xenobiotic metabolism in the olfactory epithelium as well as in the olfactory bulbs may be a component of a “nosebrain barrier.” The olfactory epithelium has a unique anatomy wherein a single receptor cell contacts the external environment in the nasal lumen and projects directly to its synapse within the CNS in the glomeruli of the olfactory bulbs. These cells, then, provide direct access for inhalants to the CNS. Several studies have demonstrated that a variety of materials instilled or surgically implanted into the nasal cavity can be transported to the olfactory bulbs (Henriksson and Tjälve, 2000; Larsson and Tjälve, 2000; McLean et al., 1989; Schultz and Gebhardt, 1934; Shipley, 1985; Tomlinson and Esiri, 1983). Generally, these studies have used concentrations of material far in excess of those encountered environmentally. Therefore, the importance of this phenomenon for inhalation of environmentally relevant concentrations of toxicants is not yet clear. A more indepth discussion of this topic can be found in Chapter 26 and in the reviews by Lewis et al. (1994a) and Tjälve and Henriksson (1999). The factors involved in a nose-brain barrier that might protect the brain from toxicant exposure have not yet been elucidated. However, nasal xenobiotic metabolism is likely to be involved. In addition, mucociliary clearance, immune responses in the olfactory or other nasal mucosa, tight junctions between epithelial cells, and the rapid death of epithelial cells following toxicant exposure are also likely to play a role (Lewis et al., 1994a). C.

Modification of Olfactory Stimuli

1.

Odorants

A third possible function for nasal metabolism is either activation of inhaled nonodorants to odorants or, conversely, clearance of odorants from the olfactory receptor cells to allow reactivation of receptors (Dahl, 1988; Getchell and Getchell, 1977). Although most biotransformation enzymes are located in the nonneuronal cells, the lipophilic substrates can quickly diffuse to all cells in the olfactory mucosa. This phenomenon of receptor reactivation has been demonstrated in lobsters (Trapido-Rosenthal et al., 1990) and in silk moths (Vogt and Riddiford, 1981), but as yet not in mammalian species. However, this possibility has been suggested for mammalian cells where nasal-specific UDPGTs have been shown to have a greater

Olfactory Mucosa

substrate specificity for odorant molecules than do the UDPGTs isolated from liver (Lazard et al., 1991). Odorant metabolites may contribute to potency and odor quality. Many odorant metabolites are more watersoluble than the parent odorants, and they may reach very high concentrations in the mucus bathing ORNs (Dahl, 1988; Price, 1984). If olfactory stimulation includes summation of signals from both parent odorant and its metabolites (Kashiwayanagi et al., 1987; Price, 1984), such metabolites could be important to the sensitivity, intensity, and quality perception of an odor. Thus, it has been hypothesized that odor quality and intensity may reflect effects of the odorant and its metabolites on ORNs (Dahl, 1988; Price, 1984, 1986). Lipophilic odorants partition favorably into the membranous structure of the neuroepithelium. Their accumulation may adversely affect many aspects of cellular function. They may saturate the odorant clearance mechanism, disturb mitochondrial energy production, and suppress or sensitize local immune systems. They may also change the biophysical properties of the plasma membrane and thus the functional capacity of ion channels and other signal transduction components. Biotransformation reactions that convert these lipophilic compounds into more water-soluble metabolites may thus be indispensable for maintaining the homeostasis of the chemosensory tissue. 2.

Steroids

Steroids are likely to represent a special case of metabolic modulation of olfactory stimuli. Inhaled steroids can serve as primary olfactory cues in the regulation of reproductive function in a number of species, as well as modulators of olfactory function. Androstenone is a steroid found in the urine and saliva of pigs and humans as well as in human sweat. In pigs, androstenone excreted by boars has been shown to initiate mating behavior in estrus sows with intact olfactory function (Beauchamp et al., 1976). Sensitivity to the odor of androstenone varies widely in humans (Dorries et al., 1989). Interaction of exogenous steroids with the olfactory system has also been demonstrated through modification of serum testosterone, testicular size, and spermatogenesis in rhesus monkeys by intranasal administration of estradiol and progesterone (Anand-Kumar et al., 1980). It is likely that the ability of the olfactory mucosa to metabolize steroids will influence responses to exogenous steroids. Mammalian olfactory mucosa has very high activities in the metabolism of all three major sex steroids (Brittebo and Rafter, 1984; Brittebo, 1985; Ding and Coon, 1990a, 1994; Hua et al., 1997, Longo et al., 1997; Marini et al., 1998). The olfactory mucosa–specific P450 2G1 metabolizes sex steroids to unique patterns of metab-

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olites when compared to those produced by other known P450s (Ding and Coon, 1990a, 1994; Hua et al., 1997). The vomeronasal organ is also capable of metabolizing sex steroids (Gu et al., 1999). Accumulation of sex steroids and other endogenous or exogenous compounds that are normally removed by P450 metabolism could affect signal transduction by competing for receptors. To that end, Rosenblum et al. (1991) reported that receptor binding of 17,20-dihydroxy-4-pregnen-3-one to goldfish olfactory mucosa is competitively inhibited by progesterone and other sex steroids. Androstenone is metabolized by the nasal mucosa in pigs (Gennings et al., 1974) and is a competitive inhibitor of steroid metabolism by CYP2G1 (Ding and Coon, 1994), which suggests that it may be metabolized by this or other P450 enzymes. The role of nasal metabolism in reproductive function has received very little attention to date, but the discovery of olfactory mucosa–specific P450s that metabolize sex steroids is likely to make this an important area of research in the future. D.

Modulation of Endogenous Signaling Molecules

The P450 isoforms identified in the olfactory mucosa are all active in the metabolism of endogenous compounds, although they are also involved in metabolizing foreign chemicals. For example, CYP1B, 2A, 2B, 2G, and 3A are active in the hydroxylation of sex steroids (Ding and Coon, 1994; Hayes et al., 1996; Liu et al., 1996; Waxman et al., 1991), CYP1A, 2B, 2C, 2E, 2J, and 4A are active in the hydroxylation or epoxygenation of arachidonic acid (Laethem et al., 1992; Luo et al., 1998; Scarborough et al., 1999), CYP1A2 and CYP2J4 are active in converting retinals to retinoic acids (RAs), and CYP1A and 2B are active in the hydroxylation of RA (Roberts et al., 1992; Zhang et al., 1998). The consequences of microsomal P450catalyzed metabolism of endogenous compounds are usually inactivation of the bioactive substance, as with hydroxylation of testosterone and RAs. However, the epoxygenated or hydroxylated products of arachidonic acid have been implicated in many biological processes, such as regulation of vascular tone, ion transport, calcium release from endoplasmic reticulum, and modification of biophysical properties of plasma membrane (Capdevila et al., 1992; Makita et al., 1996). It is believed (Nebert, 1990, 1991) that the xenobiotic-metabolizing P450s regulate steadystate levels of endogenous compounds important for growth, homeostasis, differentiation, and neuroendocrine functions. P450-catalyzed formation of arachidonic acid epoxide and hydroxides can regulate vascular tone and thus rate of blood flow (Capdevila et al., 1992; Makita et al., 1996). Decreased ability to produce these regulatory molecules

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may lead to congestion and restrictions in air flow in the nasal cavity, which could affect threshold sensitivity in odor detection. On the other hand, accumulation of arachidonic acid may lead to increased production of leukotrienes and other mediators through the lipoxygenase pathway and potentially induce airway hypersensitivity (Pinto et al., 1997). ORNs are one of the few vertebrate neuronal populations that undergo turnover and replacement throughout the life of an animal and following injury (Goldstein et al., 1998; Graziadei and Monti-Graziadei, 1983) (see Chapter 5). Such remarkable regenerative capacity may be at least partly related to the presence of the highly active biotransformation enzymes, which control the availability and level of various endogenous bioactive substances capable of regulating growth and differentiation in the target tissue. The relatively high efficiency and broad substrate specificity of the P450 enzymes toward steroid hormones (Ding and Coon, 1994) and retinoids (Roberts et al., 1992) suggest that these compounds may accumulate in olfactory mucosa when the P450s are inhibited or downregulated. RAs have powerful differentiation-promoting effects (Chambon, 1996) and induce apoptosis (e.g., Josefsen et al., 1999). RA receptors have been detected in mouse olfactory mucosa (Zhang, 1999). A role of RA in ORN differentiation has been reported (Whitesides et al., 1998), and RA has also been shown to regulate neurogenesis in adult-derived neural stem cell cultures (Takahashi et al., 1999). RAs are degraded in target tissues by microsomal P450s (Duester, 1996). RA inactivation catalyzed by an embryonic P450 isoform (P450RA) has been found to result in RA hyposensitivity in cultured cells (Fujii et al., 1997). Olfactory mucosa is also a known target tissue for steroid hormone action (Balboni, 1967; Balboni and Vannelli, 1982; Fong et al., 1999; Vannelli and Balboni, 1982). In male rats, olfactory mucosa morphology is altered by castration, and testosterone replacement counteracts these alterations (Balboni, 1967). In addition, corticosteroids may regulate olfactory secretion by modulating Na,K-ATPase (Fong et al., 1999). Thus, prolonged accumulation of these endogenous compounds may lead to changes in olfactory mucosa structure, cell biology, and functional capacity. E. Metabolic Activation and Xenobiotic Toxicity in the Nasal Mucosa The powerful biotransformation enzymes, particularly the P450 enzymes, generate reactive intermediates from inhaled or systemically derived xenobiotic substrates, which could lead to toxicity (Table 2). The activated metabolites, such as the proposed epoxide intermediates

Ding and Dahl Table 2 Instances in Which Nasal Metabolism Probably Results in Activation Substrates

Enzyme activities

2,6-dichlorobenzonitrile Coumarin Ferrocene Benzo(a)pyrene Hexamethylphosphoramide Diethylnitrosamine Organonitriles Phenacetin Esters Acetaminophen Trifluoromethylpyridine

Epoxygenase Epoxygenase Oxidases Oxidases Demethylases Deethylases Oxidases Oxidases Carboxylesterases Oxidases N-oxidases

Source: Modified from Dahl and Hadley, 1991.

from 2,6-dichlorobenzonitrile or coumarin (Ding et al., 1996; Zhuo et al., 1999), can usually be efficiently removed by phase II enzymes such as GST. However, when the dose is high or when the phase II enzymes are compromised due to chemical inhibition or genetic deficiency, the reactive metabolites would accumulate and cause cytotoxicity in the olfactory mucosa. In cases where a reactive intermediate with relatively long half-life is generated, such as the benzo(a)pyrene epoxides (Dahl et al., 1985), it may be transported to nearby organs, such as the pharynx, the esophagus, the anterior nasal cavity, and the olfactory bulb, where local biotransformation activities are much lower compared to the olfactory mucosa, and potentially cause toxicity (Dahl et al., 1985; Ghantous et al., 1990). Numerous compounds, such as ferrocene (Sun et al., 1991), 3-trifluoromethylpyridine (Gaskell, 1990), acetaminophen (Genter et al., 1998; Jeffery and Haschek, 1988), NNK (Belinsky et al., 1990), 2,6-dicholorobenzonitrile (Brittebo, 1997), and coumarin (Gu et al., 1997) are metabolized to toxicants that produce necrosis of the olfactory epithelium. This toxicity can occur following not only inhalation, but systemic administration as well. A more detailed discussion of this subject can be found in Chapter 26. Often, the relative toxicity of a compound in different species or in tissues within a given species is affected by levels of activating or detoxicating enzymes. While this probably holds for nasal toxicants as well, the relationships can be complex. For example, although activity levels in hamster nasal tissues for many enzymes known to activate toxicants are higher than those in rats, hamsters nonetheless are less susceptible than rats to the toxic effects induced by metabolites of 3-methylfuran and N-nitrosodiethylamine (Dahl and Hadley, 1991). On the other hand, a dose of 100 mg/kg of dichlobenil is needed to cause olfactory toxicity in the rat, whereas toxicities are observed in

Olfactory Mucosa

the mouse at a dose of 12 mg/kg (Brandt et al., 1990; Genter et al., 1996). There may be several reasons for such apparent discrepancies. When compounds are administered systemically, the capacity for other organs to clear the compound must be considered, thereby reducing the concentration reaching the nose. An example is the finding that although rat and mouse olfactory P450s are equally active in metabolic activation of coumarin, rats are much more sensitive to the nasal toxicity of coumarin than mice because of a lower hepatic clearance of the parent compound (Zhuo et al., 1999). When a toxicant is inhaled, differences in nasal airflow patterns, mucociliary clearance, or epithelial status may also affect the toxicity. Nasal cancers are relatively uncommon in humans, although in certain populations, notably Chinese males, the rate of occurrence is quite high (Tricker and Preussman, 1991). The occurrence of nasal tumors in laboratory animals exposed by inhalation to toxic materials, on the other hand, is a common finding. Often, the toxic materials are procarcinogens requiring metabolic activation, suggesting that differences in nasal xenobiotic metabolism between humans and laboratory animals may underlie the observed differences in nasal tumor formation. Such differences have been predictive among laboratory animal species. Thus, inhalation of the procarcinogen benzo(a)pyrene results in nasal tumors in Syrian hamsters, but not in other species such as rats (Thyssen et al., 1981). The capacity for nasal metabolism of benzo(a)pyrene in hamster is ~400 pmol/mg/min (Dahl et al., 1985), whereas in rats, the capacity to metabolize benzo(a)pyrene is only ~20 pmol/mg/min (Bond, 1983). Although such relationships are compelling as explanations of toxicity, as in the case of noncarcinogenic responses, relative metabolic capacity is not always sufficient to explain differences in toxicity. The nasal carcinogen NNK produces DNA adducts in nasal tissue via the reactive -hydroxylated N-nitrosamine metabolites. Although metabolic capacity of the tissues might lead to the prediction that the olfactory mucosa would produce comparatively more adducts than the respiratory mucosa, more adducts were actually found in the respiratory mucosa (Belinsky et al., 1990). Again, other factors, perhaps in this case route of exposure and DNA repair rates, must be taken into account to explain or predict toxicity.

VI. MODIFICATION OF OLFACTORY XENOBIOTIC METABOLISM As is the case with hepatic xenobiotic-metabolizing enzymes, nasal enzymes are susceptible to modification in their levels of activity. Specific chemical inhibitors, and in some cases inducers, can alter nasal metabolic capacity. In at

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least some instances, such an alteration has been demonstrated to subsequently alter toxicity as well. Induction of P450 activity by administration of -naphthoflavone decreased the severity of 3-methylindole–induced olfactory lesions (Turk et al., 1986), possibly as a result of lower blood levels due to enhanced liver metabolism. Conversely, treatment with dexamethasone potentiates the 3-methylindole olfactory toxicity, which could be partly due to the inducing action of dexamethasone on the P450 responsible for metabolic bioactivation of 3-methylindole in the olfactory mucosa (Kratskin et al., 1999). In addition, changes in endogenous steroid hormones may modify the biotransformation capacity of the olfactory mucosa, as demonstrated by the effects of castration on nasal metabolism of testosterone. Castrated male rats have a reduced ability to metabolize testosterone, while testosterone replacement restores metabolic capacity for the steroid (Lupo et al., 1986). However, little else is known about this potentially very interesting subject. As the following section will detail, many common environmental exposure scenarios can result in alterations of nasal enzymatic activity, thereby enhancing individual variations in responses to toxicant exposures. These alterations can result from either direct inhalation or systemic exposure to chemicals that induce or inhibit nasal enzymes or from toxicant insults that alter the histology of the tissue. For example, many toxicants cause exfoliation of the olfactory epithelium and a concomitant loss of metabolic capacity from those lost cells. Enzymatic expression also appears sensitive to hyperplastic or metaplastic alterations in the epithelium that can result from either toxicant exposures, infections, or inflammatory processes. Consideration of a patient’s exposure history may therefore be helpful in diagnosing what appears to be an atypical response to a subsequent toxicant exposure. A.

Enzyme Induction

Early reports, primarily from studies on rat nasal tissue, indicated that nasal P450s were relatively refractory to induction by a wide range of inducers effective for hepatic enzymes. Either no induction or mild induction of rat nasal P450 activity was observed following treatment with the classic inducers: phenobarbital, benzo(a)pyrene, 2,3,7,8tetrachlorodibenzo-p-dioxin, or 3-methylcholanthrene (3MC) (Baron et al., 1988; Bond, 1983; Hadley and Dahl, 1982; Longo et al., 1988). Although one study reported the induction of mouse nasal P450 activity by phenobarbital (Brittebo, 1982), the apparently increased activity may have resulted from induction of phase II enzymes occurring downstream from the P450 breakdown step, as only increased 14CO2 production was reported (Dahl and

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Hadley, 1991). Induction of phase II enzymes would be consistent with reports of induction of these enzymes in rat nasal tissue by phenobarbital (Guengerich et al., 1982; Longo et al., 1988). The phase II enzyme UDPGT is also induced by both Arochlor 1254 and 3-MC (Bond, 1983; Longo et al., 1988). Nevertheless, not all phase II enzymes are readily inducible in the olfactory mucosa. Olfactory GSTs were not induced in rats by trans-stilbene oxide, which caused a 2-fold induction in the liver (Banger et al., 1996); only a marginal induction (1.3-fold) by PB was achieved in the olfactory mucosa, while a 2.8-fold induction was found in liver (Banger et al., 1996). Rabbit nasal CYP2E1 (involved in the metabolism of ethanol and other alcohols, acetone, acetaminophen, nitrosamines, and diethyl ether) can be increased twofold by treatment with ethanol and sixfold following acetone treatment (Ding and Coon, 1990b). These data represent the first evidence of an increase in nasal xenobiotic-metabolizing capacity of a magnitude that can be considered important physiologically. Induction of CYP2E1 in the olfactory mucosa has been confirmed in other species (Genter et al., 1994; Gu et al., 1998; Longo et al., 1993). Nasal CYP2E1 and CYP1A2 can also be induced in rats by fasting (Longo et al., 2000). CYP1A1, which is active in the metabolic activation of polycyclic aromatic hydrocarbons, was not induced in the olfactory mucosa by 3-MC, but was significantly induced in Bowman’s glands and in the olfactory and respiratory epithelia following a single intraperitoneal injection of Arochlor 1254 in rats (Voigt et al., 1993); the increase in CYP1A1 protein was accompanied by dramatically enhanced benzo(a)pyrene hydroxylase activity in the same sites. More recent studies indicated an induction of CYP1A1 protein in olfactory mucosa of mainstream cigarette smoke–exposed rats, but a corresponding increase in CYP1A1 activity was not observed (Wardlaw et al., 1998). Induction of nasal P450 enzymes by tobacco smoke has been proposed as a possible mechanism for developing resistance to the environmental toxins implicated in parkinsonism and other neurological diseases (Gresham et al., 1993) (see Chapters 23 and 24). CYP2As represent major P450 isoforms in the olfactory mucosa of a number of species. A study by Beréziat et al. (1995) suggested that a CYP2A-like P450 may be induced in rats by treatment with coumarin in drinking water. However, the same results were not obtained in another study with a different strain of rats (Gu et al., 1997), and no induction of CYP2A was found following treatment of mice with several chemicals known to induce the same enzyme in the liver (Su et al., 1998). Interestingly, one of the known CYP2E1 and CYP2A5 inducers, pyrazole, was found to induce CYP2J4 in rat olfactory mucosa as well as in other tissues (Zhang

Ding and Dahl

et al., 1999). The tissue-differential inducibility of some, but not all, P450 enzymes may be related to the unique (yet still unknown) function of each P450 enzyme and the need for the olfactory mucosa to maintain certain enzymes at a relatively constant level. Alternatively, it is possible that nasal biotransformation enzymes may respond preferentially to inhaled odorants. For example, carboxylesterase is induced in the olfactory mucosa following inhalation exposure to the common solvent pyridine (Nikula et al., 1995), which is not a substrate for this enzyme. B.

Inhibition of Nasal Xenobiotic Metabolism

Unlike the case for nasal enzyme induction, inhibition of nasal xenobiotic metabolism occurs in a wide range of enzyme families. Several P450 isoforms have been inhibited in homogenates of nasal mucosa by hepatic P450 inhibitors such as metyrapone, -naphthoflavone, piperonyl butoxide, and a number of odorants, including 5-androstenone. Because these inhibitors are common ingredients in many products in everyday usage such as perfumes, cosmetics, and household insecticides, exposure to these compounds may alter nasal metabolic capacity from that observed in controlled laboratory situations (Dahl, 1982; Dahl and Brezinski, 1985; Ding and Coon, 1994; Laethem et al., 1992). Cigarette smoke is another common environmental pollutant that is known to modify olfaction (Frye et al., 1989) and to alter the capacity for nasal xenobiotic metabolism (Wardlaw et al., 1998). Alterations in nasal metabolism may be the direct result of exposure to the myriad of components of cigarette smoke known to be metabolized in the nasal epithelium including benzo(a)pyrene, N-nitrosonornicotine, and cyanide. Rhodanese, the primary enzyme of cyanide metabolism, shows nearly a 50% reduction in activity in respiratory mucosa from human smokers compared to nonsmokers (Lewis et al., 1991). Inhibitors of biotransformation enzymes have been used in vivo to demonstrate the role of local metabolism in xenobiotic toxicity. For example, treatment with metyrapone reduced or abolished cytotoxicity caused by a number of toxic chemicals, such as methimazole (Bergman and Brittebo, 1999), 2,6-dichlorothiobenzamide (Eriksson and Brittebo, 1995), 2,6-dichlorobenzonitrile (Walter et al., 1993), and IDPN (Genter et al., 1994). Metyrapone has also been used to demonstrate that inspired styrene is metabolized in nasal tissues in the rat and mouse (Morris, 2000). Other P450 inhibitors used for in vivo studies include diethyldithiocarbamate (Deamer and Genter, 1995; Eriksson and Brittebo, 1995), carbon tetrachloride (Genter et al., 1994), disulfiram (Deamer and Genter, 1995), 3-aminobenzamide (Eriksson et al., 1996),

Olfactory Mucosa

and cobalt protoporphyrin IX (Chamberlain et al., 1998); the latter depletes P450 by interfering with heme synthesis. Some inhibitors cause inactivation of a subset of P450s, such as xylene (Blanchard and Morris, 1994) and chlormethiazole (Longo et al., 2000). Inhibition of nasal GST-dependent conjugation activity has been achieved by depleting GSH with phorone (Larsson and Tjälve, 1995) or phorone plus L-buthionine sulfoximine (Chamberlain et al., 1998). In addition, the role of aldehyde dehydrogenase on nasal uptake of inspired acetaldehyde has been examined using cyanamide as an inhibitor. While these inhibitors have been useful for the initial identification of biotransformation enzymes or pathways involved in the metabolism and toxicity of a compound, they are generally not specific for any single enzyme or even a single family of enzymes (e.g., Eriksson et al., 1996). Furthermore, these inhibitors are most likely also toxicants. Therefore, caution should be exercised in interpreting the results obtained using chemical inhibitors as tools. Alternatively, mouse models with targeted gene deletion of specific P450 and other biotransformation enzymes are becoming available and have been used in limited cases to examine the role of biotransformation enzymes in nasal toxicity of xenobiotics (Genter et al., 1998). C.

Effects of Mucosal Damage on Nasal Metabolism

Expression of olfactory mucosa P450s and CPR is suppressed when ORNs undergo degeneration as a consequence of either chemical toxicity, unilateral naris closure, or olfactory bulbectomy (Gu et al., 1997; Schwob et al., 1995; Walters et al., 1992, 1993). P450 expression returns to normal following successful regeneration of ORNs, but not when degenerated olfactory mucosa was replaced by respiratory type of epithelium (Schwob et al., 1995). The suppressed expression of P450 following olfactory bulbectomy is particularly intriguing since the P450-expressing cells were apparently intact after the operation (Walters et al., 1992). This result contrasts with the report that the expression of PSTg protein is not affected in the olfactory mucosa following olfactory bulbectomy (Miyawaki et al., 1996). Decreases in GSH and GST levels have also been found in the peripheral olfactory organ of rainbow trout during retrograde olfactory nerve degeneration, which are followed by widespread recovery as the ORNs begin to repopulate the olfactory mucosa (Starcevic and Zielinski, 1997). Tissue damage resulting from chemically induced nasal toxicity may underlie some of the in vivo inhibitory effects of enzyme inhibitors described in the previous section, as well as the apparent resistance of nasal biotransformation enzymes to xenobiotic induction (Su et al., 1996). Furthermore, although it may appear logical to assume

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parallel alterations in histopathology and metabolic activity in nasal tissue, interpretation of data from these correlative studies can be complicated by the multifaceted nature of the toxic response. For example, tissue damage may lead to increased influx of immune cells, which may contribute to local metabolic activity. Another complexity in interpretation of data indicating altered metabolism following exposure to specific toxicants lies in the parallel pathological alterations to the nasal epithelium. Biochemical data are often normalized per mg protein, per mg tissue, per mg mitochondrial or microsomal protein, and so forth. If cellularity has decreased in the tissue (as is often the case in the olfactory epithelium), the normalized data may show no alteration in metabolism, but the total capacity of the tissue to metabolize inhalants may be severely reduced. Conversely, hyperplastic responses may greatly increase the metabolic capacity without altering the normalized biochemical data. Although this problem exists in other tissues as well, the structure of the nasal epithelium and the close association with cartilage and bone make it difficult to control the problem by normalizing to total tissue weight, as can readily be done in most other organs. In addition, toxicant exposure and age are both known to produce metaplastic alteration in the olfactory epithelium. Because these alterations in cell type can also affect metabolic capacity, close evaluation of both biochemical and histopathological alterations in the interpretation of data from nasal epithelium is necessary for valid extrapolations. Finally, because inhalants can contact and be metabolized in the respiratory or transitional mucosa before reaching the olfactory mucosa, metabolic processes occurring in these nasal mucosa will also affect olfactory processes.

VII.

CONCLUSIONS

Both general and research interests in the olfactory system have increased over the last two decades owing to several unique aspects of this system. Its continuous exposure to inhaled environmental toxicants, its vulnerability to cell loss resulting from toxicant insult, and its capacity to regenerate neuronal cells following this loss make it a unique neural tissue. In addition, its histological structure, with neuronal cells contacting the external environment at the nasal lumen and projecting directly to the olfactory bulb, makes it a viable portal of entry for inhaled environmental toxicants, as well as a potential route of entry for therapeutic drugs, into the CNS. Although olfaction has traditionally been thought of as a sensory system of minor importance in humans, evidence is accumulating that olfaction plays an important role in learning and memory,

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hedonic responses, and reproductive function in humans as well as in other species. Only a small fraction of the biochemical repertoire of the olfactory mucosa has been characterized to date. This will change rapidly as the new genomics and proteomics techniques are applied to the olfactory system. What has been learned so far already indicates that the nasal biotransformation enzymes are very likely to play important roles in many cellular processes in the olfactory mucosa, as supported by their high metabolic capacity, their diverse substrates of both endogenous and exogenous origins, and their tissue and cell-type specific expression. The precise nature of these roles is not yet fully understood; however, rapid progress is anticipated, as an increasing number of knockout mouse and other genetically modified animal models become available. As reviewed in this chapter, nasal biotransformation enzymes can alter inhaled toxicants either by converting them to less toxic metabolites or by activating them to reactive chemicals that result in local damage, and in some cases damage to other tissues as well. As such, the role of nasal biotransformation enzymes has historically received the most attention in the field of toxicology. The biotransformation capacity in the olfactory mucosa is vulnerable to modification by a variety of toxicant exposures, histological changes, and disease or inflammatory processes. Further, the nasal xenobiotic-metabolism activities will be influenced by genetic polymorphisms of the participating biotransformation enzymes. Thus, the capacity to detoxify or activate inhaled toxicants is likely to be a fluid system best understood with respect to an individual case history and genetic makeup. Future studies on the interactions of various components of the olfactory system, the activities of individual biotransformation enzymes and their genetic polymorphisms, and the impact of nasal xenobiotic-metabolism on other systems such as the CNS will make it possible to identify situations, periods, or individuals of increased vulnerability to inhaled xenobiotics and to reduce the risk of toxicity through targeted prevention. ACKNOWLEDGMENTS The authors acknowledge Jack Harkema for Figures 1 and 3 and Dr. Bert Menco and Virginia Carr for the transmission electron micrograph in Figure 2 in this chapter. We also thank Drs. Mary Beth Genter and Katherine Henrikson for reading the manuscript. This work was supported in part by NIH Grants ES07462 from the National Institute of Environmental Health Sciences and DC02640 from the National Institute on Deafness and Other Communication Disorders.

Ding and Dahl

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4 Molecular Neurobiology of Olfactory Transduction Cheil Moon and Gabriele V. Ronnett The Johns Hopkins University School of Medicine, Baltimore, Maryland, U.S.A.

I.

INTRODUCTION

(see Chapters 3 and 4) (Graziadei and Monti-Graziadei, 1979; Moulton and Beidler, 1967). ORNs are bipolar, extending apical dendrites to the surface of the neuroepithelium and sending unmyelinated axons through the basal lamina and cribiform plate of the ethmoid bone to terminate in glomeruli on mitral and tufted neurons in the olfactory bulb of the brain. The apical dendrites form dendritic knobs from which arise specialized, nonmotile cilia, where the initial events of olfactory transduction occur (Getchell, 1986; Labarca and Bacigalupo, 1988; Lowe and Gold, 1993a). Electrophysiological studies indicate that odorant sensitivity and the odorant-induced current are uniformly distributed along the cilia, suggesting that all the components of the immediate responses to odorants are localized to the cilia. Immunoelectron microscopic studies have confirmed the cilial localization of many of these components (Menco, 1997; Menco et al., 1992a). ORNs comprise 75–80% of the cells in the epithelium (Farbman, 1992). They are functionally homogeneous: they all detect odorants. As they mature, ORNs move apically in the epithelium, permitting determination of neuronal age by position (Roskams et al., 1998). Mature ORNs express olfactory marker protein (OMP) (Farbman and Margolis, 1980; Margolis, 1980). ORNs senesce throughout life at a regular rate and are replenished by the differentiation of globose basal cells (Caggiano et al., 1994; Graziadei, 1973; Graziadei and Metcalf, 1971). This neurogenesis can be hyperinduced by ablation of the olfactory bulb (termed bulbectomy) (Carr and Farbman, 1993; Costanzo and Graziadei, 1983; Hirsch and Margolis, 1980). Thus,

The correct interpretation of sensory information is vital to an organism’s survival. Among sensory modalities, the olfactory system has daunted many investigators seeking to understand the molecular aspects of its signal transduction and coding mechanisms (Buck, 1996; Getchell, 1986; Getchell et al., 1985). The ability of the olfactory system to discriminate among thousands of odors comprised of chemically divergent structures (odorants) posed unique challenges that have been answered only by a combination of molecular, electrophysiological, and cell biological approaches. What has emerged is that olfactory transduction combines unique receptive molecules with classical transduction cascades to detect olfactory stimuli. What is provocative is that many cascades are activated in response to odorant detection whose roles we are only beginning to be appreciated.

II. CELLULAR ANATOMY OF THE OLFACTORY EPITHELIUM The peripheral olfactory system is well adapted structurally to perform its function. The olfactory primary sensory neurons are located in a portion of the olfactory epithelium, thus facilitating their direct contact with inhaled odorants. There are several principal cell types in the olfactory epithelium, including olfactory receptor neurons (ORNs), supporting sustentacular cells, microvillar cells, Bowman gland cells, and two types of basal cells 75

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understanding of the functions of signaling components in signal transduction is facilitated by the spatial organization of ORNs. Sustentacular cells share features in common with glia. They stretch from the epithelial surface to the basal lamina, where they maintain foot processes (Getchell, 1986; Getchell et al., 1985). Sustentacular cells electrically isolate ORNs, secrete components into the mucus, and contain detoxifying enzymes (Okano, 1974). The sustentacular cells contain high concentrations of cytochrome P450–like enzymes (Lazard et al., 1991). These enzymes may modify odorants to make them less membrane permeable or inactivate them. Recent studies indicate that sustentacular cells may produce growth factors important to ORN development (Hansel et al., 2001). Neuropeptide Y (NPY) is an amidated neuropeptide that performs many functions in mammalian physiology (Baraban et al., 1997; Danger et al., 1990). NPY mRNA is upregulated following peripheral axotomy and in pheochromocytoma and ganglioneuroblastoma tissue (Adrian et al., 1983). Whereas NPY is expressed in developing ORNs during embryogenesis, it is expressed in sustentacular cells in the adult olfactory epithelium. NPY functions as a neuroproliferative factor for olfactory neuronal precursors in vivo and in vitro (Hansel et al., 2001). This is the first of potentially many growth factors that sustentacular cells contribute to ORN homeostasis. The basal cells underlie the ORNs and serve as precursors for the generation of new ORNs throughout adulthood (Caggiano et al., 1994; Graziadei and Monti Graziadei, 1979; Moulton and Beidler, 1967). Basal cells have been divided into two general classes. Horizontal cells are flat and express cytokeratin (Calof and Chikaraishi, 1989; Graziadei and Monti-Graziadei, 1979). Globose basal cells are rounded in shape and express several markers, including GBC-1, GBC-3, and GBC-5 (Goldstein and Schwob, 1996; Huard et al., 1998). Compared to other neurons, many ORNs have a relatively short survival time, in the range of several months. This may be due to the fact that ORNs are exposed to a variety of toxic or infectious agents. Thus, the function of globose basal cells in providing new ORNs is crucial to the maintenance of the sense of smell. These issues are discussed in greater detail in Chapter 4.

III. GENERAL MECHANISMS OF ODORANT TRANSDUCTION Olfactory signal transduction (Fig. 1) is initiated when odorants interact with specific receptors on cilia on ORNs (Buck, 1996; Dwyer et al., 1998; Malnic et al., 1999; Rhein

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and Cagan, 1980). Receptors subsequently couple to a Gprotein to activate adenylyl cyclase (Pace et al., 1985; Ronnett et al., 1993; Sklar et al., 1986). Electrophysiological and biochemical studies confirm that cAMP is the key messenger in the initial phase of odorant detection (Breer et al., 1990; Brunet et al., 1996; Jaworsky et al., 1995; Pace et al., 1985; Ronnett et al., 1991; Ronnet and Snyder, 1992; Sklar et al., 1986; Wong et al., 2000). cAMP levels increase and open a cyclic nucleotide-gated channel, resulting in an influx of Na and calcium (Firestein and Werblin, 1989; Nakamura and Gold, 1987). The immediate response is the generation of a graded receptor potential (Getchell and Shepherd, 1978; Ottoson, 1956). Several other second messenger cascades are activated upon odorant detection and may regulate secondary events or odorant responsivity. The increase in calcium may regulate downstream events (Frings et al., 1995; Kaupp, 1991). Odorants also increase phosphoinositide hydrolysis and the production of inositol-1,4,5-trisphosphate (IP3) (Breer and Boekhoff, 1991; Miyamoto et al., 1992; Ronnett et al., 1993; Schandar et al., 1998). Cyclic GMP production is also increased with odorant exposure (Ingi et al., 1996; Verma et al., 1993). Interestingly, the odorant-induced cGMP response is much slower than the cAMP or IP3 responses, which normally peak within 500 msec. Thus, the cGMP response does not appear to function in the immediate detection phase of olfaction, such as modulating cyclic nucleotide gated cation channels or IP3 receptors, but rather in desensitization or the modulation of the cellular response during longer exposures to odorants (Breer et al., 1992; Moon et al., 1998, 1999; Zufall and Leinders-Zufall, 1997). These messengers are discussed in subsequent sections.

IV.

ODORANT-BINDING PROTEINS

The existence of carrier proteins for odorants in the nasal mucus was predicted based upon the fact that hydrophobic odorants must travel through the aqueous mucus barrier towards the cilia of ORNs. In fact, odorant-binding proteins (OBPs) were discovered by several laboratories in early attempts to identify odorant receptors using radioactive odorants such as 3-isobutyl-2-methyloxypyrazine (Pelosi et al., 1982; Pevsner et al., 1986; Pevsner et al., 1985). Purified OBP is a homodimer comprised of two identical 19 kDa subunits and binds to odorants with affinities in the micromolar range (Pevsner et al., 1990). The molecular cloning of OBP helped to clarified its function. OBP is a member of the lipophilic molecule carrier protein family. A well-characterized member of this family is a retinol-binding protein. This protein conveys retinol from retinal pigment epithelium to rods and

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Figure 1 Model of odorant signal transduction (see text for details). Signaling cascades mediate the initial phase of odorant detection and potential long-term responses to odorant detection. Abbreviations: AC, adenylyl cyclase; bARK, beta-adrenergic receptor kinase; bARR, beta-adrenergic receptor arrestin; CaM, calmodulin, CO, carbon monoxide; CREB, camp-responsive element binding protein; GCAP, guanylyl cyclase activating protein; Golf, olfactory G protein; Gq, G protein q; HO, heme oxygenase; InsP3, inositol-1,4,5 phosphates; InsP3R, InsP3 receptor; MAPK, a mitogen-activated protein kinase, MEK, MAP or ERK kinase; OBP, odorant binding protein; oCNC, olfactory cyclic neucleotide-gated channel; OR, odorant receptor; PDE, phosphodiesterase; pGC, particulate guanylyl cyclase; PKA, cAMP-dependent protein kinase; PKG, cGMP-dependent protein kinase; PLC, phospholipase C; Raf, MEK kinase; RSK, 90 kDa ribosomal S6 kinase; sGC, soluble guanylyl cyclase.

cones where it is incorporated into rhodopsin (Heller, 1975). In situ hybridization studies of OBP mRNA in rats revealed its selective concentration in the lateral nasal gland, the largest of 20 discrete nasal glands (Pevsner et al., 1988). OBP thus appears to be secreted from this gland down a long duct to the tip of the nose, where watery secretions are atomized in order to humidify inspired air. OBP thus localized might trap odorants and carry them with inspiration to ORNs. Alternatively, OBP may function to remove odorants from sensory epithelium and cilia.

Further studies have revealed that multiple forms of OBP may be expressed in the nasal epithelium. Rabbitts and colleagues (Dear et al., 1991) identified a second form of OBP, OBPII. OBPII encodes a secretory protein with significant homology to OBPI, and it is also expressed in the lateral nasal gland, which is the site of OBP expression. Interestingly, the OBPII sequence also shows significant homology to the VEG protein, which is thought to be involved in taste transduction (Burova et al., 2000). Breer and colleagues demonstrated that rat OBPI and OBPII contain distinct ligand specificities (Lobel et al., 1998).

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Recombinant OBP proteins appear to share many structural features, but each has been shown to interact with distinct sets of odorants. OBPI binds specifically to a pyrazine derivative, 2-isobutyl-3-methoxypyrazine, whereas OBPII binds to the chromophore, 1-anilinonaphthalene 8-sulfonic acid (1,8-ANS), specifically. In other vertebrates, multiple forms of OBP have been identified. There are four OBPs in mice (Pes and Pelosi, 1995), three OBPs in rabbit (Garibotti et al., 1997), and two OBPs in cow (Bianchet et al., 1996; Dal Monte et al., 1991). OBP has also been cloned from insects (Vogt et al., 1990, 1991).

V.

ODORANT RECEPTORS

Mammals perceive a huge variety of environmental odors. The initial step in odor perception requires the interaction of odorous ligands with specific receptors on the surface of olfactory receptor neurons (Buck, 1996; Dwyer et al., 1998; Malnic et al., 1999; Rhein and Cagan, 1980). Based upon the assumption derived from biochemical evidence that odorant signal transduction involved G proteins, and thus G protein–coupled receptors, a very large gene family of closely related olfactory-specific seven transmembrane spanning domain receptors was identified by polymerase chain reaction (PCR) (Buck and Axel, 1991; Buck, 1992, 1996). In vertebrates, the family of odorant receptors (ORs) is known to encode as many as 1000 genes, suggesting that the first steps of odorant recognition are accomplished within the primary sensory neurons themselves. To date, odorant receptor genes have been isolated from 12 vertebrate species: rat, mouse, human, catfish, zebrafish, dog, frog, chicken, pig, opossum, mudpuppy, and lamprey (Mombaerts, 1999a). In humans, estimates of the size of the receptor family range from 500 to 1000 genes. Compared to the other species, human odorant receptor clones display a high frequency of pseudogenes (Mombaerts, 1999b). The expression pattern of odorant receptors in ORNs of the olfactory epithelium has an unusual spatial distribution (Ressier et al., 1993; Vassar, et al., 1993). In situ hybridization studies have shown that odorant receptor mRNAs are expressed within one of several broad, nonoverlapping zones. Within a zone, odorant receptors are expressed in a random manner. Each zone occupies about a quarter of the olfactory epithelium (Ressier et al., 1993) and is represented on the turbinates and on the septum (Mombaerts, 1999a). However, the physiological meaning of zonal expression remains unclear. While a number of studies have been done on the expression and distribution of odorant receptors at the message level, relatively little is known about the expression of

odorant receptor proteins. Polyclonal antibodies have been raised against some odorant receptors, permitting visualization of odorant receptor proteins. In rats, an odorant receptor is expressed as early as E14 in a zonally restricted pattern (Koshimoto et al., 1994). The expression of odorant receptors is restricted to the cilia and dendritic knobs of ORNs. The cilia-specific expression of odorant receptors supported a role for odorant receptors in olfactory transduction (Menco et al., 1997a, b, c). A concern with studies utilizing antibodies to identify discrete members of the odorant receptor family is the specificity of the antibodies, given the large numbers of receptors. Despite the general utility of antisera for immunohistochemical and biochemical studies, the enormous size of the odorant receptor repertoire limits the feasibility of proving the specificity of an antibody to a specific receptor. Significant difficulties with heterologous expression of odorant receptors severely limited studies designed to provide functional confirmation of the role of such receptors. The most convincing data concerning function were provided initially by genetic studies in Caenorhabditis elegans (Senhupta et al., 1996), which demonstrated that the odor 10 mutant lacked a seven transmembrane receptor and was deficient in its ability to detect acetyl (Senhupta et al., 1996). Krautwurst et al., (1998) first achieved functional heterologous expression of odorant receptors using HEK-293 cells. This group (Krautwurst et al., 1998) generated an expression library of mouse odorant receptors and identified three receptors responding to carvone, ()citronellal, and limonene using micromolar concentrations of these odorants. Firestein and colleagues also demonstrated functional expression of a cloned odorant receptor in rat nasal epithelium by using a recombinant adenovirus containing a putative odorant receptor to infect rat nasal epithelium in vivo (Zhao et al., 1998). They demonstrated that this specific odorant receptor was overexpressed in the rat olfactory epithelium and the expressed odorant receptor transduced a response to a small subset of odorants by EOG. Malnic et al., (1999) performed single cell PCR on ORNs whose odorant responses had been determined to demonstrate that a combinatorial code exists for odorant perception. These approaches to develop functional expression systems for odorant receptors can be extremely useful to screen odorant receptors on a large scale as well as to understand the molecular mechanism of odorant recognition. Besides functioning in the detection of odorants, odorant receptors are hypothesized to be involved in determining or guiding ORN axonal projections to the olfactory bulb and possibly to specific glomeruli (Mombaerts et al., 1996; Ressler et al., 1994). In rodents, the axons of ORNs expressing the same odorant receptors converge onto

Molecular Neurobiology of Olfactory Transduction

defined glomeruli in the olfactory bulb, suggesting that the rodent olfactory bulb is topographically organized and, in turn, that ORN expressing a specific odorant receptor projects to and forms a synapse with the representing glomeruli in the olfactory bulb. This represents an interesting hypothesis that an environmental odor is encoded by activation of specific glomeruli that perceive a signal from ORNs expressing a specific odorant receptor out of the odorant receptor repertoire. VI.

G-PROTEINS

The first evidence for the involvement of G-proteins in odorant transduction was provided by the observation that the odorant-induced activation of olfactory sensory cilia depended upon the presence of GTP (Rhein and Cagan, 1983). Subsequently, a G-protein was cloned from an olfactory cDNA library that was highly and almost exclusively expressed in ORNs; this G-protein was named Golf (Jones and Reed, 1987). Golf was shown to stimulate adenylyl cyclase in heterologous systems. Aside from its expression in ORNs of the olfactory epithelium, Golf is expressed in basal ganglia (Drinnan et al., 1991). As mentioned, odorants also increase IP3 production, causing many to postulate that cilia might contain olfactory-specific Gq-proteins. To date, these have not been reported. Mice with targeted disruption of the gene for Golf displayed a striking reduction in the electrophysiological response of ORNs to a wide variety of odors, supporting the hypothesis that Golf, and thus this G-protein–mediated cascade, is required for odorant signal transduction (Belluscio et al., 1998). Despite this intense attenuation in response to odors, the topographic map of ORN projections to the olfactory bulb was unaltered in Golf-deficient mice. Thus, odorant stimulation may not be an essential process in determining the targets of ORN axonal projections to the olfactory bulb. However, these studies may need to be done at higher resolution.

VII. A.

SECOND MESSENGERS cAMP

Electrophysiological studies provided some of the first evidence for the central role of cAMP in odorant detection. Patch clamp experiments on cilia demonstrated a cAMPgated conductance (Nakamura and Gold, 1987). Investigators proposed that an odorant would increase cyclic nucleotide levels to gate a cationic conductance, initiating a depolarizing response. Kinetic studies of odorant-induced currents using whole patch-clamp techniques

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(Firestein et al., 1990; Firestein and Werblin, 1989) suggested that the latency of the odorant response (several hundred milliseconds) indeed supported a role for a second messenger such as cAMP. The first direct biochemical studies reported an odorant-induced cAMP response in olfactory sensory cilia isolated from both frog and rat (Pace et al., 1985; Sklar et al., 1986). The olfactory sensory cilia were prepared by subcellular fractionation after calcium-shock of the olfactory epithelium (Rhein and Cagan, 1983). The odorant-stimulated production of cAMP was tissue-specific and occurred only in the presence of GTP, suggesting the involvement of receptors coupled to G-proteins. Further characterization using isolated rat olfactory sensory cilia showed that cAMP was best produced by fruity, floral, and herbaceous odors (Nakamura and Gold, 1987; Sklar et al., 1986). Screening many odorants at a single concentration revealed only minimal cAMP production by some, generating the hypothesis that those odorants with small or absent cAMP responses employed another cascade, perhaps inositol phosphates (Nakamura and Gold, 1987; Sklar et al., 1986). These initial measurements were made 15 minutes after the exposure of isolated cilia to odorants. To demonstrate that the production of cAMP occurs on a relevant time scale, subsecond kinetics of odorantinduced changes were analyzed by using a rapid quenchflow device (Boekhoff et al., 1990; Breer et al., 1990). In this device, cilia membranes and odorant solutions were subjected to computer-controlled mixing, with subsequent quenching of samples at intervals from 8 to 500 msec. cAMP was produced rapidly and transiently in response to odorants, with increases evident as early as 25 msec. Certain odorants such as fruity odors were able to stimulate cAMP production at concentrations as low as 10 nM, whereas others such as putrid odors had no effect, even at higher concentrations. Those odorants that did not stimulate cAMP production were hypothesized to act through the phosphoinositide (PI) cycle. High (millimolar) levels of calcium inhibited the response, but intermediate concentration ranges were not tested. The odorant-induced cAMP response was investigated further using isolated rat olfactory cilia to determine the generality of the odorant-induced cAMP response and the calcium dependence of this response (Jaworsky et al., 1995). Odorants indeed cause rapid and transient elevations of cAMP, as well as the more sustained signal, as seen by Lancet (Pace et al., 1985) and Sklar (Sklar et al., 1986). Different from the observation from Breer’s group (Boekhoff et al., 1990; Breer et al., 1990), all odorants stimulated cAMP production. Interestingly, responses were non-linear. Basal and odorant-induced cAMP levels in cilia demonstrated biphasic calcium dependence, with peak cAMP stimulation in the range of 1–10 M free

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calcium. Dose-response curves done at two calcium levels showed that the influence of calcium on odor responses was complex, suggesting the possible involvement of calcium both in signal generation and termination. To evaluate olfactory signal transduction in intact cells, primary cultures of olfactory epithelium enriched in ORNs were developed (Ronnett et al., 1991a, b, 1993). Using this primary culture system, cAMP responses to odorant stimulation were monitored in intact ORNs. Odorants were quite potent at producing cAMP, with as little as 0.1 nM isobutylmethoxypyrazine (IBMP) generating a response (Ronnett et al., 1991b, 1993). Responses were multiphasic; cAMP production increased with increasing odorant concentration, decreased at even higher odorant concentrations, and sometimes reappeared at still higher (1–10 mM) concentrations. Signals were calcium dependent, with maximal activity at 10 M free calcium and inhibition at higher calcium concentrations. Odorant induction of cAMP production was rapid, with peak effects observed at 10–15 sec, but signals continued well above baseline for minutes, confirming results from Sklar et al. (1986) and Pace and Lancet (1986). Cyclic AMP is produced by adenylyl cyclase. There are at least nine identified isoforms of adenylyl cyclases (Hanoune and Defer, 2001). A novel adenylyl cyclase, referred to as type III AC (AC3), was cloned by Bakalyar and Reed (1990). Northern blot analysis indicated that AC3 mRNA was enriched in the olfactory epithelium and that AC3 mRNA disappeared after bulbectomy. When expressed in HEK293 cells, AC3 had almost no basal activity. In contrast, AC1 and AC2 have high basal activities. Golf and AC3 have been ultrastructurally localized to olfactory cilia, indicating that Golf may mediate the activation of AC3 (Menco et al., 1992b). To evaluate the role of AC3 in the olfactory transduction, the AC3 gene has been disrupted in mice (Wong et al., 2000). Odorant-induced responses measured by electro-olfactogram (EOG) were completely eliminated in AC3-null mice. Moreover, odor-dependent learning was impaired in these mice. Interestingly, both fruity odors (transduced by cAMP) and putrid odors (formally thought to act through IP3) failed to evoke any response in these animals. This observation was mimicked by a pharmacological study that showed that adenylyl cyclase antagonists reversibly inhibit EOG responses, even to putrid odors (Chen et al., 2000). Taken together, these results confirmed earlier biochemical studies that implicated adenylyl cyclase and cAMP as essential for the initial phases of odorant transduction. IP3 was therefore postulated to play more of a modulatory role in the odorant transduction. Certain enzymes are rather broadly expressed, while others are restricted in their distribution (Mons and

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Cooper, 1995). Although AC3 is highly enriched in ORNs, other adenylyl cyclases, such as AC2 or AC4, have also been associated with olfactory neuroepithelium, raising the issue that other adenylyl cyclases may be important in different aspects of olfactory signal transduction. These other adenylyl cyclases may function in other aspects of ORN homeostasis or signaling. The various adenylyl cyclases are regulated by different mechanisms. Studies by Storm and colleagues (Choi et al. 1992, 1993; Wayman, 1995) indicated that the mechanisms of regulation of adenylyl cyclases may not only be dependent upon the specific kind of adenylyl cyclase expressed in a tissue, but by local influences and the expression of regulatory molecules in that specific cell. Thus, while ectopically expressed AC3 may be stimulated by calcium, in vivo studies in certain tissues argue for the inhibition of AC3 by calcium. Equally diverse are the effects of protein kinases on adenylyl cyclases. Phorbol esters are used to mimic the effects of protein kinase C (PKC) activation and elicit a stimulatory effect on AC2, while barely stimulating AC1 or AC8. These latter adenylyl cyclases are stimulated up to 8 times by calcium (Cooper et al., 1995). Frings (1993) has reported that activation of PKC by phorbol esters increased cAMP in frog olfactory tissue. The stimulation by calcium of AC1, AC3, and AC8 is mediated by calmodulin (Tang and Gilman, 1992); it is unclear how the calcium sensitivity of the calcium inhibition of AC5 and AC6 are achieved. There is also evidence that PKA may affect adenylyl cyclase activity. The elevation of cAMP results in the gating of the olfactory cyclic nucleotide-gated channel, OCNC, to depolarize ORNs, and thus OCNC, is an integral component in olfactory transduction (Zufall et al., 1994). The OCNC is a nonspecific cation channel, activated by both cAMP and cGMP (Zagotta, 1996). Three OCNC subunits are expressed in the olfactory epithelium: the OCNC1 and OCNC2 subunits and an olfactory-enriched splice variant of the rod photoreceptor  subunit (OCNC) (Bonigk et al., 1999; Bradley et al., 1994; Dhallan et al., 1990; Liman and Buck, 1994; Sautter et al., 1998). OCNC1 forms functional homodimers in vitro (Dhallan 1990) but in vivo is thought to be associated with either the OCNC and or OCNC2. The association with these subunits confers greater sensitivity to cyclic nucleotides and changes in single channel kinetics (Bonigk et al., 1999; Bradley et al., 1994; Liman and Buck, 1994; Sautter et al., 1998). Ngai and colleagues generated OCNC1-deficient mice and reported that these mice were anosmic and died within a few days after birth (Brunet et al., 1996). Later, Parent et al. (1998) developed a method to promote the survival of OCNC1-null mice to permit further analysis. Similar to

Molecular Neurobiology of Olfactory Transduction

AC3-null mice, the OCNC1-deficient mice showed no EOG responses to both fruity and putrid odorants, even to complex odorants such as urine. This again suggests that cAMP is the essential messenger for odorant transduction. B.

Inositol-1,4,5-trisphosphate

In the brain and peripheral tissues, receptor-mediated stimulation of phospholipase C (PLC) generates IP3, which releases calcium from endoplasmic reticulum (ER) stores by binding to specific IP3 receptors (Berridge and Irvine, 1984, 1989). Plasma membrane IP3 receptors have been identified in lymphocytes (Kuno and Gardner, 1987) and neurons (Bush et al., 1994; Fijimoto et al., 1992) to permit calcium entry from extracellular sources. Thus, calcium becomes available to modulate enzyme activities. There are now five families of IP3 receptors (Joseph, 1996; Taylor and Richardson, 1991; Taylor and Traynor, 1995). Studies in number of species implicate IP3 in olfaction. However, electrophysiological experiments have in many cases failed to demonstrate a role for IP3 in ORN depolarization. Huque and Brunch (1986) showed PLC activity in isolated catfish olfactory cilia. Restrepo et al., (1990) showed that amino acids enhanced calcium flux in isolated catfish ORNs. Utilizing the rapid mixing technique, Breer and colleagues (1990) demonstrated increases in IP3 levels in response to some odorants. Studies in primary cultures of ORNs confirmed that odorants stimulate the production of IP3. Exposure of cells to low nanomolar concentrations of odorants resulted in IP3 formation (Ronnett et al., 1993; Wood et al., 1990). All odorants were found to stimulate cAMP and IP3 production in primary culture, although with different potencies, suggesting interactions with different receptors. These responses were very sensitive to ambient calcium and odor concentrations. The enhancement by single odors of both cAMP and IP3 production affords a mechanism for increased specificity of odor detection. However, these studies were only performed at longer (1 sec and beyond) times after odor encounter. Ache and coworkers confirmed that odors differentially stimulate dual pathways in isolated lobster antennules (Boekhoff et al., 1994). Odors elevated cAMP and IP3 in the outer dendritic membranes of lobster in vitro. IP3 carried the stimulatory current, while cAMP was inhibitory, providing a mechanism for fine-tuning of responses. The relevance of IP3 to mammalian olfaction has been questioned by several groups, whose knock-outs affecting the cAMP signaling cascade failed to generate an EOG for any odor, suggesting that cAMP is the sole odorant-generated second messenger (Brunet et al., 1996). These discrepancies may be reconciled if cAMP is indeed the

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primary second messenger required for the initial events of odor detection and cellular depolarization, while IP3 is involved in secondary responses, such as adaptation or activity-driven cellular responses, not EOG generation. Using immunohistochemistry, IP3 receptors have been localized to the ciliary surface membrane (Cunningham et al., 1993), positioning IP3 to trigger the influx of extracellular calcium. There is also evidence for plasma membrane IP3-sensitive channels in lobster ORNs (Fadool and Ache, 1992; Munger et al., 2000). Kalinoski and colleagues (1992) have also demonstrated an IP3-like receptor in isolated catfish cilia, although its micromolar Kd for IP3 suggests a different form of IP3 receptor (Kalinoski et al., 1992). Several PLC isoforms are demonstrated in olfactory epithelium (Abogadie et al., 1995; Bruch et al., 1995; Munger et al., 2000). Reconciliation of the data thus far obtained for IP3 will require further work. For over 10 years, debate existed as to whether cGMP or calcium was the visual second messenger (Zuker, 1996). We now know that while cGMP is central, calcium is the major modulator of cGMP levels (Coccia and Cote, 1994; Mitchell et al., 1995; Somlyo and Walz, 1995; Udovichenko et al., 1994). Additionally, there are striking interspecies differences: while IP3 is important in amphibian phototransduction, no role has thus far been found in mammals. Olfaction may have similar complexities. C.

cGMP

Cyclic GMP is the primary second messenger in visual signal transduction. A number of studies indicate that cGMP may play an important role in the olfactory transduction. Odorants augment cGMP levels in olfactory tissues (Breer et al., 1992) and ORNs (Verma et al., 1993). Compared to the odorant-induced increase in cAMP and IP3 levels, the rise in cGMP levels occurred with a slower, sustained time course. These kinetics suggested that cGMP may not be involved in initial signaling events, but rather in long-term cellular events such as desensitization (Leinders-Zufall et al., 1996), or in the activation of neuronal activity–dependent transcription (Moon et al., 1999). cGMP levels are regulated by two distinct classes of guanylyl cyclases: soluble guanylyl cyclase and particulate guanylyl cyclase. Soluble guanylyl cyclase is activated by gaseous messengers such as NO or CO, whereas particulate guanylyl cyclase is activated by specific extracellular ligands or calcium. Both guanylyl cyclases are expressed in ORNs, implying a complex regulation of cGMP levels in olfaction (Moon et al., 1998; Verma et al., 1993). Diffusible gaseous messenger molecules such as NO or CO can stimulate soluble guanylyl cyclase by binding to

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the heme group in soluble guanylyl cyclases (Snyder, 1994). NO and CO are produced by NO synthase (NOS) and heme oxygenase (HO), respectively. In ORNs, NOS is expressed at embryonic stages and is markedly reduced at early postnatal stage, whereas HO is highly expressed after birth (Ingi and Ronnett, 1995; Roskams et al., 1994). These data suggest that NO plays an important role during development, whereas HO functions in mature ORNs. Two forms of HO have been identified: HO-1 and HO-2. HO-1 is a heat shock protein (hsp-32) induced by heme, heavy metals, stress, or hormones (Bauer et al., 1998; Beschorner et al., 2000; Ewing et al., 1994; Hirata et al., 2000; Koistinaho et al., 1996; Kutty and Maines, 1989) and is highly expressed in the spleen and liver, where it is responsible for the destruction of heme from red blood cells. HO-1 is present in rodent olfactory epithelium (J. Chen, C. Moon, and G.V. Ronnett, unpublished data), but its role and function in olfactory transduction are unclear. HO-2 is not inducible and is distributed throughout the body. HO-2 is highly expressed in the brain, especially in neurons of the olfactory epithelium and in the neuronal and granule cell layer of the olfactory bulb. In situ hybridization analysis showed that guanylyl cyclase and HO-2 are found in ORNs (Verma et al., 1993). Incubation of ORNs with the HO inhibitor, zinc protoporphyrin-9 (Zn PP-9), lowered cGMP levels in ORNs (Ingi and Ronnett, 1995). In addition, odorants augment cGMP levels in ORNs (Ingi and Ronnett, 1995; Verma et al., 1993). This odorantinduced cGMP increase could be inhibited by Zn PP-9, but not by a NOS inhibitor. Interestingly, the inhibition of HO could not entirely deplete cGMP levels in ORNs, suggesting that particulate guanylyl cyclases may also contribute to cGMP production in ORNs (Ingi and Ronnett, 1995). Exposure of isolated cilia derived from olfactory receptor neurons to various odorants increased cGMP levels (Moon et al., 1998). Thus, there was a strong suspicion that both soluble and particulate guanylyl cyclases have significant roles in olfactory signal transduction. The observation that the inhibition of HO in ORNs could not totally block the cGMP response suggested the involvement of particulate guanylyl cyclases in olfactory transduction. The fact that an NO donor and soluble guanylyl cyclase activator, sodium nitroprusside, could not alter the cGMP levels in isolated cilia supported the idea that the particulate guanylyl cyclases might play a role in olfactory cilia. An olfactory specific particulate guanylyl cyclase, guanylyl cyclase-D (GC-D), has been identified in olfactory epithelium (Fulle et al., 1995). GC-D has been suggested to function as the receptor of sensory neurons to specific odors. Other members of the particulate guanylyl cyclase family that are expressed in the olfactory epithelium have been identified by RT-PCR: GC-A, GC-B, and

Moon and Ronnett

GC-G (Simpson, Moon, and Ronnett, unpublished data). GC-B is highly expressed throughout the epithelium. These guanylyl cyclases are stimulated by specific nautriuretic peptides. At present, the role and the regulation of these guanylyl cyclases in the olfactory system are unclear. Recent studies have identified odorant-responsive particulate guanylyl cyclases in rat olfactory cilia (Moon et al., 1998). At least two particulate guanylyl cyclases exist in cilia, a low Km and a high Km isoform (Moon et al., 1998). Odorants were shown to elevate cGMP levels in cultured ORNs (Ingi and Ronnett, 1995) and in isolated olfactory cilia (Moon et al., 1998) in a calcium-dependent manner. A number of experiments suggested that calcium plays a role in odorant transduction and can fluctuate upon odorant exposure (Dhallan et al., 1990; Hatt and Ache, 1994; Yau, 1994). Hence, it was hypothesized that an olfactory particulate guanylyl cyclase could be regulated by a calcium-binding protein, such as guanylyl cyclase–activating protein (GCAP), similar to that found in the visual transduction pathway. In fact, immunohistochemical studies using anti-GCAP1 antibodies revealed that GCAP1 was highly localized to the olfactory cilia (Moon et al., 1998). Moreover, GCAP1 regulated the odorant-induced cGMP response in isolated rat olfactory cilia in a calcium-dependent manner (Moon et al., 1998). Thus, ORNs contain multiple cGMP pathways that mediate delayed and sustained cGMP responses to odorants. D.

Olfactory Phosphodiesterases

The ambient level of cAMP in a cell is dependent upon both the synthesis and degradation of cAMP. Although odorants clearly activate adenylyl cyclase, is there any effect of odorants on phosphodiesterases (PDEs)? There are at least seven different gene families of PDEs whose activities are regulated by calcium, cyclic nucleotides, and phosphorylation (Beavo, 1995; Beavo et al., 1994, Beltman et al., 1993; Burns et al., 1996). Thus, odorants could have an indirect effect on the degradation of cAMP, thus potentially providing a second site of regulation for the odorant-induced cAMP response. Several forms of cAMP-PDE are expressed in rat olfactory cilia (Borisy et al., 1991, 1993). A novel calcium/calmodulin PDE (CaM-PDE) is selectively found in ORNs, with prominent cilial expression. This novel CaM-PDE has a high affinity (Km 1.4 M) for cAMP and could be activated by odorants in response to intracilial calcium increases. Cloning of the high-affinity PDE revealed it to have a higher affinity for cAMP than any known brain isoform (Yan et al., 1995). This PDE, designated PDE1C2, is well suited for restoring the submicromolar levels of cAMP after odorant stimulation. In an ectopic expression system,

Molecular Neurobiology of Olfactory Transduction

maximum activation by calcium was reached at 10M calcium concentration. A subset of olfactory neurons expresses cGMP-stimulated phosphodiesterase (PDE2) (Juilfs et al., 1997). In these specific ORNs, GC-D is also expressed, suggesting that GC-D may play an important role in odorant transduction for a specific subset of responses. PDE2 and GC-D are both expressed in olfactory cilia of these neurons; however, only PDE2 is expressed in axons (Juilfs et al., 1997). In contrast to most other ORNs, these neurons appear to project to a distinct group of glomeruli in the olfactory bulb similar to the subset that have been termed necklace glomeruli. Furthermore, this subset of neurons are unique in that they do not contain several of the previously identified components of olfactory signal transduction cascades involving cAMP and calcium, including a calcium/calmodulindependent PDE (PDE1C2), AC3, and cAMP-specific PDE (PDE4A) (Juilfs et al., 1997; Meyer et al., 2000). Interestingly, these latter three proteins are expressed in the same neurons; however, their subcellular distributions are distinct. PDE1C2 and AC3 are expressed almost exclusively in the olfactory cilia, whereas PDE4A is present only in the cell bodies and axons. Taken together, these data strongly suggest that selective compartmentalization of different PDEs and cyclases is an important feature for the regulation of signal transduction in ORNs. E.

Calcium

Calcium regulates diverse cellular functions, and in general these functions are mediated by specific calcium-binding proteins (Baimbridge et al., 1992). Odorant stimulation of ORNs results in a calcium influx, which in turn can modulate a number of transduction pathways. Calmodulin and other calcium-binding proteins may participate in the processing of olfactory information. Therefore, study of the calcium-binding proteins may provide important background about the complex signal transduction pathway involved in olfaction. Olfactory tissues contains various calcium-binding proteins: calmodulin, calretinin, calbindin-D28k, neurocalcin, recoverin (Bastianelli et al., 1995). Another calcium-binding protein, S-100, is restricted to glial cells, primarily around the cribiform plate. Calmodulin is expressed in olfactory cilia at a concentration of about 1 µM (Anholt and Rivers, 1990). The odorant-induced intracellular elevation of calcium is thought to promote adaptation because calcium/calmodulin can reduce the affinity of the CNG channel for cAMP by 20-fold (Chen and Yau, 1994; Hsu and Molday, 1993). Extracellular calcium is absolutely required for the decay phase of the odorant-induced whole cell current, which in the absence of extracellular calcium remains at a steady state (Kurahashi

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and Shibuya, 1990). Calcium/calmodulin can also affect CNG channel activity (Kurahashi and Yau, 1993). Neurocalcin, a calcium-binding protein with three EF hand motifs, is also expressed in the rat olfactory epithelium (lino et al., 1995). Neurocalcin is localized to ORNs and distributed in the cytoplasm, where it is associated with outer mitochondrial membrane, endoplasmic reticulum, and axon fibers. The intracellular distribution of neurocalcin in ORNs suggests that this protein may participate in cytoskeletal arrangement in ORNs. The expression of neurocalcin in postnatal development was also studied (Bastianelli et al., 1995). Neurocalcin showed a gradient of expression pattern descending from the central to the lateral areas in the nasal cavity during childhood, and this expression pattern became identical to the adult profile after 20 days. Additional calcium-binding proteins have been described. A 26 kDa calcium-binding protein named p26olf was identified from the frog olfactory epithelium (Miwa et al., 1998). p26olf consists of two S-100–like regions and is localized to the cilia layer of the olfactory epithelium, suggesting that p26olf is a dimeric form of S-100 proteins and may be involved in the olfactory transduction or adaptation. Visinin-like protein (VILIP), a member of the neuronal subfamily of EF-hand calcium-sensor proteins, was found to be expressed in ORNs of the rat olfactory epithelium (Boekhoff et al., 1997). VILIP is localized prominently to cilia and dendritic knobs. In vitro recombinant VILIP attenuates odorant-induced cAMP formation in a calciumdependent manner. The observation that VILIP does not interfere with odorant-induced receptor desensitization and that VILIP inhibits the forskolin-induced cAMP production suggests that VILIP may directly affect adenylyl cyclases and in turn may play a role in adaptation of ORNs. A GCAP1-like calcium-binding protein is present in rat olfactory cilia (Moon et al., 1998). GCAP1 was initially purified and later cloned from bovine retina by Palczewski and colleagues (1994). GCAP1 is a 21 kDa cytosolic EFhand family protein and is proposed to function as a photoreceptor-specific calcium-binding protein to activate particulate guanylyl cyclase, thus restoring cGMP level in light-activated photoreceptor cells. Immunohistochemical studies using anti-GCAP1 antibodies revealed the presence of GCAP1 in rat olfactory cilia (Moon et al., 1998). Interestingly, purified GCAP1 potentiated cGMP production at high calcium concentrations in isolated rat olfactory cilia (Moon et al., 1998). In photoreceptor cells, GCAP1 activates particulate guanylyl cyclase when intracellular calcium level is low. The size of the olfactory GCAP (19 kDa) was not identical to the retinal GCAP1. Thus, the olfactory GCAP is referred to as a GCAP1-like protein. The cloning of the olfactory GCAP will answer the precise function and mechanism of the olfactory guanylyl GCAP in olfaction.

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Moon and Ronnett

A novel calcium-binding protein was recognized in the olfactory tissues by using R2D5, a mouse monoclonal antibody that labels rabbit olfactory receptor neurons (Nemoto et al., 1993). Immunoblot analysis showed that R2D5 antibody recognizes a 22 kDa protein that is abundant in the olfactory epithelium and in the olfactory bulb. This protein contains three calcium-binding EF hands and potent phosphorylation sites for calcium/calmodulin-dependent protein kinase II (CaMPK II) and cAMP-dependent protein kinase (PKA). Different from ubiquitously expressed calmodulin, this calcium-binding protein is expressed specifically in ORNs, indicating that this protein may participate in olfactory signal transduction. Calcium itself mediates Cl conductance in ORNs (Kleene and Gesteland, 1991a; Kleene, 1993; Lowe and Gold, 1993b). The odor-induced currents show little rectification. It appears that the depolarizing current has two components, an initial inward cationic conductance followed by an inward anionic Cl conductance (Kleene, 1993; Kurahashi and Yau, 1993; Lowe and Gold, 1993b). Calcium, which enters the cilia through the cyclic nucleotidegated channel, triggers a calcium-activated Cl channel in olfactory cilia membrane (Kleene and Gesteland, 1991b). This conductance may serve as a “failsafe” so that cells can depolarize, irrespective of changes in extracellular milieu.

VIII.

DESENSITIZATION

Desensitization occurs through a variety of processes, including phosphorylation, internalization, and receptoreffector uncoupling (Hausdorff et al., 1990; Huganir and Greengard, 1990; Sibley et al., 1987). The homologous desensitization of G-protein–coupled receptors is well established in 2-adrenergic receptor (AR-2) as a model (Benovic et al., 1988, 1989). Phosphorylation of receptors by a specific receptor kinase termed -adrenergic receptor kinase (ARK) mediates homologous desensitization. Complete quenching of signal transduction requires the binding of a protein called -arrestin (ARR) to phosphorylated receptor (Lohse et al., 1990). Specific isoforms of ARK and ARR, ARK-2 and ARR-2 were localized to olfactory neurons, specifically to olfactory cilia and dendritic knobs (Dawson et al., 1993). Other isoforms of ARK or ARR were not present in these regions. Functional studies of ARK-2 and ARR-2 in the olfactory cilia were performed (Dawson et al., 1993; Schleicher et al., 1993). The odorant-induced cAMP production was monitored in the presence or absence of neutralizing antibodies against specific isoforms of ARK and ARR. Preincubation of isolated olfactory cilia with neutralizing

antibodies to ARK-2 and ARR-2 increased the absolute levels of odorant-induced cAMP as much as four fold and completely blocked desensitization. Later mice targeted disrupted of ARK-2 have been available, and cilia preparations derived from the ARK-2–deficient mice showed lack of the agonist-induced desensitization (Peppel et al., 1997). Taken together, the expression of ARK-2 and ARR-2 within the olfactory cilia, the inhibition of desensitization with ARK-2–and ARR-2–neutralizing antibodies, and the lack of the agonist-induced desensitization in the ARK-2–deficient mice suggest that ARK-2 and ARR-2 mediate the odorant-dependent desensitization in olfaction. In addition, it has been suggested that PKA or PKC may play a role in olfactory desensitization (Boekhoff and Breer, 1992). PKA has been implicated in olfactory desensitization following increase in cAMP by odorant stimulation, and PKC may mediate desensitization following Pl cycle activation by odorant stimulation. However, these results need to be reexamined, given more recent data using knockout animals that indicate that cAMP mediates odorant detection. Cyclic GMP may also be involved in desensitization. Zufall and Leinders-Zufall (1997) showed that cGMP mediated a long-lasting form of odor response adaptation in tiger salamander. The long-lasting adaptation lasted for several minutes and was attributable to cyclic nucleotidegated channel modulation by cGMP. They also showed that this form of long-lasting adaptation was abolished selectively by HO inhibitors (thus preventing CO release and cGMP formation), whereas odor excitation was unaffected. The results suggest that endogenous CO/cGMP signals contribute to olfactory desensitization.

IX. LONG-TERM RESPONSES TO ODORANT DETECTION The theory that extracellular signals, such as hormones, growth factors, and neuronal activity, modulate transcriptional events to produce long-term changes in cellular activity is well established (Hill and Treisman, 1995). However, the long-lasting effects of odorant stimulation in ORNs are unknown. A delayed cAMP response upon odorant stimulation was characterized and was mediated by cGMP via activation of a cGMP-dependent protein kinase (PKG) (Moon et al., 1998). Based on the kinetics of the delayed cAMP response previously discussed, it was postulated that cGMP might mediate a delayed cAMP response to regulate longterm cellular responses to odorant detection, including gene expression. Recent work supports this idea. Odorant stimulation can result in transcriptional changes via CREB activation (Moon et al., 1999). While incubation with either

Molecular Neurobiology of Olfactory Transduction

8-Br-cGMP or a soluble guanylyl cyclase activator (sodium nitroprusside) increased CREB activation, PKG could not directly phosphorylate CREB in ORNs. Thus, cGMP produced upon odorant stimulation may generate a sustained cAMP signal capable of activating CREB. Involvement of the Ras-MAPK (mitogen-activated protein kinase) signal transduction pathway in olfaction was recently reported in C. elegans (Hirotsu et al., 2000). The Ras-MAPK pathway plays important roles in cellular proliferation and differentiation in response to extracellular signals. Mutational inactivation and hyperactivation of this pathway impaired efficiency of chemotaxis to a set of odorants. The activation of MAPK upon odorant stimulation was dependent on calcium via the nucleotide-gated channel and the voltage-activated calcium channel. More recently, Storm and colleagues demonstrated that odorants activate MAPK in rodent ORNs (Watt and Storm, 2001). The odorant-activation of MAPK pathway led to the activation of cAMP response element (CRE)–mediated transcription. The odorant stimulation of MAPK activation was ablated by inhibition of CaM-dependent protein kinase II (CaMKII), suggesting that odorant activation of MAPK is mediated through CaMKII. Moreover, discrete populations of ORNs display CRE-mediated gene transcription when stimulated by odorants in mice. Taken together, these data suggest that ORNs may undergo long-term adaptive changes mediated through CRE-mediated transcription. X.

CONCLUSIONS

Olfaction is an essential sensory modality that influences the quality and in many cases the survival of an organism. Tremendous progress has been made in the last decade regarding our understanding of odorant transduction. Challenges remain. Understanding the olfactory code will allow us to manipulate olfactory perception in both health and disease. Our appreciation of the ability of odor perception to influence long-term neuronal responses, and potentially neuronal survival, may provide clues to understanding this process in other neuronal systems. Given the tools that we have available, it is clear that the olfactory system is an excellent model for signal transduction and neuronal homeostasis. The cytoarchitecture, life cycle, availability of in vitro models, and straightforward axonal projections of ORNs make them amenable to future studies that seek to investigate stimulus processing. REFERENCES Abogadie, F. C., Bruch, R. C., Wurzburger, R., Margolis, F. L., and Farbman, A. I. (1995). Molecular cloning of a phospho-

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5 Neurogenesis in the Adult Olfactory Neuroepithelium Alan Mackay-Sim Griffith University, Brisbane, Queensland, Australia

I.

II.

INTRODUCTION

OVERVIEW

After the early reports of basal cell mitosis in mouse olfactory epithelium (Nagahara, 1940) and regeneration of olfactory sensory neurons after zinc sulfate lesion in monkey (Schultz, 1941), there followed numerous reports confirming these observations in a variety of vertebrates: frog (Smith, 1951), fish (Westerman and Baumgarten, 1964), cat and dog (Andres, 1966), lamprey (Thornhill, 1970), and mouse (Smart, 1971). These early histological observations were supported by analyses using tritiated thymidine to label cells during S-phase (DNA replication) of the cell cycle (Graziadei and Metcalf, 1971; Moulton et al., 1970; Thornhill, 1970). The field of olfactory neurogenesis was greatly expanded in the 1970s and 1980s by intensive efforts to document and understand the morphological features of neurogenesis and especially the stimulus to neurogenesis brought about by destruction of the sensory neurons. The quantitative 3[H]-thymidine analyses and electron microscopic investigations led to the oft-repeated view that neurogenesis in the adult olfactory epithelium is unique in the adult nervous system, now known to be untrue. Another oftrepeated view is that the olfactory sensory neurons live for only 30 days and are more-or-less automatically replaced by division and differentiation of the basal cells (Graziadei and Monti Graziadei, 1979; Moulton, 1975). This model of shortlived sensory neurons and “automatic” replacement became the “orthodoxy” and is cited in primary papers, reviews, and textbooks. This model was challenged by evidence that some sensory neurons may live for at least one year (Hinds et al.,

Neurogenesis has long been recognized as a property of the adult olfactory epithelium. Over 50 years ago mitotic activity was first observed in the olfactory epithelium of adult mice (Nagahara, 1940). Olfactory sensory neurons regenerate in monkey (Graziadei et al., 1980; Schultz, 1941) and human (Murrell et al., 1996; Wolozin et al., 1992). Human olfactory neurogenesis continues into old age, making the olfactory system one of the most continually variable regions of the nervous system. It is now recognized that neurogenesis occurs in a number of sites within the adult brain. A recent study has even identified newly formed neurons in the brain of aged humans (Eriksson et al., 1998). Sites of neurogenesis in the brain include the dentate gyrus and the subventricular zone of the forebrain (recently reviewed in Scheffler et al., 1999). Neurogenesis in the subventricular zone gives rise to neurons which migrate forward to populate the olfactory bulb, providing interneurons in the periglomerular and granule cell layers (Luskin, 1993). This chapter presents a review of investigations of neurogenesis in the adult olfactory epithelium. This process is shown to be regulated by endocrine, autocrine, and paracrine factors and modulated by environment factors presented in the inspired air. Current hypotheses for the lineage and regulation of neurogenesis are discussed and explored to provide a cellular and molecular model of this unusual and interesting “embryonic” feature of adult olfactory epithelium. 93

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1984). It was also challenged by evidence that the rate of basal cell mitosis may be inversely proportional to epithelial thickness, indicating regulatory mechanisms at work within the epithelium (Mackay-Sim and Patel, 1984). These and other data led to an alternative model, which proposed that olfactory sensory neurons do not die “automatically” after 30 days— that their lifespan is regulated by extrinsic factors, such as the odorous environment in the nose, rather than immutable, cellintrinsic factors and that olfactory neurogenesis is a process regulated by endocrine, autocrine, and paracrine factors similar to those operating during embryonic organogenesis (Breipohl et al., 1986). A model of regulated neurogenesis is more in keeping with current views of the development of cells and tissues during embryogenesis. Research in the last 10 years has centered on the factors that regulate olfactory neurogenesis, and many growth factors have now been implicated. Although it is recognized that basal cells give rise to neurons, less obvious are the identities of the cells in the lineage hierarchy from uncommitted stem cell to precursor cells to neurons in the adult olfactory epithelium. These investigations are reviewed in this chapter, and the implications for cell therapy based on olfactory epithelium are discussed.

III. OLFACTORY SENSORY NEURONS: DISPOSABLE OR REPLACEABLE? Injection of 3[H]-thymidine into the adult mouse labels many dividing cells in the olfactory epithelium. At early survival periods after injection the dividing cells are located in two places—most of them are among the basal cells, close to the basement membrane, with a few located apically, among the supporting cells (Graziadei and Monti Graziadei, 1979; Moulton et al., 1970). A similar distribution of labeled cells is observed in amphibian (Graziadei and Metcalf, 1971; Graziadei, 1973; Mackay-Sim and Patel, 1984). With increasing periods after injection of 3[H]-thymidine, the labeled basal cells migrate away from the basement membrane until their nuclei lie in the midzone of the epithelium in the region of the sensory neuron nuclei (Graziadei and Monti Graziadei, 1979; Moulton et al., 1970). These observations are consistent with basal cells giving rise to neurons, and under the electron microscope there appear to be transitional cell types whose morphology suggests that they are immature neurons (Graziadei, 1973; Graziadei and Monti Graziadei, 1979). By 30 days after injection of 3[H]-thymidine, the labeled cells have either disappeared from the epithelium (Graziadei and Monti Graziadei, 1979) or are reduced in number (Mackay-Sim and Kittel, 1991a; Moulton et al., 1970). In combination with the migration of labeled cells from the

Mackay-Sim

basal region into the midzone containing sensory neuron nuclei, the loss of labeled cells by 30 days was interpreted to mean that the neurons “remain in the epithelium as mature functional elements for approximately 25 days” (Graziadei and Monti Graziadei, 1979). This view was reinforced by a quantitative analysis, which indicated that the “turnover time of the entire population” of cells in the olfactory epithelium is 28.6 days and that the “turnover time of the receptor cells should approximate that of the entire population” (Moulton, 1975). A quantitative study of regeneration of the hamster olfactory epithelium also led to an estimate of the life span of receptor neurons of 25–35 days (Samanen and Forbes, 1984). These conclusions—that mature olfactory sensory neurons live for about 30 days—were based on the assumption that cells entered the population via division of basal cells and left it as mature neurons (Fig. 1). This assumption was later shown to be false. Nevertheless, there was no doubt that new neurons arise in the olfactory epithelium from division of the basal cells. Along with the concept of a short-lived sensory neuron, there came to be an assumption that turnover of sensory neurons from basal cell mitosis was a “predetermined . . . genetic characteristic” (Graziadei and Monti Graziadei, 1978). The prevailing model thus came to be one of disposable neurons in the olfactory epithelium, similar to cells in other epithelia such as the epidermis and the intestinal epithelium in which the neurons seemed to be inherently obsolescent. It was believed that the olfactory epithelium was unique in (1) having short-lived neurons and (2) having the ability to replace them (Graziadei and Monti Graziadei, 1978). The notion that mature olfactory sensory neurons live for only about 30 days was first challenged by evidence of labeled neurons present one year after injection (Hinds et al., 1984). This evidence questioned the prevailing view of a short-lived, disposable neuron, but it was only one of several lines of evidence that olfactory neurogenesis may be actually a highly regulated process, rather than being driven by an genetically predetermined, “clock-like” process (Breipohl et al., 1986). This “regulated” model places adult olfactory neurogenesis as an extension of the same processes occurring during embryonic development of the nervous system in general (MackaySim and Kittel, 1991a) with the difference being that the mature olfactory neurons are directly exposed to the external environment and at risk of damage by it (Breipohl et al., 1986; Hinds et al., 1984; Mackay-Sim and Kittel, 1991b). Significant predictions of this “regulated” model were (1) that the majority of dying cells in the olfactory epithelium would be developing, immature neurons, rather than mature sensory neurons and (2) that mature sensory neurons would remain alive and connected to the olfactory bulb unless damaged by the environment (Breipohl et al., 1986).

Neurogenesis in the Adult Olfactory Neuroepithelium

95

Figure 1 Disposable neuron model of the genesis of the olfactory sensory neuron. The sensory neuron arises from division and differentiation of the basal cell, lives for about 1 month, and is automatically lost from the epithelium.

Many investigations bear out these predictions. Retrograde labeling by injection of colloidal gold provided direct evidence that olfactory neurons remain connected to the olfactory bulb for at least 3 months (Mackay-Sim and Kittel, 1991b), supporting 3[H]-thymidine evidence for long-lived neurons (Hinds et al., 1984; Mackay-Sim and Kittel, 1991a; Moulton et al., 1970). Quantitative analyses after 3[H]-thymidine injection show that 70–80% of the labeled cells are lost from the epithelium between 14 and 21 days, after migrating into the neuronal layer (Mackay-Sim and Kittel, 1991a; Moulton et al., 1970). The surviving labeled cells survive for at least 3 months (Mackay-Sim and Kittel, 1991a; Moulton et al., 1970) or longer (Hinds et al., 1984). The most parsimonious explanation for this is that the cells lost early are immature neurons which were not successful in making connections in the olfactory bulb, whereas the surviving cells are those neurons that found synaptic space at the bulb and dendritic space at the epithelial surface (Breipohl et al., 1986; Mackay-Sim and Kittel, 1991a). In other parts of the nervous system during development, immature neurons pass through a “critical period” during which they must make the correct connections. This is a period of intense competition for synaptic space. For example, 80% of retinal ganglion cells die during development (Williams and Herrup, 1988). It is probable that competition for space in the bulb and at the epithelial surface is a major determinant of whether immature neurons survive beyond 2–3 weeks. Cell death is an integral part of neurogenesis during embryonic development, and analyses of cell death in the adult olfactory epithelium indicate that all cell types undergo apoptosis, not just

mature sensory neurons (Mahalik, 1996). Cell death can occur within one day of birth (Carr and Farbman, 1993), indicating that apoptosis in the olfactory epithelium is an integral and early part of neurogenesis in the adult olfactory epithelium. In summary, there are two complementary hypotheses supported by the published data. The first of these is that olfactory neurogenesis in the adult reflects ontogeny in other parts of the nervous system except that adult olfactory neurogenesis is an ongoing process. In the embryo, neuronal precursors are born and developing neurons reach stages of differentiation within a limited time period so that the developmental events in the population are reflected in the molecular events guiding the differentiation of each cell. In contrast, all stages of development are seen simultaneously in the adult olfactory epithelium. The second hypothesis supported by the published data is that adult olfactory neurogenesis is a regulated process with fine controls over cell proliferation, cell differentiation, and cell death. According to these models of olfactory neurogenesis, the emphasis shifts from considering the olfactory sensory neuron being unusual for its alleged short lifespan to investigating the cellular mechanisms of the regulation of neurogenesis (Breipohl et al., 1986; Mackay-Sim and Kittel, 1991a, 1991b).

IV. REGULATION OF OLFACTORY NEUROGENESIS IN VIVO Olfactory neurogenesis has now been studied for about 60 years, and it is appropriate to bring together all the available

96

Mackay-Sim

Figure 2 Replaceable neuron model of the genesis of the olfactory sensory neuron. Like other neurons, the olfactory sensory neuron lives until damaged by its environment. It is replaced by a dynamic process involving many paracrine and autocrine signals, which eventually select a few neurons to undergo final maturation.

information to develop hypotheses that can help direct future research. A working hypothesis is that neurogenesis in the adult olfactory system is similar to embryonic development, following similar developmental rules that govern the development of other sensory systems, such as the retina (Breipohl et al., 1986). Most of the data described above can be interpreted to support this “developmental” hypothesis: (1) cell death occurs at all stages of neuronal development, (2) developing neurons are overproduced, (3) developing neurons pass through a “critical period” during which they must find synaptic space at their target, (4) successful neurons are dependent on their target for survival, and (5) mature neurons are not programmed to die but may die from external influence. Figure 2 summarizes the cycle of neurogenesis and some of the regulating factors described below. A.

Stimulation of Neurogenesis by Death of the Sensory Neurons

Olfactory sensory neurons are lost and then regenerate after the olfactory nerve is cut (Graziadei, 1973; Nagahara, 1940) or the epithelium is washed with zinc sulfate (Margolis et al., 1974; Schultz, 1941; Smith, 1951). There is a decrease in epithelial thickness and a decrease in the number of nuclei in the epithelium and sensory dendrites at the epithelial surface (Costanzo and Graziadei, 1983; Samanen and Forbes, 1984). The loss of cells after nerve section is confined to the basal cell and sensory neuron layers of the epithelium, with the supporting cell numbers

unaffected (Costanzo and Graziadei, 1983). The cell death observed after olfactory nerve section is apoptotic (Deckner, 1997; Holcomb et al., 1995; Michel et al., 1994) and reaches a peak at about 1.5–2 days (Costanzo and Graziadei, 1983; Deckner, 1997; Michel et al., 1994), declining to control levels at 4 days after nerve section (Deckner, 1997). The loss of neurons is accompanied by an increase in basal cell mitosis (Graziadei, 1973), which reaches its peak 4 days after nerve section (Camara and Harding, 1984). The sensory neuron population recovers in number following olfactory nerve section, but the cell numbers and epithelial thickness reach only 60% of control levels (Costanzo and Graziadei, 1983; Samanen and Forbes, 1984), although functional recovery is observed (Costanzo, 1985). When the sensory neurons are destroyed by application of zinc sulfate to the nose, the recovery in epithelial thickness is more complete, reaching control levels after about 1 month (Matulionis, 1975) followed by restoration of olfactory function (Harding et al., 1978). B. Synaptic Contact with the Olfactory Bulb and Sensory Neuron Survival The olfactory nerve is most commonly sectioned by complete removal of the olfactory bulb, their synaptic target. When this occurs, the subsequent development of the olfactory axons is seriously disrupted, and their aberrant growth can lead to neuromas within and below the olfactory epithelium (Schwob et al., 1994b). This can also occur when the olfactory nerve is simply transected without removal of the olfactory bulb (Schwob et al., 1994b). Thus, although there can be some functional recovery after olfactory nerve transection (Costanzo, 1985), the epithelium still fails to recover to control levels (Costanzo, 1984). When the olfactory bulb is removed, the many newly formed neurons fail to fully differentiate and die within 2 weeks (Carr and Farbman, 1992; Schwob et al., 1992). Death of newly formed cells is maximal death at 6–8 days after cell birth even when the animal is killed 12 days or 7 weeks after olfactory bulbectomy (Carr and Farbman, 1993). Taken together these observations suggest that the developing sensory neurons require contact with cells in the olfactory bulb for their survival, perhaps because bulb cells release some trophic factor (Schwob et al., 1992). This was tested directly when the mitral cells, the main target for olfactory sensory axons, were reduced in number by sectioning of their axons in the lateral olfactory tract (Weiler and Farbman, 1999). This reduction in mitral cells in the olfactory bulb stimulated basal cell proliferation in the olfactory epithelium at all time points (up to 14 months) (Weiler and Farbman, 1999).

Neurogenesis in the Adult Olfactory Neuroepithelium

The importance of the olfactory bulb for sensory neuron differentiation was shown in organ cultures of embryonic olfactory epithelium, cultured with or without an olfactory bulb (Chuah and Farbman, 1983). Sensory neuron differentiation was assessed by measuring the amount of olfactory marker protein (OMP) in the cultured olfactory epithelium. Under these conditions the olfactory bulb increased the amount of OMP only if it was co-cultured in contact with the olfactory epithelium, indicating that physical contact between the tissues was necessary for the effect (Chuah and Farbman, 1983). Similarly, contact co-culture increased the numbers of OMP-positive cells (Chuah and Farbman, 1983) and the numbers of ciliated dendritic knobs at the surface of the differentiating epithelium (Chuah et al., 1985). The induction of sensory neuron maturation was tissuespecific: culture of the olfactory epithelium with brain, spinal cord, or heart did not increase OMP levels (Chuah and Farbman, 1983). Taken together with the in vivo observations, there seems no doubt that the survival of olfactory sensory neurons depends on synaptic contact with mitral cells in the olfactory bulb, probably due to a nondiffusible trophic factor provided by contact with mitral or other cells in the olfactory bulb. C.

Neurogenesis is Regulated by Usage

If one naris is closed during early development, there are marked differences in the olfactory epithelia from the control and occluded sides (Farbman et al., 1988) (see Chapter 29). The thickness of the epithelium is reduced on the occluded side, accompanied by a reduction in cell number and a reduction in the number of proliferating basal cells (Farbman et al., 1988). Despite these differences there was no effect on the number of sensory neurons, indicated by the numbers of olfactory dendrites at the epithelial surface (Farbman et al., 1988). These observations suggest that the rate of basal cell proliferation was reduced because of a protective effect of naris occlusion with a reduction in the access of infectious or toxic agents (Farbman et al., 1988). A lack of toxic environmental effects was also proposed for the observation of long-lived sensory neurons living in a clean air environment (Hinds et al., 1984). When the naris is closed in adult mice, it is the open side that is reduced in thickness (Maruniak et al., 1989). The reduction in thickness is associated with loss of sensory neurons (Maruniak et al., 1989) in marked contrast to the effects of naris occlusion during development in which the mature neurons are unaffected (Farbman et al., 1988). The loss of sensory neurons was not accompanied by loss of other cell types in the epithelium, leading to the conclusion that naris occlusion had an effect specifically on the sensory

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neurons (Maruniak et al., 1989, 1990). This effect was greater rostrally than caudally, leading to the conclusion that all of the inspired air passing through the one side of the nose leads to accelerated sensory neuron death and a lack of the regenerative ability to maintain their numbers (Maruniak et al., 1989, 1990). The mechanism for the sensory neuron loss is unknown, but speculations are that it is due to overuse or to toxins or to infections, supported by evidence for a large number of polymorphonuclear leukocytes on the open side after 7 and 8 months of closure (Maruniak et al., 1990). D. Regulation of Neurogenesis by the Density of Immature Neurons When basal cell proliferation is observed using 3[H]thymidine, it is evident that the density of proliferating cells is not constant across the epithelial sheet. There are obvious regions where proliferation is more active (Graziadei and Monti Graziadei, 1978; Moulton et al., 1970; Weiler and Farbman, 1997). This suggests that in the normal epithelium neurogenesis is under local control mechanisms. Another example of this is the observation that in the salamander the rate of basal cell proliferation is inversely proportional to the thickness of the epithelium (Mackay-Sim and Patel, 1984). In this species the olfactory epithelium varies in thickness from anterior to posterior, being thicker anteriorly. Quantitative analysis of the cell types in this epithelium demonstrated that the only cell type whose numbers increased with epithelial thickness were the immature neurons (Mackay-Sim et al., 1988). The numbers of mature sensory neurons, supporting cells, and basal cells were all constant and independent of epithelial thickness. Therefore, the rate of basal cell proliferation was effectively inversely proportional to the number of immature sensory neurons, leading to the conclusion that the developing neurons exert an inhibitory influence basal cell proliferation (Mackay-Sim et al., 1988). This conclusion is supported by a report that the rate of proliferation in vitro was reduced when precursor cells were co-cultured with sensory neurons (Mumm et al., 1996). E.

Regulation of Neurogenesis by Thyroxine

Adult mice made hypothyroid exhibit olfactory dysfunction, from which they recover if normal thyroxine levels are restored (Beard and Mackay-Sim, 1987). After 7 weeks of hypothyroidism there is a reduction in epithelial thickness without loss of sensory neurons (Mackay-Sim and Beard, 1987). The reduction in epithelial thickness is due to loss of immature neurons (Mackay-Sim and Beard, 1987).

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F.

Mackay-Sim

Cell Death as an Integral Part of Neurogenesis

As indicated above, a single intraperitoneal injection of 3[H]-thymidine labels a large number of basal cells in the olfactory epithelium (Graziadei and Monti Graziadei, 1978; Hinds et al., 1984; Mackay-Sim and Kittel, 1991a; Moulton et al., 1970) indicating a high rate of proliferation in the normal, undisturbed epithelium. Despite this continual proliferation of neuronal precursors, there is no continual increase in epithelial thickness in the adult rat from 60–330 days of age (Hinds and McNelly, 1981; Weiler and Farbman, 1997). In rat there is an increase in surface area of the epithelium in adulthood (Hinds and McNelly, 1981; Paternostro and Meisami, 1993; Weiler and Farbman, 1997), and it is probable that basal cell proliferation at the edges of the epithelium could contribute to its expansion. There is no evidence that dividing basal cells of their progeny migrate very far laterally to populate new regions. Given the lack of increase in epithelial thickness, it follows that the constant proliferation must be balanced by a concomitant constant cell death. This is supported by evidence that from 1 to 17 weeks of age, the rates of basal cell proliferation and of cell death in the epithelium show a similar age-related decline (Fung et al., 1997). It is now evident that cell death occurs at all stages of development after basal cell division. This cell death in the normal undisturbed epithelium is apoptotic (Magrassi and Graziadei, 1995), similar to death induced by olfactory nerve section. By labeling dividing cells with 3[H]-thymidine and looking for thymidine-labeled pyknotic nuclei, it was shown that cell death can occur as early as one day after birth (Carr and Farbman, 1993). When cells were identified with cell-type specific markers and doublelabeled to identify dying cells, it was clear that the apoptotic cells could be horizontal basal cells, globose basal cells, immature neurons, or mature neurons (Holcomb et al., 1995; Mahalik, 1996). Apoptotic cell death is a highly regulated process (Vaux and Strasser, 1996). Evidence for the involvement of regulatory genes in olfactory neurogenesis are that overexpression of the bcl-2 gene protects the adult animal from apoptotic death after olfactory nerve transection (Jourdan et al., 1998). Further evidence for apoptotic regulation of neurogenesis is given by experiments describing the presence of apoptotic regulatory molecules in the adult olfactory epithelium. The apoptotic cascade can be induced by activation of the cell surface receptors Fas and tumor necrosis factor (TNF) receptor-1 by their ligands FasL and TNF-. Both the receptors and their ligands were observed in olfactory epithelium in vivo (Farbman et al., 1999), and addition of either FasL and TNF- induced

apoptosis in vitro (Farbman et al., 1999). Several enzymes of the caspase family, enzymes known to be involved in TNF-–induced cell death in other cell types, are present in the adult olfactory epithelium, and inhibition of these enzymes blocks apoptosis in a dose-dependent manner in olfactory epithelial cultures (Suzuki and Farbman, 2000). In summary, there is strong evidence that the cell death seen in the undisturbed olfactory epithelium and after olfactory nerve section is apoptosis, or programmed cell death, and there is evidence for autocrine or paracrine signaling pathways involved in apoptosis in the olfactory epithelium. These observations further confirm that olfactory neurogenesis is a regulated process with an intricate balance between production of new neurons and death of all cell types to maintain equilibrium within the epithelium. G.

Neurogenesis and Aging

Olfactory neurogenesis continues throughout adult life, observed in aged rat (Loo et al., 1996; Weiler and Farbman, 1997) and human (Murrell et al., 1996; Wolozin et al., 1992). As rats and humans age, there are histopathological changes that suggest that neurogenesis is not able to fully maintain the epithelium. In rat the anterodorsal region of the epithelium shows a greater average number of proliferating basal cells but also a greater level of intraand interanimal variability of basal cell proliferation (Loo et al., 1996; Weiler and Farbman, 1997). Histologically, this region also appears disordered with a reduction of lamination, a loss of neurons, and increased proliferation of supporting cells (Loo et al., 1996). These changes are consistent with damage to this area, with a concomitant attempt to reconstitute the sensory neuron population (Loo et al., 1996). Similar changes are observed in olfactory epithelium sampled from adult humans. In aged humans the olfactory epithelium may show a reduction in thickness, a reduction in sensory neuron number, and patchy distribution of olfactory epithelium within the respiratory epithelium (Naessen, 1971; Nakashima et al., 1984, 1985). It is suggested that the pathology seen in the aged olfactory epithelium resembles the changes induced in the open side of adult animals subject to unilateral naris occlusion (Loo et al., 1996). When one naris is closed, sensory neurons are lost on the open side, resulting, in places, in an epithelium composed of supporting cells only (Maruniak et al., 1989, 1990; Walters et al., 1992). These observations suggest that a similar mechanism may act during aging, which is accelerated by naris occlusion, that is, the most exposed regions of the olfactory epithelium (e.g., the anterodorsal region in the rat) may be subject to overusage or airborne factors that continually stimulate neurogenesis.

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With age the level of neurogenesis may not be able to be maintained, thus leading to a reduced capacity for repair and replacement of olfactory epithelium for respiratory epithelium. H.

Summary

As the discussion above indicates, there is increasing evidence for various regulatory controls on olfactory neurogenesis in the adult. In the normal epithelium these controls act to maintain the epithelial thickness and the number of sensory neurons and to balance the rate of cell birth with the rate of cell death. From the manipulations of olfactory neurogenesis in vivo, the action of various unknown but potential regulatory factors can be implied. Table 1 summarizes these. The regulatory pathways suggested in Table 1 are only speculative, but they can explain the observations. Clearly basal cell proliferation can be regulated up and down by the density of sensory neurons and immature neurons. Such regulation could be achieved by the release of a stimulatory factor when neurons die and an inhibitory factor while they live. Regulation could be achieved with a single stimulatory or inhibitory factor, but two would provide finer control and would allow a greater variation in local control mechanisms. Similarly, multiple stimulatory or inhibitory factors would provide even greater variation and some redundancy. In addition to factors regulating basal cell proliferation, the evidence suggests that there are also factors promoting survival of the sensory neurons, as shown by the continuing death of neurons unable to make contact with their target, the olfactory bulb. Similarly, the loss of immature neurons during

hypothyroidism suggests that thyroxine is involved in promoting survival of these cells. Another form of “regulation” of neurogenesis occurs via the influence of the environment on the sensory neurons. Already discussed is the use of nasal lavage of zinc sulfate to experimentally destroy the sensory neurons. Inhaled toxic gases can also destroy the olfactory epithelium and the neurons within it. Inhalation of N-methyl-formiminomethylester and methyl bromide led to a temporary loss of smell and a reversible loss of the sensory neurons (Hurtt et al., 1988; Rehn et al., 1981; Schmidt et al., 1984). Even nasal lavage with a large protein conjugate, wheat germ agglutinin-horseradish peroxidase, led to loss of sensory neurons from the epithelium and stimulated basal cell proliferation (Moon and Baker, 1998). Taken together these observations indicate that the sensory neurons are vulnerable to damage from inhaled molecules leading to sensory neuron death and stimulation of neurogenesis. It is informative to speculate that the high rate of proliferation of neuronal precursors, the high mortality of the immature neurons, and the low rate of replacement of sensory neurons may be related to the requirement to balance the birth and death of sensory neurons expressing individual receptor genes. Each olfactory neuron appears to expresses a single odorant receptor gene (Buck and Axel, 1991), which is involved in targeting the axon to restricted glomeruli in the olfactory bulb (Vassar et al., 1994). The receptor gene is expressed early in differentiation before the sensory neuron establishes connections with the olfactory bulb (Leibovici et al., 1996), and its expression in the nasal cavity is independent of the presence of the olfactory bulb in

Table 1 Regulation of Basal Cell Proliferation In Vivo Experiment

Stimulus

Effect on basal cell proliferation

Olfactory nerve cut

Death of mature neurons

Increased

Chemical destruction of neurons Destruction of mitral cells

Death of neurons

Increased

Death of neurons

Increased

Increase in usage/ loss of neurons Reduction in usage/ no loss of sensory neurons Increased density of immature neurons

Increased

Naris occlusion open side Naris occlusion closed side Epithelial thickness

Decreased

Decreased

Possible regulatory pathway Proliferating factor released from dying sensory neurons Proliferating factor released from dying sensory neurons Lack of trophic factor in olfactory bulb leads to death of sensory neurons and release of proliferating factor Proliferating factor released from dying sensory neurons Lack of proliferating factor

Lack of proliferating factor or of anti-proliferative factor

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the adult (Konzelmann et al., 1998; Margalit and Lancet, 1993; Strotmann et al., 1995; Sullivan et al., 1995). Cells expressing each receptor gene are expressed stochastically within restricted regions of the olfactory epithelium (Buck and Axel, 1991; Ressler et al., 1993; Strotmann et al., 1996), so the question arises as to how the numbers of neurons expressing each gene are maintained. Perhaps the most critical property of a developing neuron in the adult is whether its receptor gene matches the receptor gene of the dying neuron it replaces. With approximately 1000 genes distributed in four epithelial zones, dying sensory neurons could express one of 250 receptor genes. Is it possible that 250 developing neurons are necessary for each dying neuron to be replaced by a cell expressing the correct receptor gene? The selection of the successful developing neuron may be regulated by its finding synaptic space in target glomeruli in the olfactory bulb, although all successful neurons would also require dendritic space at the epithelial surface. According to this argument, there could be 249 unsuccessful neuronal precursors for every cell that accomplishes differentiation into a functioning sensory neuron.

V. MOLECULAR REGULATION OF OLFACTORY NEUROGENESIS Presumably the principles by which the olfactory epithelium is maintained in the adult animal are similar to the principles by which it develops in the embryo, namely, the cells are subject to autocrine and paracrine signals as well as cell-cell contact signals, which maintain or induce different cells types. In the olfactory epithelium it is possible that signals arise from any or all of the cell types (horizontal and globose basal cells, immature and mature neurons, supporting cells), but signaling molecules will not be confined to the epithelium. In addition to the putative signals from the olfactory bulb, there may be signals from the olfactory nerve ensheathing cells. Furthermore, because the surface density of sensory neuron dendrites is controlled and stable (Mackay-Sim and Kittel, 1991b), it is possible that signals may be present in the mucus, acting as target-derived factors for the dendrites. In that case signaling molecules may arise from the Bowman’s glands and other cells that produce the olfactory mucus. In considering the signaling molecules that regulate the different aspects of olfactory neurogenesis (proliferation, differentiation, survival, death), it is important to be open to the possibilities of multiple factors operating in multiple pathways. For example, when sensory neurons die they may release a factor that stimulates proliferation of the basal precursor cells, but that is not the only possible

Mackay-Sim

regulatory pathway. Another pathway could act via the supporting cells. The supporting cells surround the dendrites of the sensory neurons (Breipohl et al., 1974; Graziadei and Monti Graziadei, 1979) with which they make tight junctions close to the surface (Menco, 1980). Therefore, the supporting cells are in a position to monitor the local density of sensory neurons and release factors to regulate proliferation of basal cells or differentiation of neuronal precursors and immature neurons. Other cells that could be important for the survival and differentiation of the developing neurons are the horizontal basal cell and the olfactory nerve ensheathing cell. The horizontal basal cell wraps around the axons before they leave the epithelium (Holbrook et al., 1995), and the ensheathing cells do so when they enter the lamina propria and guide them to the olfactory bulb (Doucette, 1984; Gong et al., 1994). Either of these cell types could regulate neuronal survival and differentiation. Broadly speaking, there are two types of signals that could regulate olfactory neurogenesis at the local or cellular level: diffusible and fixed. Growth factors are diffusible signals which can have paracrine or autocrine actions. Fixed signals include physical interactions via direct cell surface contacts and indirect contacts through the extracellular matrix. Such signals act via cell surface integrin receptors, and cells can be switched from growth to apoptosis simply by changing their shape (Chen et al., 1997). Extracellular signals have not been extensively investigated in olfactory neurogenesis; much more is known about growth factors. A.

Growth Factors and Receptors Present in Olfactory Epithelium

Growth factors are proteins or peptides found in tissues which exert highly specific effects at very low concentrations. Each growth factor acts through a specific cellsurface receptor or set of receptors, which convey signals via kinases and other second-messenger systems. In the nervous system the first growth factor to be isolated was nerve growth factor (NGF), and its initially defined effect was the promotion of neuron survival (Levi-Montalcini, 1987). “Growth factor” is now a term for increasing numbers of molecules that regulate cell proliferation, cell differentiation, and cell death. Growth factors may have multiple actions on multiple cell types. For example, platelet-derived growth factor (PDGF), named for the cell type it was originally identified in, can act on fibroblasts, smooth muscle, and neuroglia. In other parts of the nervous system it is evident that the function of growth factors and their influence on individual cells can vary with stages of development and the actions of single growth factors can

Neurogenesis in the Adult Olfactory Neuroepithelium

be different in the presence of others. The overall picture of the functions of growth factors is increasingly complex: neurons can require different growth factors at specific stages of development and can require several growth factors simultaneously. A recent review presents a fuller discussion of growth factors and their roles in the olfactory system (Mackay-Sim and Chuah, 2000).

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A large number of growth factors and their receptors have been identified in the olfactory epithelium (Table 2). Although dopamine is not a peptide or protein, nor is it usually classified as a growth factor, it is included here because of its growth factor–like effects in vitro (see below). For many of growth factors the cell types that express them are not identified. The exceptions are ciliary

Table 2 Growth Factors and Receptors in the Olfactory Mucosa Growth factor family

Ligands

Cytokines

CNTF

Dopamine

EGF family

FGF family

GDNF family

IGF family

Neurotrophins

Ref.

Receptors

DA

Buckland and Cunningham, 1999 Lucero and Squires, 1998

D2

TGF

Farbman and Buchholz, 1996

EGFR

NDF

Salehi-Ashtiani and Farbman, 1996

ErbB2

FGF2

GDNF

IGF-I IGF-II

NGF

BDNF

Chuah and Teague, 1999; Goldstein et al., 1997; Hsu et al., 2001

Buckland and Cunningham, 1999; Nosrat et al., 1996; Woodhall et al., 2001

Ayer-LeLievre et al., 1991 Ayer-LeLievre et al., 1991

Ayer-LeLievre et al., 1983; Williams and Rush, 1988; Woodhall et al., 2001 Buckland and Cunningham, 1999; Woodhall et al., 2001

PDGF family

PDGFA

Orr-Urtreger and Lonai, 1992

TGF family

BMP2,4,7

Shou et al., 2000

Ref.

ErbB3 ErbB4 FGFR1

Coronas et al., 1997b; Féron et al., 1999c; Koster et al., 1999 Farbman et al., 1994; Holbrook et al., 1995; Rama Krishna et al., 1996; Salehi-Ashtiani and Farbman, 1996 Salehi-Ashtiani and Farbman, 1996 Perroteau et al., 1998 Perroteau et al., 1998 DeHamer et al., 1994

FGFR1b,c FGFR2 FGFR2b,c FGFR3b,c Ret

Hsu et al., 2001 DeHamer et al., 1994 Hsu et al., 2001 Hsu et al., 2001 Nosrat et al., 1997

GFR1

Nosrat et al., 1997; Woodhall et al., 2001 Woodhall et al., 2001 Pixley et al., 1998 Bondy and Lee, 1993; Federico et al., 1999 Federico et al., 1999 Federico et al., 1999 Bondy and Lee, 1993 Miwa et al.,1998; Roskams et al., 1996

GFR2 IGFR-I IGFBP-2 IGFBP-3 IGFBP-4 IGFBP-5 TrkA

TrkB TrkC PDGFR BMPR-Ib ActR-Ib

Roskams et al., 1996; Woodhall et al., 2001 Roskams et al., 1996 Lee et al., 1990; OrrUrtreger and Lonai, 1992 Zhang et al., 1998 Verscheuren et al., 1995

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neurotrophic factor (CNTF) (basal cells and neurons) (Buckland and Cunningham, 1999), dopamine (mucus) (Lucero and Squires, 1998), dopamine D2 receptor (basal cells and neurons) (Féron et al., 1999c; Koster et al., 1999), epidermal growth factor (EGF) receptor and transforming growth factor alpha (TGF) (horizontal basal cells and supporting cells) (Farbman and Buchholz, 1996; Farbman et al., 1994; Holbrook et al., 1995; Rama Krishna et al., 1996), fibroblast growth factor 2(FGF2) (supporting cells, neurons, basal cells) (Chuah and Teague, 1999; Gall et al., 1994; Goldstein et al., 1997; Hsu et al., 2001; Matsuyama et al., 1992), glial cell line–derived growth factor (GDNF) (neurons) (Buckland and Cunningham, 1999), insulin-like growth factor I (IGF-I) and binding proteins 2–4 (mucus) (Federico et al., 1999), tyrosine kinase A (TrkA) (horizontal basal cells) (Miwa et al., 1998; Roskams et al., 1996), TrkB and TrkC (neurons) (Roskams et al., 1996), nerve growth factor (NGF) (neurons) (Aiba et al., 1993; Roskams et al., 1996; Williams and Rush, 1988), and brain-derived growth factor (BDNF) (horizontal basal cells) (Buckland and Cunningham, 1999). The variety of growth factors and receptors present in the olfactory epithelium and the variation in expression by the different olfactory cell types suggests a rich complexity in the regulation of olfactory neurogenesis. B. Growth Factor Function in Olfactory Epithelium Defining the actions of growth factors can be very difficult. In order to be certain that a specific growth factor functions in olfactory neurogenesis, the growth factor must be shown to be available to the putative target cells, the target cell must be shown to express the appropriate receptors, and the

growth factor must be shown to have a specific action on the target cell. In defining the actions of growth factors, in vitro techniques are used because the simplification of cell and tissue culture allows more variables to be controlled. The causative pathway becomes less obvious as the system increases in complexity. For example, does the growth factor act directly or via a neighboring cell? Even in vitro, under relatively simple conditions, it can be difficult to distinguish between the possible actions of a growth factor. For example, an increase in the number of neurons in a culture (or in the tissue) induced by an added growth factor may be caused by increased proliferation, increased differentiation, increased survival, or a combination of these. Table 3 summarizes the existing data on growth factor function in olfactory neurogenesis. These data derive from culture systems of different complexity with variations in the cell types present and in the culture media. Consequently the conclusions drawn from these studies should be considered as working hypotheses. Nonetheless, converging data suggest distinct and definable roles for TGF, FGF2, and TGF2 in olfactory neurogenesis. Proliferation of the horizontal basal cells is stimulated by EGF and the related TGF (Farbman and Buchholz, 1996; Féron et al., 1999a; Satoh and Takeuchi, 1995). Note that TGF, but not EGF, is expressed in the olfactory epithelium, and their cognate receptor (EGFR) is also present (see above). FGF2 stimulates proliferation of a “stem cell” (DeHamer et al., 1994) and the globose basal cell (Newman et al., 2000). In a globose basal cell–like cell line FGF2 stimulated proliferation and inhibited differentiation (Goldstein et al., 1997). In contrast, in a human olfactory cell line, which also produced FGF2, this growth factor stimulated proliferation and induced differentiation

Table 3 Growth Factors Active in Olfactory Epithelium Growth factor

Cell type

Action

BMP2/4/7 BMP4 Dopamine EGF/TGF

Neuronal precursors Immature neurons Immature neurons Horizontal basal cells

Inhibit proliferation Promotes survival Stimulates differentiation Stimulates proliferation

EGF/TGF FGF2

Supporting cells Globose basal cells/ neuronal precursors Mature neurons Globose basal cells/ neuronal precursors

Stimulates proliferation Stimulates proliferation

PDGF TGF2

Promotes survival Stimulates differentiation

Ref. Shou et al., 1999, 2000 Shou et al., 2000 Féron et al., 1999c Farbman and Buchholz, 1996; Farbman et al., 1994 Farbman and Buchholz, 1996 DeHamer et al., 1994; Newman et al., 2000 Newman et al., 2000 Mahanthappa and Schwarting, 1993; Newman et al., 2000

Neurogenesis in the Adult Olfactory Neuroepithelium

(Ensoli et al., 1998). FGF2 also induced differentiation in explant cultures of mouse and human olfactory epithelium (MacDonald et al., 1996; Murrell et al., 1996), although we now believe this to have been an indirect effect via stimulation of globose basal cell proliferation (Newman et al., 2000). All studies are in agreement that FGF2 stimulates proliferation of a neuronal precursor, both in primary culture and as a cell line; it remains to be proven whether FGF2 also has a differentiating effect. In vivo, appropriate FGF receptor subtypes (FGFR1 and FGFR2) are present and FGF2 immunoreactivity is also present in a number of cell types (Hsu et al., 2001). TGF2 induces differentiation of neuronal precursors (Mahanthappa and Schwarting, 1993), a keratin-positive basal cell line (Satoh and Takeuchi, 1995) and the globose basal cell (Newman et al., 2000). We have identified mRNA for TGF-receptor subtypes I, II, and III in the olfactory epithelium, although the cellular distribution is currently unknown (P. Hsu and A. Mackay-Sim, unpublished). In vivo (Mackay-Sim and Patel, 1984) and in vitro experiments (Mumm et al., 1996) indicated that neurons or immature neurons exert an inhibitory effect on basal cell proliferation. It is possible that this inhibition is mediated via BMPs and their receptors. Recent experiments indicate that the bone morphogenic proteins (BMPs) 2, 4, and 7 can inhibit proliferation of neuronal precursors in vitro (Shou et al., 1999). BMP receptor subtype Ib is present in embryonic olfactory epithelium (Zhang et al., 1998), and we have identified mRNA for BMP receptor subtypes Ia, Ib, and II in adult olfactory epithelium (P. Hsu and A. Mackay-Sim, unpublished). In the embryo, BMPs 2, 4, and 7 are expressed by cells in the lamina propria beneath the olfactory epithelium and noggin, a BMP antagonist, inhibited olfactory neurogenesis in embryonic cultures (Shou et al., 2000). In these cultures low concentrations of BMP4 but not BMP7 promoted survival of newly generated olfactory receptor neurons (Shou et al., 2000). These results suggest both antiproliferative and neuronal survival roles for BMPs, at least during embryogenesis. Their roles in adult olfactory epithelium remain to be defined. Dopamine, although it is not a traditional growth factor, was shown to induce apoptosis and differentiation of an olfactory cell line (Coronas et al., 1997a) and to promote differentiation in explant culture of olfactory epithelium of adult mouse (Féron et al., 1999c). In human explant cultures dopamine inhibited mitosis and induced apoptosis (Féron et al., 1996b). These effects were mediated via the dopamine D2 receptor (Féron et al., 1999c), which has been identified in the neuronal layer of the olfactory epithelium (Féron et al., 1999b; Koster et al., 1999). Dopamine is present in the mucus above the olfactory epithelium (Lucero and Squires,

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1998), and it modulates an inwardly rectifying current in sensory neurons (Vargas and Lucero, 1999) via adenylyl cyclase (Coronas et al., 1999; Mania-Farnell et al., 1993). These observations suggest that dopamine present in the mucus could act as signal to the developing neuron that its dendrite has reached the epithelial surface, thereby triggering cessation of dendritic extension and initiation of cilial growth. Dopamine is also present in the glomerulus, the site of axon termination (Davis and Macrides, 1983; Halasz et al., 1977), and it could act there as a signal to the developing neuron that its axon has reached it target. There is evidently a positive feedback when the axon makes connection with the bulb because dopamine and its synthetic enzyme, tyrosine hydroxylase, are selectively downregulated by chemical destruction of the sensory neurons and upregulated when sensory innervation returns (Baker et al., 1983; Nadi et al., 1981). Even occlusion of the naris can reduce tyrosine hydroxylase and dopamine expression in the olfactory bulb (Baker et al., 1993; Philpot et al., 1998). In vitro experiments indicate that the upregulation of tyrosine hydroxylase by sensory neurons acts via odorant-stimulated glutamate release by the sensory neuron terminals (Puche and Shipley, 1999). A model emerging from all these data is that dopamine may signal that the dendrite and axon have reached their targets and are active. This is then reinforced by odorant-stimulated activity in the dendrite and subsequent synaptic activity in the bulb, leading to dopamine synthesis in the periglomerular cells. In the epithelium the level of dopamine is regulated by activity in the trigeminal nerve (Lucero and Squires, 1998), whose activity is stimulated by odorant stimulation (Cain, 1974; Doty, 1975; Silver and Moulton, 1982). It is possible, therefore, that dopamine may act continually as a trophic factor at both ends of the active sensory neuron. Other growth factors have been implicated in olfactory neurogenesis, although their functions are less well defined. IGF-I is present in human olfactory mucus (Federico et al., 1999), and infusion of IGF-I into the external naris increased the thickness of the olfactory epithelium and increased the number of proliferating cells (Pixley et al., 1998). Of the neurotrophins, BDNF and neurotrophin 3 (NT-3) but not NGF, increased the numbers of immature neurons in primary cultures of olfactory neurons (Holcomb et al., 1995; Liu et al., 1998; Roskams et al., 1996). Given the distribution of the neurotrophin receptors (see above), it is not surprising that sensory neurons were not affected by the presence of NGF. The increased cell numbers may have resulted from the survival-promoting effects of BDNF and NT-3. It is interesting to note that in co-cultures of

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Mackay-Sim

Figure 3 Growth factor regulation of cell dynamics in the olfactory mucosa. Most of these functions have been demonstrated in vitro. The functions of the neurotrophins (NGF, BDNF, and NT3) and IGF-I are inferred from the presence of their receptors on the cell types indicated. A transition from horizontal basal cell to globose basal cell is disputed.

neurons and ensheathing cells, withdrawal of NGF resulted in a dramatic decrease in neuron number (Bakardjiev, 1997). This may have been an indirect effect via loss of the ensheathing cells, which have the low-affinity NGF receptor (see below). Finally, the cytokine leukemia inhibitory factor (LIF) stimulated proliferation of a neuronal precursor population (Satoh and Yoshida, 1997). Because of the importance of olfactory ensheathing cells in the promotion of sensory neuron differentiation (see below), it is interesting to consider the evidence for growth factor regulation of ensheathing cell growth and development. Of the growth factors investigated, the activity of the neuregulins is the most well defined. The neu differentiation factors (NDF -1, -2, and -3) stimulate proliferation of olfactory ensheathing cells and the ensheathing cells express ErbB2 receptors (Pollock et al., 1999). FGF2 also stimulates proliferation of ensheathing cells (Chuah and Teague, 1999). Another neuregulin, glial growth factor 2 (GGF2), is weakly proliferative (Chuah et al., 2000). GGF2 induces differentiation of ensheathing cells and is expressed by them (Chuah et al., 2000), and FGF1 also

stimulates ensheathing cell differentiation (Key et al., 1996). Figure 3 summarizes the actions of various growth factors in the olfactory epithelium and lamina propria. C.

Regulation of Neurogenesis by Ensheathing Cells

The remarkable capacity of the olfactory epithelium to regenerate lies, in part, in the properties of the olfactory nerve ensheathing glia that accompany the sensory axons from the epithelium to the bulb. Olfactory ensheathing glia derive from the olfactory placode (Chuah and Au, 1991; Doucette, 1989; Farbman and Squinto, 1985) and are present in the olfactory nerve and the outer region of the olfactory bulb in the adult (Doucette, 1984; Franceschini and Barnett, 1996; Marin-Padilla and Amieva, 1989; Valverde and Lopez-Mascaraque, 1991). In the adult the morphology of the ensheathing cells appears homogeneous (Doucette, 1991), whereas during development two morphotypes are evident (Cuschieri and Bannister, 1975; Doucette, 1989; Valverde et al., 1993). Two types of ensheathing cells are seen in cultures from embryonic (Kafitz and Greer, 1999) and newborn animals (Pixley, 1992).

Neurogenesis in the Adult Olfactory Neuroepithelium

Olfactory ensheathing glia have properties that are similar both to peripheral Schwann cells and to astroglia of the central nervous system. Although they do not myelinate the olfactory nerve, like Schwann cells, they evidently allow and promote axon growth and can myelinate dorsal root neurites in vitro (Devon and Doucette, 1992). Unlike Schwann cells but like astroglia, they exist in the central nervous system. The olfactory ensheathing glia can be recognized, and distinguished from astrocytes and Schwann cells, by the expression of a combination of proteins. Like Schwann cells they express the calciumbinding protein S-100 (Pixley, 1992) and the p75 lowaffinity neurotrophin receptor (p75NTR Gong et al., 1994; Pixley, 1992; Roskams et al., 1996; Turner and PerezPolo, 1992; Vickland et al., 1991). Like astroglia and nonmyelinating Schwann cells, they express the glial acidic fibrillary protein (GFAP) (Pixley, 1992). There appear to be two types of olfactory ensheathing glia, one of which expresses both GFAP and S-100, the other only GEAP, detected at high antibody dilution (Pixley, 1992). In vitro these two glial types have distinguishable morphologies, the former being spindly and bipolar, and the latter, flatter (Pixley, 1992). Others have defined two types of olfactory ensheathing glia based in their expression of p75NTR and polysialated neural cell adhesion molecule (ENCAM) (Franceschini and Barnett, 1996). During development S-100 immunoreactivity emerges before GFAP immunoreactivity (Astic et al., 1998), but in the adult olfactory nerve and bulb, all four antigens (S-100, GFAP, E-NCAM, and p75NTR) are present (Franceschini and Barnett, 1996). Curiously, in vitro p75NTR-immunoreactive cells were reported to be GFAP-immunoreactive but not S-100–immunoreactive, even though GFAP and S-100 are co-expressed in many cells (Kafitz and Greer, 1999). This is an unusual finding considering that in some cultures virtually all cells appear to express all three antigens (Franceschini and Barnett, 1996; Tisay and Key, 1999). These discrepancies may be explained by the ages of the rats from which the ensheathing cells arise: embryonic day 15 (Kafitz and Greer, 1999), a stage at which S-100 but not GFAP is expressed in vivo (Astic et al., 1998), and postnatal day 7 (Tisay and Key, 1999) and adult (Franceschini and Barnett, 1996), by which time both antigens are expressed in the olfactory nerve (Astic et al., 1998). Culture conditions can also affect the differentiation of ensheathing cells (Franceschini and Barnett, 1996). Olfactory sensory neurons, when given the choice in vitro, preferentially grow on ensheathing glia (Chuah and Au, 1994; Tisay and Key, 1999). The ensheathing glia extend processes around neurites in vitro (Chuah and Au,

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1994) and promote their extension via soluble factors (Kafitz and Greer, 1999) and extracellular matrix (Tisay and Key, 1999). Ensheathing glia promote axonal extension of retinal ganglion cells (Goodman et al., 1993) and can myelinate neurites from the dorsal root ganglion (Devon and Doucette, 1992). These observation indicate that the supportive role of olfactory ensheathing glia is attributable not simply to specific interactions with olfactory sensory axons, but to interactions with growing or regenerating axons in general. This ability is in evidence in their ability to assist the regeneration of dorsal root axons to reenter the dorsal horns (Ramon-Cueto and NietoSampedro, 1994), to assist remyelination in the descending motor axons after nerve crush (Imaizumi et al., 1998) and electrolytic lesions (Li et al., 1997; Li et al., 1998), and, remarkably, to promote spinal regrowth and behavioral recovery after complete spinal transection (Ramon-Cueto et al., 1998, 2000). The regenerative properties of olfactory ensheathing glia probably arise from the variety of growth factors and extracellular matrix molecules that they secrete (Liesi, 1985). In addition to laminin, ensheathing cells express cell-surface antigens and extracellular matrix–associated molecules such as L1, laminin, collagen IV, NCAM, heparan-sulfate proteoglycans, and gliaderived nexin (Chuah and Au, 1992; Doucette, 1990; Liesi, 1985; Miragall and Dermietzel, 1992; Miragall et al., 1988, 1989, 1992; Reinhard et al., 1988; Scotti et al., 1994; Treloar et al., 1996; Whitesides and LaMantia, 1996). Several of these extracellular molecules are important for survival and differentiation of sensory neurons in vitro. Olfactory ensheathing cells are also a rich source of growth factors: NGF, (Woodhall et al., 2001) BDNF (Woodhall et al., 2001), FGF1 (Key et al., 1996), FGF2 (Chuah and Teague, 1999; Gall et al., 1994; Matsuyama et al., 1992), GDNF (Woodhall et al., 2001), and CNTF (Guthrie et al., 1997). This list is not exhaustive and no doubt has more members because several growth factors have been identified in the lamina propria of the olfactory mucosa without identifying the expressing cells (for a recent review, see Mackay-Sim and Chuah, 2000). D.

Summary

Recent research is beginning to flesh out the molecular signals that regulate proliferation, differentiation, survival, and death in olfactory neurogenesis. There are now several growth factors whose roles have been defined in vitro and whose presence and the presence of whose receptors are confirmed in the epithelium. The paracrine and autocrine

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pathways by which these growth factors act are still being established but will include cell-cell communication both within the epithelium and between sensory neurons and the ensheathing cells within the lamina propria, as well as sensory neurons and their synaptic targets in the olfactory bulb. As in other parts of the nervous system, extracellular factors and physical cell surface interactions are also expected to play important roles as well.

VI. CELL LINEAGE IN OLFACTORY NEUROGENESIS The olfactory epithelium is a pseudo-stratified, columnar epithelium containing four cell types: the sensory neuron; the supporting cell, a glial-like cell; the globose basal cell; and the horizontal basal cell. These cells can be identified immunologically. The supporting cell is identified with the antibody SUS1 (Hempstead and Morgan, 1983). Mature olfactory sensory neurons are distinguished from immature neurons by their expression of olfactory marker protein (OMP) (Margolis, 1985). Immature and mature neurons express neuron-specific -tubulin and some isoforms of NCAM, while immature neurons express other isoforms of NCAM, GAP43, as well as -tubulin (Goldstein and Schwob, 1996). All globose basal cells are identified with the antibodies GBC1–3 (Goldstein and Schwob, 1996). The horizontal basal cell expresses keratin and a surface glycoprotein, which binds to the lectin BS-I (Holbrook et al., 1995). The olfactory epithelium contains a stem cell that gives rise to sensory neurons, but is it multipotent? Can it give rise to other cell types? There is evidence that it can. After destruction of the olfactory epithelium with methyl bromide, the epithelium regenerates all cell types including neurons, supporting cells, basal cells, and duct cells of Bowman’s glands (Schwob et al., 1995). Retroviral lineage analysis of the regenerating epithelium indicated that there may be two multipotent progenitors, one of which gave rise to nonneuronal cells only (supporting cells, Bowman’s gland cells and duct cells) and another that gave rise to basal cells, neurons, and supporting cells (Huard et al., 1998). It is generally accepted that new neurons arise in the olfactory epithelium from proliferation and differentiation of basal cells, but the roles of globose versus horizontal basal cells are in dispute. Early in vivo quantitative analysis suggested that the stem cell resides on the basement membrane, in the location of the horizontal basal cell (Mackay-Sim and Kittel, 1991a). In agreement with these conclusions, in vitro experiments also suggested that neurons can arise from horizontal basal cells (Mahanthappa and Schwarting, 1993; Satoh and

Mackay-Sim

Takeuchi, 1995). In contrast, experiments using in vivo retroviral labeling of proliferating cells in undisturbed epithelium suggest that neurons arise only from globose basal cells (Caggiano et al., 1994; Schwob et al., 1994a). There is no direct evidence linking immunological phenotype with a role as progenitor or stem cell. Mackay-Sim and Kittel (Mackay-Sim and Kittel, 1991a) identified the stem cell as an asymmetrically dividing cell, located on the basement membrane, but did not identify these cells immunologically or morphologically as horizontal basal cells. Both Mahanthappa and Schwarting (1993), and Satoh and Takeuchi (1995) used only one basal cell antibody (keratin) and one neuronal antibody (NCAM) to characterize the cells in vitro. They did not account for either globose basal cells or supporting cells in their cultures. In other words, the observed neurons may have arisen from unidentified cells but ascribed to the keratin-positive horizontal basal cells. In the retroviral labeling experiments of undisturbed epithelium (Caggiano et al., 1994; Schwob et al., 1994a), the animals were killed too early to observe the division of a horizontal basal cell with a cell cycle period of about 50 days (Mackay-Sim and Kittel, 1991a). The data from the retroviral lineage study of methyl bromide–treated epithelium also do not allow one to distinguish the direction of lineage relations between the globose and horizontal basal cells, although the authors favor the hypothesis that horizontal basal cells arose from globose basal cells (Huard et al., 1998). There can be no doubt now that the immediate neuronal precursor is a globose basal cell and that the globose basal cells can proliferate (Calof and Chikaraishi, 1989; Goldstein and Schwob, 1996; Graziadei and Monti Graziadei, 1979; Newman et al., 1999). These properties define the globose basal cell at least as a proliferating precursor. They may be a committed precursor population because they reduced in numbers with time as the neuronal population was reconstituted after methyl bromide damage (Huard et al., 1998). Still in doubt is whether the globose basal cell population contains an undifferentiated, uncommitted stem cell (Huard et al., 1998) or whether such a stem cell resides among the horizontal basal cells (Mackay-Sim and Kittel, 1991a).

VII. NEUROGENESIS IN THE ADULT OLFACTORY EPITHELIUM This review has drawn together observations from in vivo and in vitro studies of neurogenesis in the adult. The case is

Neurogenesis in the Adult Olfactory Neuroepithelium

made here that olfactory neurogenesis is a continuing, regulated process, which is similar in many respects to neurogenesis in the embryonic nervous system. Obviously neurogenesis in the adult olfactory epithelium cannot mimic all aspects of neurogenesis in other parts of the nervous system. There are peculiarities of this tissue that define it; for example, olfactory sensory neurons are exposed to the atmosphere, hence they might be more prone to die by external influences than neurons in other parts of the nervous system. Perhaps the evolutionary selection pressure to preserve olfactory neurogenesis into adulthood can be due to a number of interrelated factors. On the one hand, olfaction evolved as essential for survival of the individual through food finding and selection and essential to survival of the species through social, sexual, and predator selection. On the other hand, the transduction mechanism required that sensory neurons be exposed to the odorous air, which could also contain toxic substances, viruses, and bacteria, whose actions could destroy the sensory neurons or pass via the sensory neurons to the brain. Under these conditions there might be selection pressure to evolve labile sensory neurons, which can be replaced through a continued neurogenesis occurring through the reproductive life of the organism. It is noteworthy that although neurogenesis continues throughout adult life, the rate of basal cell proliferation declines in old age in rodents (Weiler and Farbman, 1997) and there is a significant decrease in olfactory function in aging humans (Doty et al., 1984). The regulation of olfactory neurogenesis is still not well understood. There are a large number of candidate factors whose function is unknown but whose presence is inferred by the expression of appropriate receptors in the epithelium (Mackay-Sim and Chuah, 2000). The olfactory epithelium provides a useful model tissue to investigate neurogenesis and has the advantages of relatively large volume and accessibility compared to the embryo. Apart from its intrinsic interest in scientific terms, understanding olfactory neurogenesis is becoming important now for its potential clinical applications in diseases of brain development and in nervous system repair. Differences in olfactory neurogenesis were observed in persons with schizophrenia (Féron et al., 1999b). These differences may lead to a better understanding of the neurodevelopmental origins of the disease. Additionally, it is evident that the olfactory nerve–ensheathing glia are quite different from any others in the nervous system (Doucette, 1990; Ramon-Cueto and Nieto-Sampedro, 1992), and they show great promise in promoting repair and recovery after spinal damage (Lu et al., 2001; Ramon-Cueto et al., 2000). Additionally, when cells from the olfactory mucosa were transplanted into the embryonic brain, some developed into nonolfactory neurons (Magrassi and Graziadei,

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1996), thus raising the possibility that olfactory tissues could be used for transplantation repair of the brain.

ACKNOWLEDGMENTS Alan Mackay-Sim is supported by the Garnett Passe and Rodney Williams Memorial Foundation.

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6 Developmental Anatomy of the Olfactory System Meng Inn Chuah University of Tasmania, Hobart, Australia

James E. Schwob Tufts University School of Medicine, Boston, Massachusetts, U.S.A.

Albert I. Farbman Northwestern University, Evanston, Illinois, U.S.A.

placode had been removed, showed considerable differences in the size of the two hemispheres, the operated side being the smaller.” Thus, Burr (1916) was the first to demonstrate that development of the olfactory bulb was, in some way, dependent on the nerves growing out of the olfactory placode and reaching the forebrain. In this chapter we review current knowledge about the major steps in development of the sensory epithelium and the bulb and describe some of the experimental studies used to examine the developing olfactory system.

The descriptive anatomy of olfactory system development had been done by nineteenth-century anatomists, who had shown that in vertebrates the paired nasal (olfactory) placodes on the anterolateral region of the embryonic head were the precursors of the nasal cavity, which contained the olfactory sensory epithelium. The first successful experimental study on development of the olfactory system was done in 1916 by Burr, who surgically removed one or both placodes from the larval form of Amblystoma, a salamander, and showed that the olfactory bulb(s) on the operated side(s) did not develop. A previous effort to do the same experiment in the frog, Hyla esculenta, had failed because the placode regenerated and development of the bulb was not compromised (Bell, 1907). Burr’s success in his experiments was dependent on the fact that in Amblystoma the olfactory placode was sharply outlined and easily distinguishable from the surrounding epidermis, whereas in the frog, the experimental animal used by Bell, the outline of the placode was difficult to discern. Indeed, Burr showed that partial removal of the placode in the frog permitted regeneration of the placode, as Bell (1907) had reported, but complete removal resulted in failure of the bulb to develop. Burr concluded: “The removal of the nasal epithelium deprives the developing forebrain of a stimulus necessary for its complete development. This is evidenced by the fact that the forebrain of the six months old larva from which one

I. DEVELOPMENT OF THE NASAL CAVITY AND CONCHAE The structures of the human nose develop from the nasal/olfactory placodes, which emerge bilaterally as thickenings of the surface ectoderm on the anterolateral sides of the head at stage 11 (24 days gestation and the formation of 21 somites), i.e., just after closure of the anterior neuropore (Bossy, 1980; Verwoerd and van Oostrum, 1979). Fate mapping has determined that the paired placodes derive from cells located in the anterior neural ridge just lateral to the midline at the neural-fold stage of embryonic development in chicks and mammals (Couly and Le Douarin, 1985; Verwoerd and van Oostrum, 1979), although in fish there may be a different origin (Whitlock 115

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and Westerfield, 1998). Experimental embryological analyses of transplantation experiments indicate that the development of the olfactory placode is induced and/or maintained via the action of two distinct organizing centers, identified as the prechordal plate and the posteriorly adjacent neural plate (Jacobson, 1963). Several molecular candidates have been proposed for inducers/organizers that act to specify the anterior end of the neural plate and anterior neural ridge. The process of anterior neural specification begins with the invagination of the prospective endoemesoderm through the primitive node during gastrulation and the secretion of the neural inducers Noggin, Chordin, Follistatin, and Cerberus. These inducers impart an anterior character to the neuroectoderm if unopposed by posteriorizing factors like FGFs and Wnts that derive from the regressing node (Sasai and De Robertis, 1997). A number of homeobox-containing transcription factors are expressed in the anterior neural ridge at a stage subsequent to gastrulation, including members of the Anf class, the empty spiracles homolog Emx2, Dlx5, BF-1, Vaxl, Pax6, Six3, etc. (Zaraisky et al., 1992; Simeone et al., 1992; Yang et al., 1998; Tao and Lai, 1992; Hallonet et al., 1998; Puschel et al., 1992; Oliver et al., 1995, respectively). Likewise, secreted factors, such as FGF8, and members of signaling cascades, such as the Wnt receptor frizzled7, are also expressed in the anterior neural fold region of the developing nervous system in advance of placodal emergence (Shanmugalingam et al., 2000; Stark et al., 2000). It is important to note that mutation of several genes will disrupt the peripheral olfactory system along with forebrain or other anterior structures, e.g., empty spiracles in Drosphila and Emx2 in mice (Cecchi et al., 1999; Hirth et al., 1995), likewise, Pax-6 (small-eye mouse mutant) (Hill et al., 1991; Hogan et al., 1986), Otx2 (Acampora et al., 1995), and BF-1 (Hatini et al., 1999). However, it is also fair to say that we cannot yet

Chuah et al.

identify with any confidence the molecular pathways that direct that part of the anterior neural ridge to form the olfactory placode. The nature and role of the prechordal plate is somewhat better understood. At stages subsequent to neural fold formation the prechordal plate probably does not participate in induction of placodal tissue and eventually olfactory epithelium, per se. However, it is absolutely critical to the separation of anterior neurectoderm into two placodes (Jacobson, 1963; Macdonald et al., 1995). It is likely that the molecular factor that is responsible for splitting the anterior end of the embryo into symmetrical sides is sonic hedgehog (sHH), whose action is opposed by members of the bone morphogenetic protein (BMP) family (Roelink, 1996). Thus, in the absence of sHH or the overexpression of BMP, the eye and olfactory placode fields, marked by the expression of the paired box transcription factor Pax6, remain continuous across the anterior midline resulting in cylcopia, holoprosencephaly, and proboscis formation, with an area within the proboscis that is recognizable as olfactory epithelium although reduced in size (Chiang et al., 1996; Golden et al., 1999). The appearance of the nasal/olfactory placode is followed by the rapid growth of the mesenchyme around the placode, forming a horseshoe-shaped ridge, the sides of which are called the medial and lateral nasal prominences (Fig. 1). The shallow depression between the lateral and medial prominences is known as the nasal pit. The medial nasal prominences are separated from one another by the frontonasal prominence, which contributes to the formation of the nasal septum. Mesenchymal growth appears to be tightly regulated and may also impact and/or reflect neural development in the periphery. Mutation of zinc finger transcription factors expressed at later stages of nasal development, such as the cubitus interruptus homolog Gli3 (which is truncated in the mouse mutant known as

Figure 1 Frontal and side views of a human embryo head, approximately 33 days old. A horseshoe-shaped rim of mesenchyme surrounds the nasal pit. The lateral part of the rim forms the lateral nasal prominence; the medial part forms the medial nasal prominence. (Adapted from Moore, 1988.)

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Figure 2 Sketches of coronal sections of the human embryonic head from weeks 6 through 12, illustrating development of the palate. (A) The nasal septum becomes established and grows ventrally. (B) The conchae (turbinates) develop as elevations on the lateral walls of the nasal cavity, while the lateral palatine processes extend medially to meet with each other and the nasal septum. (C) Fusion of the lateral palatine processes with one another and with the nasal septum completes the formation of the palate. As a result, the oral cavity is separated from the nasal cavities. (Adapted from Moore, 1988.)

extra-toes), and members of the Dlx family (vertebrate homologs of the fly gene distal-less, a homeobox-containing gene downstream of the homeotic selector genes), affect mesenchymal structures (Hui and Joyner, 1993; Schimmang et al., 1992). Mutation of these genes also disrupts the formation of axonal connections between the epithelium and telencephalon (Johnson, 1967; Qiu et al., 1995). Thus, in extra-toes mutant mice, the epithelium apparently differentiates and grows axons as in normal animals (Sullivan et al., 1995) and even contacts the telencephalon long enough to permit migration of LHRH() cells from the placode to the basal forebrain (S. Wray, personal communication), but the bulb does not form because the contact between the olfactory axons and the telencephalon is not maintained. Whether the disruption is primarily a consequence of excessive growth of branchial arch–derived mesenchymal tissues or an abnormality in the telencephalic target area remains to be determined.

The later stages of nasal and craniofacial development can be disrupted in human populations, resulting, for example, in cleft lip and palate. However, much less is known about the molecular controls on these morphogenetic events. During the later stages, the nasal pits deepen into nasal sacs, which grow dorsocaudally ventral to the developing forebrain. Initially, these sacs are separated from the oral cavity by the oronasal membrane, but this membrane soon ruptures, thus establishing continuity between the nasal and oral cavities. The opening between these two regions is called the posterior choana, and the midline piece of tissue anterior to it is the median palatine process. As the lateral part of the embryonic head grows rapidly, the anterior nasal openings are shifted relatively closer to the midline. A palate forms, which separates the oral and nasal passages (Fig. 2). Lateral palatine processes extend from the maxillary processes and fuse with each other in the midline. Anteriorly they fuse with the median

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palatine process, and superiorly with the nasal septum, resulting in the separation of right and left nasal cavities. Palate formation enables the neonate to feed and breathe at the same time (Moore, 1988). While these events are occurring, the superior, middle, and inferior turbinates (conchae) develop as elevations on the lateral wall of each nasal cavity in the human embryo. They are supported by a cartilaginous framework, which is gradually replaced by bone. In macrosmatic mammals, i.e., those that have a relatively powerful sense of smell as compared with the microsmatic human, several elaborately scrolled turbinates form and become more complex as the snout grows in postnatal life, thus dramatically expanding the surface area of the olfactory epithelium. For example, in the rat there is an eightfold increase in the olfactory area during the first postnatal month (Meisami, 1989). In contrast, olfactory epithelium of the human is restricted to the roof of the nasal cavity, the adjacent superior part of the nasal septum, and the superior concha.

II.

DIFFERENTIATION OF OLFACTORY EPITHELIAL CELLS

In the adult, the olfactory epithelium is a pseudostratified columnar epithelium overlying a lamina propria. It is composed of five basic cell types, distinguishable on morphological and biochemical grounds, which are arrayed in stereotyped layers in the epithelium (Fig. 3). From the apical surface deep, they are the sustentacular cells (of which the microvillar cells are a variant), olfactory sensory neurons (mature ones positioned superficial to immature ones), globose basal cells, and horizontal basal cells (which are tightly apposed to the basal lamina) (Carr et al., 1991; Graziadei and Monti Graziadei, 1978, 1979; Holbrook et al., 1995; Schwartz Levey et al., 1991). The supporting cell nuclei are arranged in a single layer near the surface, the several layers of olfactory neuron nuclei are deep to it, and the basal cell nuclei are closely related to the basal lamina (Fig. 3). The olfactory neurons make up 80–85% of the epithelial cells, the supporting cells 12–15%, and the basal cells about 5%, although those proportions vary depending on location (neurons being densest in the posterior dorsal part of the olfactory epithelium) (Farbman et al., 1988; Youngentob et al., 1997). The fifth element is the Bowman’s gland/duct complex that extends from the glands in the lamina propria to the ducts within the epithelium, which carry the secretions to the apical epithelial surface. All of these elements derive from the nasal/olfactory placode, as does the respiratory epithelium lining more rostral and ventral portions of the nasal cavity.

Figure 3 Histological section through olfactory mucosa of a newborn rat. The supporting cell nuclei stain slightly darker than the others and are arranged in a single layer nearest to the surface of the epithelium (arrows). The basal cell nuclei are immediately above the basal lamina and appear indistinct in this specimen. The sensory cell nuclei are located between the supporting cell nuclei and the basal cell nuclei. Nerve bundles (NB) consisting of olfactory axons can be found in the lamina propria. Bar
25 m.

A.

Cell Division

The tissue lining the nasal pit is also a pseudostratified columnar epithelium. In human and other mammalian embryos, a dramatic increase of mitotic figures is observed in the olfactory epithelium immediately before the first emergence of olfactory axons at stage 13 (28 days) (Bossy, 1980; Cuschieri and Bannister, 1975a; Smart, 1971). At this early stage, mitosis in the olfactory epithelium is similar to the process of cell division in the developing neural tube. Thus, epithelial cells undergo interkinetic nuclear migration (Sauer, 1937): G1- and S-phases take place in deeper parts of the epithelium and the cells migrate superficially to complete mitosis at the apical epithelial surface (homologous to the ventricular surface of the neural tube) (Cuschieri and Bannister, 1975a; Smart, 1971). The mitotic pattern changes in older fetuses when the basal cell layer is established; there is a progressive shift of mitotic activity to the base of the epithelium (Smart, 1971). The precise relationship between the two spatially distinct precursor populations has not been clarified, but it may be amenable to better understanding given the recent isolation of markers that label basal cells in the postnatal epithelium (Goldstein and Schwob, 1996; Goldstein et al., 1997). Nonetheless, neurons are being generated even at the times when mitoses are concentrated apically. The dividing basal cells in the older embryo are predominantly of the globose

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variety, as in the adult. Indeed, the horizontal basal cell population emerges somewhat later in development after the translocation of the proliferating population basalward (Holbrook et al., 1995). Genesis of olfactory neurons occurs continuously throughout the life of the animal (Graziadei and Monti Graziadei, 1978), promoting anatomical and functional recovery after injury either to the epithelium or the olfactory nerve (Costanzo, 1991). We have a better understanding of the process of neurogenesis during postnatal life than during embryonic development (Chapter 5). A variety of experimental approaches (including pulse-chase studies with 3H-thymidine in vivo and in vitro and application of retroviral or other markers for tracing lineage in vivo) have indicated that the immediate neuronal precursor cell, i.e., the dividing cell whose daughter(s) differentiate into neurons, resides among the population of globose basal cells (Caggiano et al., 1994; Calof and Chikaraishi, 1989; Graziadei and Monti Grazeidei, 1979; Schwartz Levey et al., 1991; Schwob et al., 1994). The distribution and density (number per unit length of epithelium) of proliferating globose basal cells declines dramatically with increasing age of the animal (Weiler and Farbman, 1997). In younger animals (up to about 40 days postnatal) proliferating neuronal precursors are evenly distributed in the basal epithelium (Weiler and Farbman, 1997), whereas in older animals they are unevenly distributed and form roughly circular patches that are distributed across the tangential extent of the adult epithelium (Graziadei and Monti Graziadei, 1979; Loo et al., 1996; Weiler and Farbman, 1997). The surrounding areas are populated with fewer globose basal cells and are described as quiescent, containing mainly mature olfactory neurons. Although it has been suggested that horizontal basal cells serve as stem cells that support ongoing neurogenesis, direct evidence for that notion is lacking (Graziadei and Monti Graziadei, 1979; Holbrook et al., 1995; Mackay-Sim and Kittel, 1991). The molecular regulation of basal cell division remains largely unexplored. Analysis of explants of embryonic/neonatal olfactory epithelium or epitheliumderived cell lines provides a few clues. FGF2 has modest effects on the proliferation of neuronal precursors in vitro (Calof and Chikaraishi, 1989; DeHamer et al., 1994) and of olfactory-derived cell lines (Goldstein et al., 1997; Vawter et al., 1996). TGF- also stimulates basal cell proliferation in explants and cell lines; in particular, it is a potent stimulus for proliferation of the horizontal basal cells, which express the EGF receptor (Farbman and Buchholz, 1996; Getchell et al., 2000; Mahanthappa and Schwarting, 1993). Some of the analysis on cell lines suggests that FGF-2 suspends neuronal differentiation, holding the cells in a more globose basal cell-like pheno-

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type (Goldstein et al., 1996), although other investigators have suggested an alternative role for FGF-2 (Mackay-Sim and Chuah, 2000). In muscle development, FGF-2 has the effect of blocking myocyte differentiation, which is more analogous to the effect on basal cell differentiation noted by Schwob’s lab (Goldstein et al., 1996). B.

Cellular Differentiation

The most detailed studies on the ontogeny of the various cell types of the olfactory epithelium have been done in rat and mouse fetuses. The first cells to differentiate are neurons, ca. E10.25 in mice and E12 in rats, which is shortly after the placode begins to invaginate (Cau et al., 2000; Cuschieri and Bannister, 1975a,b). (The terminology used here indicates embryonic development in terms of gestational age; in rodents conception is assumed to occur at midnight preceding the morning when the dam is found to be sperm-positive.) Slightly later there is a major transition in the appearance of the epithelium, which is coincident with the shift of mitotic profiles from the apical epithelium into the basal compartment. At this time the characteristic distribution of the three major cell types––the lamination of supporting cells, olfactory neurons, and basal cells in the olfactory epithelium into relatively distinct, progressively deeper epithelial zones––emerges, and the apical aspects of the neurons and supporting cells begin to elaborate the adult-like complement of dendrites and microvilli (Farbman, 1991; Menco and Farbman, 1985a). 1.

Olfactory Neurons

In olfactory neuron development, genesis of the axon precedes differentiation of the dendritic process (Cuschieri and Bannister, 1975a,b; Farbman and Squinto, 1985). Each sensory cell body gives rise to a small-diameter axon, which fasciculates with other axons, and the bundles project into the lamina propria, at E12 in rats (Farbman and Squinto, 1985) and at E10.5 in mice (Cuschieri and Bannister, 1975a,b). These bundles are the fila olfactoria of the olfactory nerve (Fig. 4), and they are surrounded by ensheathing cells (see Sec. II. C). The fibers from each nasal cavity can be roughly divided into two projections: a medial projection composed of fibers from the nasal septum and a lateral one from the turbinates. The olfactory nerve fibers reach the presumptive olfactory bulb roughly one day later, and morphologically distinct synapses are demonstrable 2–3 days after that (Farbman, 1986; Gong and Shipley, 1995; Hinds, 1972a,b). In the human embryo, axonogenesis also takes place relatively early in gestation. At stage 15 (33 days gestation), olfactory axon bundles are seen in the lamina propria (Pyatkina, 1982); they reach the

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Figure 4 Specimen from 15-day embryonic rat in which the nasal pits containing olfactory placode cells (OP) were impregnated with a fluorescent dye that traces the olfactory nerves (arrows) projecting medially and laterally towards the olfactory bulb. Bar
50 m.

forebrain in the region where the olfactory bulb forms within a week (Bossy, 1980). The formation of dendrites marks another stage of olfactory neuron development. At the time that neurons first appear, the primitive olfactory dendritic processes terminate as cytoplasmic expansions at the surface of the epithelium and contain several centrioles, which have migrated from the perikaryon (Menco and Farbman, 1985a; Mulvaney and Heist, 1971). The cells go through a stage when they elaborate a primary cilium. Eventually, the centrioles become basal bodies and give rise to multiple cilia (Fig. 5), which are first seen at E15 to E16. Cilia increase in number and length until about the second or

Figure 5 An electron micrograph of rat olfactory epithelium showing dendritic knobs (D) of olfactory sensory neurons. Arrows indicate cross sections of cilia. Bar
0.5 m.

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third postnatal week, when they reach the adult value of an average of 11 per knob in the rat (Menco and Farbman, 1985b). About this time, freeze-fracture electron microscopy reveals also that increasing numbers of intramembranous particles are inserted into the ciliary membrane (Menco, 1988). In human embryos, ciliogenesis begins at the ninth week of gestation, and a dramatic increase in ciliary number occurs in the subsequent 2 weeks (Pyatkina, 1982). When maturation is complete, it is estimated that the number of cilia on each dendritic knob is between 10 and 50 (Chuah and Zheng, 1992; Ohno et al., 1981). During ciliogenesis the perikaryon of the human olfactory neuron also undergoes differentiation. The rough endoplasmic reticulum in the perinuclear region becomes more elaborate, and free ribosomes are organized increasingly into polyribosomes (Pyatkina, 1982). In vitro studies show that although ciliogenesis can occur to a limited extent in the absence of the bulb (Chuah et al., 1985; Farbman, 1977), it is enhanced in the presence of the presumptive olfactory bulb. This suggests that the final maturation of olfactory neurons may be regulated by the establishment of contact with its target tissue (Chuah et al., 1985). However, no cause-and-effect relationship between these two events has been established definitively, as discussed more thoroughly below. Indeed, the evidence in the adult rat suggests that the relative lack of mature neurons in the absence of the bulb can be explained by the premature death of the neurons that occurs in the absence of the trophic influence exerted by the bulb on sensory neuron survival (Schwob et al., 1992). In humans, ciliogenesis lags genesis of axons significantly; olfactory axons in humans reach their target tissue 3 weeks before the first olfactory neurons begin to sprout cilia (Bossy, 1980; Pyatkina, 1982). Roughly coincident with the elaboration of cilia is the expression of the elements that constitute the signal transduction cascade for olfactory stimuli. Members of the odorant receptor (OR) gene family are expressed by rare neurons ca. E11.5 in mice (Sullivan et al., 1995), lagging slightly the morphological emergence of neurons at the nasal pit stage (E10.5). Probe-positive cells become more abundant at the time of the aforementioned morphological transition in the lamination of proliferating cells, the differentiation of the surface of epithelium, and ciliogenesis (E12.5) (Royal and Key, 1999; Sullivan et al., 1995). Similar timing is observed in rats (Saito et al., 1998; Strotmann et al., 1995). Initial contact of olfactory axons with the olfactory bulb occurs about the time that odorant receptors are first expressed (Hinds, 1972a,b; Gong and Shipley, 1995). As a consequence, it is unlikely that a retrograde signal from the bulb to the epithelium is responsible for eliciting the pattern of receptor expression. Indeed, receptor genes are expressed with a spatial distrib-

Developmental Anatomy of the Olfactory System

ution that is indistinguishable from normal in the extratoes mouse mutant in which the olfactory bulb does not develop normally due to truncation of Gli3, a zinc finger protein that participates in the sHH signal transduction cascade (also described above). G-proteins, which are involved in transduction of the olfactory stimulus, are expressed in cilia shortly after they are formed (ManiaFarnell and Farbman, 1990; Sullivan et al., 1995). Finally, a newly cloned member of the NCAM family of Ig cell adhesion molecules termed OCAM or mamFas II (for the mammalian homolog of Fasciclin II) is expressed around the same time as the first appearance of odorant receptors (Yoshihara et al., 1997). OCAM/mamFas II is differentially expressed according to zone of origin in the olfactory epithelium, i.e., there are high levels on axons of neurons derived from ventrolateral epithelium (Zone 2 and higher) and very low levels on axons from dorsomedial epithelium (Zone 1) in developing and adult olfactory system (Mori et al., 1985; Schwob, 1992; Schwob and Gottlieb, 1986, 1988; Yoshihara et al., 1997). Like ORs, differential OCAM expression is maintained independent of the bulb in both settings. Additional components of the signal transduction cascade and the multitude of adhesion and matrix molecules that accompany genesis and growth of axons are detailed further below. At a molecular level, we know little about the events that accompany the differentiation of neurons from the daughters of immediate neuronal precursors. As for other neuronal precursors and their neuronal descendants, members of the basic helix-loop-helix (bHLH) family of transcription factors are expressed in the olfactory epithelium and seem to participate in the process of olfactory neuronal differentiation. Mash1 and its cognate protein MASH1, a mammalian homolog of the Drosophila proneural gene, Achaete-Scute, is expressed by basal cells that seem to function as transit amplifying cells, i.e., cells that proliferate to expand the population of precursors and do not give rise to neuronal daughters directly, yet are committed to neuronal differentiation (Gordon et al., 1995; Guillemot et al., 1993). In keeping with that role, elimination of Mash1 by homologous recombination produces an epithelium that is largely depleted of neurons by birth (Guillemot et al., 1993). Interestingly, some differentiating neurons are observed at the nasal placode/pit stages in Mash1 mutant mice before the onset of massive cell death eliminates the neuronal population (Cau et al., 1997), suggesting that not all neurons are equivalent in the early placode. Indeed, a distinct population of pioneer neurons is responsible for establishing the olfactory nerve in zebrafish, which then disappear (Whitlock and Westerfield, 1998). Several other bHLH neuronal differentiation genes are expressed in the embryonic epithelium.

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The timing of their expression allows them to be assigned to a particular stage in the process of neuronal differentiation. Thus, neurogenin1/Math4C is expressed by precursors that are downstream of the Mash1-expressing transit amplifying cells, and NeuroD is seen in the differentiating neurons of the olfactory epithelium (Cau et al., 1997). Still other members of the bHLH superfamily, Hes1 and Hes5, vertebrate homologs of the Hairy-enhancer of Split family in Drosophila, seem to operate to hold in check the elaboration of Mash1-expressing precursors (Hes1) and, later on, the differentiation of immature neurons (both Hes1 and Hes5 acting together) (Cau et al., 2000). The regulation of early neuronal differentiation is little understood beyond these few items. Cell lines and neurons in explants can be pushed to differentiate by application with members of the TGF- superfamily, either alone or in combination with other growth factors and cytokines (Mahanthappa and Schwarting, 1989; Vawter et al., 1996).

2.

Supporting Cells

Supporting cells are easily distinguished ultrastructurally from olfactory neurons in the early human embryo, but the literature on their differentiation is scarce. In the 7week-old human embryo they are cylindrical in shape, with numerous microvilli projecting from the domeshaped apical surface (Pyatkina, 1982). A layer of glycocalyx is deposited on the microvilli. The perikaryon characteristically contains short profiles of rough endoplasmic reticulum, abundant microfilaments, and a large number of glycogen granules. By the ninth week of gestation in humans, a morphologically different supporting cell appears. This cell is considerably narrower, lacks glycogen granules, and possesses bundles of filaments along its longitudinal axis. However, it resembles the predominant type of supporting cell by the presence of vesicular inclusions in the perinuclear cytoplasm. It is likely that this second type of supporting cell is actually a morphologically differentiated state of the first because ultrastructural and immunohistochemical studies show that the supporting cells in the adult human contain large bundles of filaments, particularly in the lower two thirds of the cell (Graziadei and Monti Graziadei, 1979; Holbrook et al., 1995; Moran et al., 1982a,b). Maturation of supporting cells in the perinatal period is marked by a decrease in the electron density of the cytoplasm and a conspicuous increase in the amounts of smooth and rough endoplasmic reticulum. A nonneuronal microvillar cell has been identified in rat olfactory epithelium on the basis of its immunoreactivity with a specific monoclonal antibody, 1A-6 (Carr et al.,

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1991). This microvillar cell type is believed to be nonneural because (1) it has no identifiable axonal process, (2) it is not reactive with an antibody against olfactory marker protein, and (3) it survives ablation of the olfactory bulb. These cells fail to react with a supporting cell-specific monoclonal antibody (Hempstead and Morgan, 1983) and consequently are thought to be different from ordinary supporting cells. It is not clear whether this cell type is the same as that thought to be a microvillar sensory neuron (Moran et al., 1982b). However the latter investigators showed no convincing experimental evidence that the microvillar cell they described had an axon and was connected to the olfactory bulb. 3. Horizontal Basal Cells As early as the ninth week of gestation in humans, two types of basal cells are recognizable in the human olfactory epithelium: one with electron-dense cytoplasm, corresponding to the horizontal basal cells, the other with lighter cytoplasm, corresponding to the globose basal cells (Pyatkina, 1982). A similar delay in the emergence of phenotypically distinct horizontal basal cells relative to the differentiation of neurons or sustentacular cells is observed in rodent epithelium as well (Holbrook et al., 1995). C.

Figure 6 A photomicrograph showing a group of cells (arrows) migrating out of the fetal rat olfactory epithelium. These cells accompany the growing olfactory axons as they project toward the olfactory bulb. Bar
20 m.

ble of giving rise to typical olfactory neurons also migrate out of the epithelium along the fascicles of the olfactory nerve during embryonic development as well as after injury in the adult (Monti Graziadei, 1992; Schwob et al, 1995). Hence, the cells of the original olfactory placode give rise not only to intrinsic epithelial cells, but also to those that eventually function at distant sites.

Extraepithelial Cell Migration

During ontogeny, groups of cells migrate out of the epithelium before the outgrowth of the first axons from the olfactory epithelium (Fig. 6) (e.g., Bossy, 1980; Farbman and Squinto, 1985; Mendoza et al., 1982; Schwanzel-Fukuda and Pfaff, 1989; Wray et al., 1989). Recent evidence indicates that these cells are functionally heterogeneous. A large mass of cells derived from the epithelium occupies the region between the epithelium and the telencephalon in advance of contact with the telencephalon. The cell mass may be serving as an intermediate target for the olfactory nerve, given the sharp bend that the fibers take on contacting the mass (Drapkin and Silverman, 1999; Gong and Shipley, 1995). Some of the cells migrating from the medial side of the olfactory epithelium contain luteinizing hormone-releasing hormone (LHRH); some LHRH-positive cells eventually reside in the hypothalamus (Schwanzel-Fukuda and Pfaff, 1989), whereas others become ganglion cells of the terminal nerve (Schwanzel-Fukuda and Silverman, 1980). Still other migrating cells become the ensheathing cells of the olfactory nerve (Fig. 7) (Chuah and Au, 1991a) and of the nerve bundles in the outermost layer of the bulb (Doucette, 1989). Finally, precursor cells capa-

Figure 7 A photomicrograph of ensheathing cells that had been isolated from the olfactory nerve layer of the newborn rat olfactory bulb and grown in culture. Most of the ensheathing cells are spindle-like and bipolar in shape, but some have a few processes. Bar
20 m.

Developmental Anatomy of the Olfactory System

III. BIOCHEMICAL AND FUNCTIONAL ASPECTS OF DIFFERENTIATION Immunochemical and biochemical methods have also been used to assess maturation of olfactory neurons. In the E12 rat embryo, adenosine deaminase immunoreactivity can be demonstrated in the olfactory epithelium cells (Senba et al., 1987). With the first axon growth at E13, certain membrane-related antigens are expressed on the olfactory neurons. A monoclonal antibody that binds N-CAM–like moieties, Neu-5, is reactive with rat olfactory axons at E13 and with the perikarya the next day (Carr et al., 1989). Members of the N-CAM family are known to mediate neuron-neuron adhesion in vivo and neurite growth and fasciculation in vitro (for review, see Rutishauser and Jessell, 1988). In the mouse, N-CAM expression in the olfactory placode is detected at E9, while OCAM/mamFasII, a recently isolated member of the N-CAM family, is associated with subsets of olfactory axons at E13 (Yoshihara et al., 1997). Similarly early onset was noted in rats in vivo (Schwob, 1992). Another cell adhesion molecule, L1, appears at E11 (Miragall et al., 1989). In the mouse, L1 is thought to be involved in interactions between neurons and extracellular matrix, neuroglia, and other neurons (see Persohn and Schachner, 1987; Seilheimer and Schachner, 1988). More recently, it has been shown that growing olfactory axons express retinoic acid–binding protein (CRABP I) as early as the 12th day of gestation in the mouse (Gustafson et al., 1999). It is uncertain when N-CAM is first synthesized in the human olfactory system, although it is present as early as the 17th week of gestation. At this time, N-cadherin, another cell adhesion molecule, is also demonstrable in the olfactory epithelium (Chuah and Au, 1991b). The expression of these membrane-related molecules at a period of extensive olfactory nerve growth suggests that these molecules are involved in modulating axonal growth. Interestingly, ensheathing cells that accompany the growing axons also express N-CAM and L1 (Gong and Shipley, 1996; Miragall et al., 1989). Several lines of evidence from morphological, behavioral, and electrophysiological studies indicate that the mammalian olfactory system may be functional before birth (Cuschieri and Bannister, 1975a,b; Hinds and Hinds, 1976; Gesteland et al., 1982; Stickrod et al., 1982). Given the early onset of receptor expression that has been documented in the rat and mouse (as described above), it is not surprising that at E14 rat olfactory neurons begin to exhibit odorant-induced electrical activity (Gesteland et al., 1982). At this stage, molecules involved in olfactory signal transduction, such as olfactory cyclic nucleotidegated channel subunit 1 (OcNC1) and Gb, can be demonstrated immunohistochemically in select populations of

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olfactory neurons (Matsuzaki et al., 1999; Saito et al., 1998). Two days later, at E16, the first action potential in single olfactory neurons can be recorded. At E18 to E19, when synaptogenesis between olfactory axons and mitral dendrites is first observed, an increasing number of olfactory neurons become responsive only to specific types of stimuli, suggesting that they can discriminate among odorants (Gesteland et al., 1982). Interestingly, it is also at E18 that distinct immunoreactivity for OcNCI at the level of the cilia can be demonstrated ultrastructurally (Matsuzaki et al., 1999). Consistent with the notion of a functioning fetal olfactory system, behavioral studies have shown that fetal rats at 20 days of gestation are sensitive to odor molecules dissolved in amniotic fluid and are able to undergo odor aversion conditioning (Stickrod et al., 1982). The time of onset of the electro-olfactogram (EOG) in the rat at E14 coincides with the first expression of the olfactory marker protein (OMP) in the olfactory neurons (Allen and Akeson, 1985a). Olfactory marker protein is a cytosolic, acidic protein, molecular weight about 18 KDa (Margolis, 1972; Margolis, 1982), found throughout the olfactory neuron, from the dendritic knob to the axon terminal (e.g., Farbman and Margolis, 1980; Monti Graziadei et al., 1980). OMP is widely accepted as a marker for mature olfactory neurons (Farbman and Margolis, 1980), although its function has not been clearly defined. Studies with OMP-null mice suggest that OMP may play a modulatory role in odor detection or olfactory signal transduction (Buiakova et al., 1996) and /or in neurogenesis (Carr et al., 1998). The appearance of OMP in humans is first apparent at about 24 weeks postconception, i.e., about 1 month after synaptogenesis has occurred (Chuah and Zheng, 1987; Johnson et al., 1995). In addition to OMP, blood group antigens have been detected by immunostaining in rat olfactory neurons (Astic et al., 1989). H antigen immunoreactivity appears on E14 in the cell body, dendrite, and axon terminal, and the B antigen is detectable on E16 in some of the cells expressing the H antigen. Blood group antigens are normally found in low amounts on a very small number of CNS or PNS neurons; the significance of the presence in olfactory neurons is not known. Another cell surface glycoprotein expressed on olfactory neurons is recognized by a monoclonal antibody referred to as 2B-8 (Allen and Akeson, 1985b). In adult rats, about 25% of OMP-positive olfactory neurons are immunoreactive with 2B-8. The relationship between this antigen and early differentiation is yet to be elucidated. Carnosine synthetase activity has been detected as early as E16 in the embryonic rat (Margolis et al., 1985). This enzyme is involved in the synthesis of the dipeptide carnosine (β-alanylhistidine), a major constituent of mature

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olfactory cells and their terminals in the olfactory bulb (Margolis, 1980). The biological function of carnosine remains unknown, although a neurotransmitter role has been hypothesized (Margolis, 1980). Biochemical and neurochemical data are consistent with this idea (Hirsch and Margolis, 1979; Margolis, 1974, 1980; Margolis et al., 1979), but electrophysiological studies have produced conflicting results (Frosch and Dichter, 1984; GonzalesEstrada and Freeman, 1980; Macleod and Straughan, 1979; Tonosaki and Shibuya, 1979). During ontogeny, carnosine can be demonstrated immunohistochemically in rat olfactory neurons at E17 (Biffo et al., 1992). Carnosinelike immunoreactivity is also present in human olfactory neurons (Sakai et al., 1990). One marker that olfactory neurons have in common with mature neurons in other systems is neuron-specific enolase (NSE). Following axotomy of olfactory nerves in the guinea pig, mature neurons die and within 7 days are replaced by newly differentiating neurons which show immunoreactivity for NSE (Yamagishi et al., 1989). In the human embryo, NSE is already present at the end of the first trimester (Takahashi et al., 1984). More complicated is the question of which type IV intermediate filament proteins are expressed by olfactory neurons. The predominant intermediate filament proteins expressed in the olfactory axons in the rodent are vimentin and peripherin (Escurat et al., 1990; Gorham et al., 1991; Schwob et al., 1986). Vimentin is also seen in the olfactory nerve fibers of other species (Ophir and Lancet, 1988). Different research groups have produced conflicting reports of whether neurofilaments are expressed in olfactory neurons (Bruch and Carr, 1991; Ophir and Lancet, 1988; Schwob et al., 1986; Takahashi et al., 1984; Talamo et al., 1989; Vollrath et al., 1985; Yamagishi et al., 1989). The evaluations have all been immunohistochemical and have employed a number of different polyclonal and monoclonal antibodies. Vollrath and coworkers (1985) failed to demonstrate any neurofilament staining in rat olfactory epithelium, whereas Schwob and colleagues (1986), using several affinitypurified polyclonal antisera, demonstrated that expression of neurofilament proteins is limited to a small subpopulation of neurons in the lateral olfactory epithelium and their axons projecting via the lateral side of the olfactory nerve layer of the bulb. In contrast, Bruch and Carr (1991) found that prominent immunostaining for the 200 kDa neurofilament was present in the cell bodies of rodent olfactory neurons. In humans, the 145 kDa neurofilament is demonstrable histologically in the olfactory neuron cell bodies at the 16th week of gestation (Takahashi et al., 1984). In the adult this molecule is located only in the axons

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(Talamo et al., 1989). The reasons for the shift in location of this neurofilament subunit are unclear. The 73 and 200 kDa neurofilament subunits are not detectable in human olfactory tissue. However, the 200 kDa subunit has been convincingly demonstrated in cell lines derived from the human olfactory epithelium (Vawter et al., 1996).

IV. EXTRINSIC INFLUENCE ON OLFACTORY NEURON MATURATION A.

Influence from the Olfactory Bulb—Maturation and Survival

In many parts of the nervous system, the target organ of the growing axon has a major effect on the survival or maturation of the neurons. For example, embryonic motoneurons are unable to survive beyond a certain period without connections to muscle cells (e.g., Oppenheim et al., 1978). Similarly, neurons of the ciliary ganglion are dependent upon their target tissue for survival (Pilar and Landmesser, 1976). Superior cervical ganglia grown with target salivary glands show greater elaboration and directionality of nerve fiber outgrowth than control explants (Coughlin et al., 1978). In 1975 Cuschieri and Bannister hypothesized that the olfactory bulb had an influence over the maturation of olfactory neurons because ciliogenesis was complete only after the axons had reached their target. Support for this hypothesis comes from results of organ culture experiments and degeneration/reconstitution experiments. When olfactory mucosa is explanted alone, the olfactory neurons differentiate to some extent but fail to reach full maturation. The differentiating olfactory neurons grow axons and express some cilia on the dendritic knobs; EOGs can be recorded from these cells, and some of them synthesize OMP (Chuah and Farbman, 1983; Chuah et al., 1985; Farbman, 1977; Farbman and Gesteland, 1975). When the olfactory mucosa is cocultured with the bulb, twice as many olfactory neurons contain OMP, and the number of cells with ciliated dendritic knobs also increases twofold (Chuah and Farbman, 1983; Chuah et al., 1985). This enhancing influence appears to be mediated by interaction between the bulb and olfactory mucosa; however, the target tissue does not appear to control directly the trajectory of the olfactory axons (Gonzales et al., 1985). After unilateral ablation of the olfactory bulb in mammals, the olfactory neurons undergo degeneration and new neurons are generated from globose basal cells to repopulate the olfactory epithelium. However, reconstitution of

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the epithelium is incomplete as it does not usually reach its preoperative thickness (Costanzo and Graziadei, 1983). The number of mature olfactory neurons, as evidenced by the presence of OMP, is greatly reduced. On the other hand, the number of immature elements is greatly increased compared to the control side (Monti Graziadei, 1983; Monti Graziadei and Graziadei, 1992; Schwob et al., 1992; Verhaagen et al., 1990). Interestingly, a recent study has shown that the remaining OMP-positive neurons present after bulbectomy demonstrate a significant elevation in their OMP level (Carr et al., 1998). A possible explanation for the failure of the epithelium to be fully reconstituted is that the sensory neurons are trophically dependent on the bulb for their prolonged survival (Schwob et al., 1992; Weiler and Farbman, 1999). In the absence of the bulb, neurons are produced at a twofold greater rate than in a control situation (Carr and Farbman, 1992; Schwob et al., 1992), but nearly 90% of the neurons on the operated side die before they reach the age of 2 weeks (Schwob et al., 1992). That is shorter by far than any determinations of life span in the normal epithelium (Graziadei and Monti Graziadei, 1978; Hinds et al., 1984; Mackay-Sim and Kittel, 1991). In other words, the data suggest that if an olfactory sensory neuron does not receive a required trophic factor from the bulb at a critical stage in development, it is very likely to die before reaching full maturity. Consequently, most of the cells in the reconstituted epithelium are relatively immature. These data have led to the suggestion that olfactory neurons are trophically dependent on the bulb not only for maturation as discussed above (Chuah and Farbman, 1983; Chuah et al., 1985), but also for their survival (Schwob et al., 1992). Indeed, it is difficult to separate a direct effect on maturation per se from a failure to mature due to abbreviated neuronal survival. A recent study has in fact shown that mitral cells, the major postsynaptic target, are crucial in maintaining the survival of olfactory neurons (Weiler and Farbman, 1999). It was found that depletion of mitral cells resulting from transection of their axons in the lateral olfactory tract led to increased numbers of proliferating neurons in the olfactory epithelium, presumably related to and resulting from an increased level of cell death. B.

Regulation of Olfactory Axonal Growth

In the process of attaining full functional maturity, olfactory neurons need to extend axons from the epithelium to the olfactory bulb and to make accurate contact with the appropriate dendrites in specific glomeruli. This type of precise target recognition is probably regulated by a series of distinct guidance cues, arranged in a hierarchical manner (Lin and Ngai, 1999).

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In the early stages of axon growth during ontogeny or reconstitution of the olfactory epithelium, axons are enveloped by cytoplasmic processes of ensheathing cells. Doucette (1990) has suggested that the ensheathing cells guide the olfactory axons to their target and that the guidance is probably modulated by cell adhesion molecules, extracellular matrix molecules, and chemotropic substances. This notion is supported by the positive immunohistochemical staining for the neuronal cell adhesion molecules, N-CAMs and L1, on olfactory neurons, ensheathing cells, and in the mesenchyme surrounding the developing olfactory pathway in rodents (Gong and Shipley, 1996; Miragall et al., 1988, 1989; Whitesides and LaMantia, 1996). The use of antibodies to adhesion molecules on cultures of olfactory neurons growing on astrocyte monolayers has provided some insight into the functional role of these molecules in olfactory axonal growth. Neurite outgrowth from cultured olfactory neurons is inhibited by antibodies to NCAM, N-cadherin, and L1 (Chuah et al., 1991). The fact that OCAM, a member of the N-CAM family, is expressed by olfactory neurons in the early stage of prenatal development and is also present in restricted zones of the olfactory epithelial sheet suggests that OCAM may be involved in broad segregation of nerve fascicles (Yoshihara et al., 1997). In humans, N-CAM immunoreactivity in the olfactory nerve bundles can be demonstrated as early as the 18th week of gestation. Western blot analysis shows that the N-CAMs of the human olfactory nerve consist of all three molecular isoforms: N-CAM 180, N-CAM 140, and N-CAM 120 (Chuah and Au, 1992). In addition to adhesion molecules, extracellular matrix has also been implicated in stimulating and sorting olfactory axons as they course towards the olfactory bulb. In the rat, laminin and heparan sulfate proteoglycans, which are expressed by ensheathing cells, are associated with the developing nerve pathway as early as E13 (Liesi, 1985; Treloar et al., 1996). It is thought that these two molecules provide a conducive substrate on which olfactory axons elongate. In contrast, chondroitin sulfate proteoglycans, which are selectively present in the mesenchyme and marginal zone of the telencephalon, may contribute to restricting axonal growth along a particular trajectory (Treloar et al., 1996). Concerning the effect of laminin on olfactory nerve outgrowth, cell cultures of dissociated olfactory neurons on laminin have produced conflicting results. Although some studies showed that olfactory neurons were able to adhere to laminin and subsequently differentiate into bipolar cells (Pevsner et al., 1988; Pixley and Pun, 1990), Chuah and colleagues found that the percentage of olfactory neurons growing neurites when placed on

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laminin was negligible (Chuah et al., 1991). Recent in vitro studies show that exogenous laminin enhances the spreading and migration of ensheathing cells, and this probably facilitates their function as a conduit of axonal elongation (Tisay and Key, 1999). Other classes of molecules have been implicated in the sorting out and fasciculation of axons during development. A candidate is the carbohydrate-binding protein galectin-1, which is first apparent in the mesenchyme surrounding the nasal cavity at E15 and which can be localized distinctly to ensheathing cells at E17 when the first nerve fibers have reached the olfactory bulb (St. John and Key, 1999). Expression of galectin-1 is maintained throughout development, and in the postnatal rat it can be observed in the ensheathing cells surrounding the axon bundles in the lamina propria as well as those residing in the olfactory nerve layer of the bulb (St. John and Key, 1999). In summary, the data are consistent with the notion that ensheathing cells express specific surface molecules that promote axonal elongation. Ultrastructural observations reveal that these cells are intimately related to the early formation of olfactory axons—their processes are always present ahead of growing axonal terminals (Tennent and Chuah, 1996). In addition to the expression of extracellular matrix molecules, ensheathing cells may also be a rich source of growth factors. It has been shown that these cells produce brain-derived neurotrophic factor, glial cell line–derived neurotrophic factor, and neuregulins such as glial growth factor 2 (Chuah et al., 2000; Salehi-Ashtiani and Farbman, 1996; Woodhall et al., 2001). Whether the secreted growth factors play a role in promoting axon growth is yet to be determined. The olfactory bulb represents another candidate for directional guidance of olfactory axons. In vitro studies show that the olfactory bulb secretes soluble molecules that act as chemoattractants to ensheathing cells (Liu et al., 1995). The identity of these chemoattractants has not been elucidated, and they could well be any number of the growth factors that are known to be present in the olfactory bulb (Mackay-Sim and Chuah, 2000). Once the olfactory axons enter the olfactory bulb, additional cues may be required to fine-tune target recognition and ensure that axons terminate in the appropriate glomeruli. Experiments with genetically engineered mice show that expression of an odorant receptor is required for convergence to specific glomeruli (Mombaerts et al., 1996; Wang et al., 1998). Although the exact mechanisms are not known, receptor-swap experiments have shown that odorant receptors play an instructive role in glomerular targeting and establishing a topographic map of the projection onto the bulb (Mombaerts et al., 1996; Wang et al., 1998).

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Recently, the isolation of several members of the OR37 subfamily of receptors has elucidated the extent of the topographic specificity. OR37A-E are each expressed in a patch in the posterodorsal epithelium and are not found throughout the full anteroposterior extent of the epithelium, in contrast to the expression patterns of more typical receptors (Kubick et al., 1997). Each set of neurons of the OR37 subfamily project to a single, different glomerulus at the ventral margin of the bulb (Strotmann et al., 2000). In contrast to the absolute positional specificity reported for zebrafish glomeruli (Dynes and Ngai, 1998; Friedrich and Korsching, 1997), the position of the OR37 subgroups of glomeruli are not fixed but vary relative to each other (Strotmann et al., 2000). The means by which receptor choice directs targeting and glomerular convergence remains a subject of intense investigation. Recent evidence suggests that activity, and more importantly coordinate activity by a set of olfactory neurons, is crucial for the acquisition and maintenance of glomerular territory. The experiments take advantage of the fact that elimination of the OcNC1 subunit of the cyclic nucleotide receptor abolishes stimulus-evoked olfactory neuronal activity (Brunet et al., 1996). Some OR-defined classes of neurons innervate multiple glomeruli in the region where they would normally terminate, rather than the single glomerulus that is typical (Zheng et al., 2000). For other neuron types, targeting appears normal (Lin et al., 2000; Zheng et al., 2000). Intriguingly, Zhao and Reed (2001) have taken advantage of the fact that roughly half of the neurons in a female mouse heterozygous for the channel mutation are silenced via allelic inactivation of genes, such as OcNC1, on the X chromosome. In this case, their lab has shown that silenced neurons are gradually excluded from glomeruli and eventually from the epithelium as a whole.

V.

DEVELOPMENT OF THE OLFACTORY BULB

A.

Anatomy

The general anatomical features of bulb development in all mammals are similar to one another. Most of the descriptive and experimental studies have been done on rodent embryos, and most of what follows is based on the results of these studies. Where information is available, we have included the stages of human olfactory bulb development. The bulb is derived from the rostral region of the cerebral (telencephalic) vesicle of the early mammalian embryo. Before the bulb becomes apparent as an entity, the cerebral vesicle is a fluid-filled cavity lined with an

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epithelium divisible into two regions: a highly cellular ventricular region bordering the ventricle and an acellular marginal region. In the rat, between the 11th and 14th embryonic days, an increasing number of axons from the olfactory epithelium reach the cerebral vesicle and align themselves parallel to the vesicle surface. A subpopulation of these axons penetrate the epithelium lining the most rostral and inferior part of the cerebral vesicle and extend past the marginal layer to end deep in the ventricular layer. The entry of the first or “pioneer” olfactory axons into the bulb precursor is correlated with an average increase in the length of the cell cycle in the ventricular region of the bulb precursor, compared to that in the ventricular region of the adjacent cerebral vesicle (Gong and Shipley, 1995). The significance of this increase in the average cell cycle length is that, in the bulb precursor, more postmitotic cells exit from the cell cycle and begin their differentiation into presumptive mitral/tufted cells than is the case in the adjacent cerebral vesicle epithelium. Thus, in the forebrain the mitral/tufted cell precursors are among the first to begin their differentiation. At E15-E16 in rats (at E13-14 in mice) the presumptive bulb becomes more obvious as an evagination of the rostral end of the telencephalic vesicle. In the human embryo, this stage begins at 37–41 days after conception. At the same time, the sensory epithelium is expanding. Along with this expansion, more olfactory axons project from the sensory epithelium to the presumptive bulb and add to the numbers of parallel axons along the bulb surface, outside of the marginal layer. These axons form what will later become the outer nerve layer of the bulb. For a few days axons do not penetrate into the presumptive bulb (see Bailey et al., 1999). This raises the question of how molecular interactions between these axons and the bulb precursor might differ from those of the pioneer axons which, at an earlier stage, do have the ability to penetrate into the lining of the cerebral vesicle. The bulb increases in size and takes on a more definitive shape after a constriction forms in the ventricular cavity. The constriction occurs between what will become the olfactory ventricle, a transient central cavity, and the remainder of the lateral ventricle that persists in the adult cerebrum. As the bulb increases in size, it grows rostrally and expands in diameter. The growth pattern results in the relative caudal displacement of the ventricle so that it is no longer seen in histological sections of the bulb proper. In humans this occurs by the 19th week (Humphrey, 1940). In rats and mice, the ventricle recedes caudally during the first 2–3 weeks following birth. Associated with the expansion in size is the loss of the embryonic marginal layer and the appearance of the adult laminae.

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Maintained contact between sensory epithelium and the telencepalon is required for induction and proper formation of the olfactory bulb. We have already noted that ablation of the placode prevents contact, telencephalic evagination, and bulbar differentiation (Burr, 1916; Stout and Graziadei, 1980). A variety of malformations and teratogens, including the extra-toes mouse mutation mentioned above (Johnson, 1967) and the small eye mutation (Dellovade et al., 1998), may also cause induction to fail. In the majority of these cases, the olfactory epithelium has formed and has elaborated axons. One of the more instructive examples is the human disorder termed Kallmann syndrome. Patients with Kallmann syndrome are identified on the basis of anosmia and hypogonadotrophic hypogonadism (Kallmann et al., 1944). The olfactory bulb is absent (Yousem et al., 1993), but the olfactory epithelium forms and generates olfactory neurons (Schwanzel-Fukuda et al., 1989; Schwob et al., 1993). The mutated gene, KAL1, has been identified as mutated in the X-linked variant of the disease and encodes anosmin-1, a cell adhesion protein expressed by the presumptive telencephalic anlage of the bulb around the time that it is contacted by axons from the epithelium (Franco et al., 1991; Legouis et al., 1991, 1993; Rugarli, 1999). It has been suggested that the contact between the olfactory nerve and bulbar anlage breaks down due to the lack of anosmin-1, leading to the failure of bulb induction (Legouis et al., 1993; Rugarli, 1999). As a likely consequence of the absence of the bulb, the olfactory epithelium is highly abnormal in biopsy specimens from a Kallmann patient and from another individual with congenital anosmia without accompanying endocrinologic abnormalities. Only immature olfactory neurons, which lack cilia on the dendritic knobs, are seen (Leopold et al., 1992; Schwob et al., 1993). Some of the axon bundles in the lamina propria are devoid of ensheathing cells and form dense neuromas. Ultrastructural observations show that the abnormal axons are either swollen or their membranes are fragmented, suggesting that the axons are degenerating (Leopold et al., 1992; Schwob et al, 1993). B.

Proliferation of Neuron Precursors and Formation of Laminae

Before the evagination of the bulb from the cerebral vesicle, many of the first cells to exit from the cell cycle are the forerunners of mitral cells. In human embryos (Fig. 8) mitral cells are first noticeable in the presumptive bulb at stage 21 (~52 days), and they become significantly larger at stage 22 (~60 days) (Humphrey, 1940; Humphrey and Crosby, 1938). After bulb evagination (10 weeks in human embryos), mitral

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Figure 8 Embryonic development of the human olfactory bulb. (A) At about 41 days gestation, olfactory nerve fibers form a rudimentary olfactory nerve layer (ONL) along the ventral side of the presumptive olfactory bulb. Lamination is absent at this stage. (B) At about 60 days gestation the mitral cell layer (MCL), which consists of the large cell bodies of mitral cells, appears most distinct. The external plexiform layer (EPL) can be distinguished as a thin lamina immediately deep to the external granular layer (EGL), a cellular region sandwiched between the developing EPL and ONL. (C) At about 70 days of gestation, all the laminae of the olfactory bulb are formed, with the glomerular layer (GmL) developing last. The MCL appears less distinct as the distance between adjacent mitral cells increases. The thickness of the EPL has increased dramatically. GL, granular layer.

cells become larger and have migrated away from the ventricular zone, where they were generated, to a more peripheral location (Chuah and Zheng, 1992), where they begin to form a mitral layer. However, in the human bulb the mitral layer is never as clearly defined as that in rodents, so that it is difficult to distinguish a clear boundary between it and the adjacent external plexiform layer. The time of origin of the various neuron types has been well studied in the mouse embryo (Hinds, 1968a,b). At E12 (the 12th embryonic day; the mouse has a gestation period of about 19 days) precursors to mitral cells undergo their final cell divisions in the ventricular layer and migrate peripherally. They reach their definitive locations about 3 days later where they form a discrete layer, the mitral cell layer, much more obvious and clearly defined than in the human bulb. Generally the smaller and more superficially located tufted cells arise in the ventricular layer later than the mitral cells and they migrate past the mitral cells to the presumptive external plexiform layer where they are found at all levels, some close to the mitral cell layer, some intermediate and others more superficial, near the glomerular layer. Tufted cells appear to be formed in an inside-out gradient, i.e., those nearest the mitral layer are formed first, and those that take up residence higher in the external plexiform layer are formed later. In the mouse, and in other mammals, the last neurons to form are the interneurons, the small periglomerular and granule neurons. In rodents, the earliest interneurons are generated in the subventricular (subependymal) layer a few days before birth, but most are generated within the first 2 or 3 weeks after birth (see Bayer, 1983; Hinds, 1968a;

Rosselli-Austin and Altmann, 1979). The ventricular zone as a site of neurogenesis disappears at E18 in mice, one day before birth. The interneurons generated after this time are generated in a subventricular (subependymal) zone, first in the olfactory ventricle, but later, after the ventricle recedes from the olfactory bulb, they are generated in the subventricular zone of the anterior region of the lateral ventricle. Although most––perhaps as many as 90%—of these new neurons die soon after they are produced (Morshead and Van der Kooy, 1992), some do survive and migrate via the rostral migratory stream into the bulb, where they become granule or periglomerular neurons and are incorporated into the neural network within the bulb (Altman, 1969; Lois et al., 1996; Luskin, 1993). Neuronal precursors migrate as chains of cells passing through “tunnels” of glial cells in the rostral migratory stream (Lois et al., 1996). These neuronal precursors continue to divide as they migrate. Migration appears to be dependent on the presence of polysialic acid–rich N-CAM (neural cell adhesion molecule) on the surfaces of the neuronal precursors (Bonfanti and Theodosis 1994; Hu et al. 1996). In development of the neuronal populations in the bulb, then, the mitral cells develop first and establish a one-cellthick layer. Tufted cells arise later and come to rest at varying levels between the mitral cell and the nerve layer of the bulb. Because most of the granule and periglomerular cells are not present at birth, the granule cell layer and the external plexiform layers are rather thin, and glomeruli are not outlined by a ring of periglomerular cells as they are in the mature bulb. Most of the growth of the granule cell layer and the external plexiform layer occurs in the

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layer between E17 and postnatal day 10 in the mouse (Hinds 1968a,b). These probably include both astrocytes and ensheathing cells originating from the olfactory placode. The stages are similar in the rat, but they begin at E13-E16 (Bayer, 1983). The number of the various neuronal types in the developing olfactory bulb can be influenced by physical and chemical teratogens, such as x-irradiation (Bayer and Altman, 1975), phenobarbital (Rosselli-Austin and Yanai, 1989), and alcohol (Bonthius and West, 1991). In experimental animals postnatal administration of alcohol appears to induce cell death or interfere with neurogenesis or migration of granule cells. Although some degree of recovery in the granule cell population occurs following cessation of alcohol treatment, the effect on mitral cells is permanent (Bonthius and West, 1991). This is probably because all of the mitral and tufted cells are produced during a brief window of development, whereas granule and periglomerular neurons are produced continually throughout life.

C.

Figure 9 Photomicrographs demonstrating changes in the laminae of the rat olfactory bulb from the time of birth (A) until 10 days postnatal (B). Most granule cells are generated postnatally; consequently the granular layer (GL) undergoes extensive growth after birth. The external plexiform layer (EPL) also increases in thickness because of the more complex dendritic arborization of mitral cells and the arrival of centripetal inputs. The glomerular layer (GmL) is better defined as the number and size of the individual glomeruli increase. MCL, mitral cell layer; ONL, olfactory nerve layer. Bar
30 m.

first weeks after birth in rodents (Fig. 9) (reviewed by Brunjes and Frazier, 1986). Neuroglia arise from scattered proliferating glioblasts originally derived from the subventricular germinal layer. Many proliferating cells are found in the olfactory nerve

Formation of Glomeruli

Within the presumptive bulb (rat, E11-12) a population of glial cells is organized radially from the ventricular to the marginal layer. By E15, the proximal and distal ends of these radial glia form two plexuses: one in the marginal zone of the presumptive bulb, the other in the subventricular zone (Bailey and Shipley, 1993; Bailey et al., 1999). The plexus in the marginal (sub-pial) layer may act as a temporary barrier, of sorts, against invasion of olfactory axons in the nerve layer (Bailey et al., 1999; Treloar et al., 1999; Valverde et al., 1992). The details of glomerulus formation have been studied most intensively in the developing rat brain and in an invertebrate, the moth. The principles of construction appear to be similar. After the radial glia and the presumptive nerve layer of the bulb become established at E16 in the rat, axon bundles from the nerve layer begin to penetrate into the presumptive bulb (in the moth, the antennal lobe), forming knots of neuropil or “protoglomeruli” just beneath the bulbar surface (Graziadei and Samanen, 1980; Graziadei et al., 1979; Malun and Brunjes, 1996) as they intermingle with the radial glia (Bailey et al., 1999; Treloar et al., 1999; Valverde et al., 1992) [see also development in the moth (Oland and Tolbert, 1996; Oland et al., 1990)]. These protoglomeruli are the glomerular precursors and are demarcated by glial processes. Some sorting process must occur before olfactory axons form the protoglomeruli because all of the axons from cells expressing a particular olfactory receptor go to only two glomeruli

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in the bulb—one located laterally and one medially (Mombaerts et al., 1996; Ressler et al., 1994; Vassar et al., 1994). Experiments with genetically manipulated mice have shown that deletion of a particular receptor gene results in spreading of axons across a wider expanse of bulb, whereas insertion of an “incorrect” receptor gene into an olfactory neuron results in the axons terminating in a different glomerulus than in the unperturbed animal (Mombaerts et al., 1996). Soon after the axons reach the target glomerular region, they form extensive branches and begin to form synaptic relationships with dendrites of mitral and tufted cells (Halaász and Greer, 1993; Kasowski et al., 1999, Klenoff and Greer, 1998; Treloar et al., 1999). On a molecular level, the expression of the gene, sonic hedgehog, by mitral and tufted cells is associated with branching of axons within glomeruli and glomerular formation (Gong and Farbman, 1999). Axons form their synapses on the dendrites of mitral cells and on periglomerular cell dendrites. It has been shown that as the olfactory epithelium expands and more axons grow to the bulb, the newly arrived axons, which express GAP-43, a marker for young olfactory neurons, occupy the central region of the glomerulus, whereas those that had arrived previously, more mature axons that express OMP, are pushed to the lateral regions of the glomerulus (Treloar et al., 1999). The synapse-specific molecule synaptophysin is expressed in GAP-43-positive axons, suggesting that synapses are formed by these axons before becoming fully mature, i.e., before expressing OMP (Kim and Greer, 2000; Treloar et al., 1999). However, beginning at around the 12th postnatal day in rats, immature axons entering glomeruli were distributed in the periphery and moved toward the center as they matured (Kim and Greer, 2000). This change in trajectories of axons into glomeruli suggested that different rules may be followed in establishing glomeruli and maintaining them. Early in glomerular development a mitral cell may project apical dendrites to more than one glomerulus, but during maturation all but one apical dendrite are withdrawn, so that each mitral cell projects to only one glomerulus in the adult. Thus, given that (1) sensory neurons each express a single receptor and (2) all neurons expressing a given odorant receptor project to only two or three glomeruli in the bulb (Ressler et al., 1994; Vassar et al., 1994), the fact that each mitral cell is postsynaptic to a single glomerulus indicates that it is responsive to a very narrow range of odorant stimuli (reviewed in Mori et al., 1999). The organization of glomeruli into a distinct lamina seems to depend largely on an interaction between olfactory axons and radial glia (Bailey et al., 1999; Oland and Tolbert, 1990). Glial cells form boundaries delimiting the

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edges that restrict olfactory axons inside protoglomeruli (González et al., 1996; Valverde et al., 1992). It has been shown that experimentally induced reduction of the number of glial cells causes disruption or the complete disappearance of glomeruli in the olfactory lobe of the moth (Oland et al., 1988). Efforts to determine whether the actual presence of the postsynaptic partners of olfactory axons, i.e., periglomerular or mitral/tufted cells, are necessary for glomerular formation have indicated that they are not. As shown in the moth (Oland and Tolbert, 1998) and in mutant mice (Buffone et al., 1998), olfactory glomeruli develop in the absence of target cells. Olfactory axons clearly play a key role in development of glomeruli. Indeed, as indicated above, if afferent input is experimentally removed by extirpating the olfactory placode, the vertebrate olfactory bulb does not form at all (e.g., Burr, 1916; Stout and Graziadei, 1980; Venneman et al., 1982). However, glomeruli do form in mutant mice in which the axons are rendered unable to conduct an impulse, by deleting either a gene coding for a channel subunit (Brunet et al., 1996) or a gene coding for a G-protein necessary for signal transduction (Belluscio et al., 1998). These experiments show that neural activity of olfactory axons is not required for glomerulus formation but the presence of the axons is. Undoubtedly axons either produce a factor or carry a factor on their surface that is important in glomerular formation. The ability of olfactory axons to induce formation of glomeruli is not restricted to the glomerular layer of the bulb, but under experimental conditions can also induce glomeruli at ectopic sites in the bulb, in other parts of the telencephalon, in the diencephalon, midbrain, and hindbrain (Graziadei and Kaplan, 1980; Graziadei and Samanen, 1980; Magrassi and Graziadei, 1985; Monti Graziadei and Graziadei, 1992). In some of these experiments fragments of cerebral or cerebellar cortex were transplanted into the space following partial or complete bulbectomy. The ingrowing axons were able to reorganize components of these “inappropriate” target tissues to form glomeruli. However, when fragments of olfactory epithelium are excised and transplanted to other parts of the brain, the axons invade the host tissue but fail to form glomeruli (Monti Graziadei and Graziadei, 1983; Morrison and Graziadei, 1983). Graziadei and Monti Graziadei (1986) have suggested that the glomerulus can form in any part of the brain only if it receives axonal contributions from a broad region of the olfactory epithelial sheet. This is consistent with the fact that innervation of individual glomeruli is derived from the convergence of several thousand individual olfactory axons derived from cells broadly distributed within four zones of the olfactory sheet (Astic and Saucier, 1986; Astic et al., 1987; Ressler et al., 1993).

Developmental Anatomy of the Olfactory System

Olfactory sensory neurons may also play a role in the initiation and maintenance of neurotransmitter expression in some bulbar neurons. Embryonic olfactory bulbs co-cultured with olfactory epithelium possess more tyrosine hydroxylase–containing neurons than if they are cultured alone (Baker and Farbman, 1993). In deafferented olfactory bulbs, dopamine in periglomerular, external, and middle tufted cells is severely reduced (Kawano and Margolis, 1982; Kream et al., 1984); so is the number of periglomerular cells expressing tyrosine hydroxylase and its messenger RNA (Baker et al., 1983). Following reinnervation of the bulb, dopamine levels and the number of tyrosine hydroxylase immunoreactive cells return to control values, indicating that olfactory neurons are crucial in the maintenance of neurotransmitter synthesis in bulbar cells (Baker et al., 1983; Kawano and Margolis, 1982).

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7 Anatomy and Neurochemistry of the Olfactory Bulb Igor L. Kratskin University of Pennsylvania, Philadelphia, Pennsylvania, U.S.A.

Ottorino Belluzzi University of Ferrara, Ferrara, Italy

The olfactory system is able to recognize a great variety of odorous substances and discriminate between chemicals that have subtle differences in their structural properties. Sensory neurons of the olfactory epithelium perceive odor molecules, transduce and encode that information, and transmit impulse responses to the primary olfactory center, the main olfactory bulb. After processing in the olfactory bulb, physiological signals are delivered directly to the secondary sensory centers in the primary olfactory cortex. There is “ample evidence that the olfactory bulb is not merely a ‘ganglion’ in which the olfactory pathway is synaptically interrupted, but is indeed a centre of great complexity containing associative connections at several levels, intrinsic neuronal circuits of varying length, and a ‘centrifugal’ as well as the sensory input” (Nieuwenhuys, 1967). This chapter focuses on the anatomical organization and the neurotransmitters of the olfactory bulb, with an emphasis on afferent projections from brain sources. An overview of neural and molecular mechanisms underlying odor coding in the olfactory bulb is also provided. Most of the information comes from rodents; human data are presented, whenever possible. A number of original papers could not be

cited due to space limitations; readers are referred to other reviews for this information (Halász and Shepherd, 1983; Macrides and Davis, 1983; Brunjes and Frazier, 1986; Mori, 1987; Scott and Harrison, 1987; Halász, 1990; Trombley and Shepherd, 1993; Ressler et al., 1994a; Mori and Yoshihara, 1995; Sullivan et al., 1995; Buck, 1996; Shipley and Ennis, 1996; Shepherd and Greer, 1998).

I.

ANATOMICAL ORGANIZATION

The olfactory bulbs are paired, ovoid-shaped structures forming the rostral end of the telencephalon. In many mammals, they occupy the foremost position in the skull and are quite large. In humans and other primates, the bulbs, displaced by the enlarged cerebrum, are relatively small and located under the ventral surface of frontal lobes. The olfactory bulb is a cortical structure and has a characteristic laminar organization (Fig. 1). The first anatomical descriptions of the olfactory bulb, made in the second half of the nineteenth century and summarized in the classic book of Ramón y Cajal (1911), were significantly expanded by subsequent studies. In the olfactory bulb, like in other brain centers, an input fiber, a principal cell, and an intrinsic neuron form a triad of neuronal elements (Shepherd and Koch, 1998). Two major sets of input fibers come to the bulb: axons of olfactory sensory neurons, or olfactory axons, which trans-

This chapter is dedicated to the memory of Dr. A. A. Bronstein (1927–1976), an extraordinary olfactory scientist from the Sechenov Institute of Evolutionary Physiology and Biochemistry, St. Petersburg, Russia.

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form the olfactory nerve layer. Within a single bundle, olfactory axons are packed very tightly (5–20 nm from one another), allowing ephaptic interactions between neighboring axons (Eng and Kocsis, 1987). Olfactory axons do not branch before entering the bulb, and their number corresponds to the number of OSNs that, in rabbits, was estimated to be around 50,000,000 on each side of the nasal cavity (Allison and Warwick, 1949). In adult humans, this number may be about 6000,000 (Moran et al., 1982). Unique glial cells, called olfactory ensheathing cells, surround axon bundles on their way to the bulb and within the olfactory nerve layer (Doucette, 1991). These cells have common features with astrocytes and Schwann cells and express a series of neurotrophic factors (Bartolomei and Greer, 2000; Mackay-Sim and Chuah, 2000). Ensheathing cells may promote axonal regeneration after traumatic injury (Bartolomei and Greer, 2000). 2.

Figure 1 Transverse vibratome section of rat olfactory bulb illustrating the basic laminar organization (staining with Giemsa dye). The olfactory nerve layer and glomeruli are stained due to anterograde labeling of olfactory axons with horseradish peroxidase. ONL, olfactory nerve layer; GL, glomerular layer; EPL, external plexiform layer; MCL, mitral cell layer; IPL, internal plexiform layer; GRL, granule cell layer. Scale bar-50 µm.

mit information about odor molecules, and axons of brain neurons, or centrifugal axons, which exert modulatory influences on bulbar microcircuits (Fig. 2A). There are two classes of principal cells in the olfactory bulb: mitral cells and tufted cells (Fig. 2B). Intrinsic neurons, or local interneurons, fall into three categories: periglomerular cells, granule cells, and short axon cells (Fig. 2C). A.

Layers and Neuron Types

1.

Olfactory Nerve Layer

Unmyelinated axons (mean diameter ~0.3 m) of olfactory sensory neurons (OSNs) constitute the primary olfactory projection. The axons come to the olfactory bulb in discrete bundles that interweave on its surface and

Glomerular Layer

Axons of OSNs run from the olfactory nerve layer to spherical neuropil regions termed olfactory glomeruli. In different vertebrate species, the glomeruli vary from 30 to 200 m in diameter and constitute the glomerular layer, one or two glomeruli thick. Each olfactory axon innervates only a single glomerulus. After entering the glomerulus, the olfactory axon gives rise to an arbor of branches (mean branch length 170 m) with terminal boutons and en passant varicosities; the branch arbor occupies about 14% of the glomerular area (Halász and Greer, 1993; Klenoff and Greer, 1998). Within glomeruli, olfactory axons make synapses onto dendrites of principal and intrinsic cells, so each glomerulus is a complex structure consisting of axonal and dendritic compartments (Kosaka et al., 1997; Kasowski et al., 1999). A number of light and electron microscopic studies have described in eloquent detail neurons in the glomerular layer (Pinching and Powell, 1971a; Macrides and Davis, 1983; Halász, 1990). Each glomerulus is surrounded by numerous small somata (long axis 6–8 m) of periglomerular (PG) cells. A PG cell dendrite ramifies and terminates within one or two glomeruli, intermingling with terminals of olfactory axons and dendrites of principal cells, whereas a PG cell axon extends across three to five glomeruli. The population of PG cells comprises neurons differing in their neurochemical, morphological, and physiological features (Kosaka et al., 1995, 1997; Puopolo and Belluzzi, 1998a; Toida et al., 1998, 2000). About 10% of PG cells, composing a chemically distinct neuronal group, have no synapses from olfactory axons (Kosaka et al., 1997; Toida et al., 1998).

Anatomy and Neurochemistry of the Olfactory Bulb

Figure 2 Schematic representation of afferent fibers, principal cells, and local interneurons in the olfactory bulb. Layers of the bulb are indicated as in Figure 1. (A) ON(m) and ON(l), medial and lateral groups of olfactory axons. Centrifugal fibers originate in the ipsilateral and contralateral anterior olfactory nucleus (iAON and cAON), taenia tecta (TT), olfactory cortex (OC), nucleus of the horizontal limb of the diagonal band (HDB), locus coeruleus (LC), and raphe nucleus (Ra); pE, pars externa of the AON; pM, pars medialis of the AON. (B) The axons (a), axon collaterals, and dendrites (d) of a mitral cell (M), displaced mitral or internal tufted cell Md/Ti, middle tufted cell Tm, and external tufted cell Te; LOT, lateral olfactory tract. (C) GI, GII, and GIII designate three types of granule cells; PG, periglomerular cell. Various short axon cells are shown: SA(B), Blanes’ cell; SA(C), Cajal’s cell; SA(G), Golgi cell; SA(H), Hensen’s cell; SA(S), Schwann cell; SA(V), Van Gehuchten cell. (From Shepherd and Greer, 1998.)

PG cells are intermixed with external tufted cells and short axon cells. A short axon cell has an oval cell body (long axis 12 m in rats), dendrites that ramify between or around glomeruli, and an axon extending to one to three glomeruli. Dendrites of PG cells and short axon cells pos-

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sess spines, in contrast to smooth dendrites of tufted cells. An external tufted cell (long axis 10–15 m) has a short apical dendrite, terminating within a glomerulus, and one to three basal dendrites extending immediately below glomeruli. Some external tufted cells send axons to the olfactory cortex, whereas others, whose axons terminate within the bulb, represent intrinsic cells. Intrinsic tufted cells either have connections in the glomerular layer or engage in point-to-point reciprocal projections between opposite regions of the olfactory bulb, forming a topographically organized “intrabulbar associational system” (Schoenfeld et al., 1985). The glomeruli are the most distinctive feature of the olfactory bulb and illustrate “the principle of grouping neural elements and synapses into anatomically defined modules” (Shepherd and Greer, 1998). Glomeruli are not functionally uniform and play a key role in odor coding in the olfactory bulb. An example of glomerular specificity is the so-called “modified glomerular complex,” which is a group of glomeruli in the caudal dorsomedial part of the bulb (Teicher et al., 1980; Greer et al., 1982). These glomeruli have atypical structural features and likely process information related to a specific odor cue important for suckling behavior. Development of the glomeruli depends upon influences exerted by OSNs (see Chapter 6). There is a unique plasticity in the olfactory system: although the OSNs are replaced by newly generated cells throughout adult life (Graziadei and Monti Graziadei, 1978), a constancy of zonal projections from the olfactory epithelium to glomeruli is maintained (Schoenfield et al., 1994). Extracellular matrix/neuronal cell adhesion molecules are one of the factors providing the basic guidance of olfactory axons to the glomeruli (Kafitz and Greer, 1998).

3.

External Plexiform Layer

A very dense neuropil, formed by dendrites of principal neurons and granule cells, and a relatively low density of cell bodies are characteristic features of the external plexiform layer (EPL). Most neurons in the EPL are middle and internal tufted cells, and some represent short axon cells. Middle tufted cells have their somata (long axis 15–20 m) near the middle of the EPL; each cell gives rise to thin basal dendrites and an apical dendrite that terminates within a single glomerulus. Axons of these cells give off collaterals and project to the primary olfactory cortex. Internal tufted cells (long axis about 27 m), located in the deeper one third of the EPL, are similar in their morphology to displaced mitral cells. Importantly, it is within this layer that basal dendrites of principal cells have synaptic contacts with peripheral dendrites of granule cells.

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4. Mitral Cell Layer The mitral cell layer is thin and contains relatively large somata (long axis 20–33 m) of mitral cells. These cells have one apical dendrite (diameter 2–12 m; length 200–800 m) that runs through the EPL and terminates within a single glomerulus. Each mitral cell also has two to nine basal dendrites (diameter 1–8 m; length up to 1300 m) that branch and terminate in the EPL, within a field with a radius of about 900 m. Two types of mitral cells have been identified (Macrides and Schneider, 1982; Mori et al., 1983; Orona et al., 1984). Type I mitral cells, which are more numerous, have basal dendrites in the deep portion of the EPL, whereas type II, or displaced, mitral cells, whose somata lie more superficially, have basal dendrites near the middle of the EPL. Myelinated axons of mitral cells (diameter 0.5–3.0 m) give off collaterals, which terminate deep within the bulb, and run to the secondary olfactory centers. The mitral cell number has been estimated to be about 51,000 in persons in their mid-twenties (Bhatnagar et al., 1987). The mitral and tufted cells exhibit multiple differences, including the position of cell bodies, distribution of basal dendrites, and the transmitter specificity. These neurons also have distinct genetic determinants of cell differentiation (Greer and Shepherd, 1982). Moreover, the mitral and tufted cells are likely connected to particular groups of granule cells (see below) and have differing projection pattern in the olfactory cortex (Schoenfeld and Macrides, 1984; Schoenfeld et al., 1985; Scott et al., 1985). It thus appears that the two classes of principal cells are involved in segregated and overlapping circuits via intrinsic and extrinsic connections (Macrides et al., 1985). 5.

Internal Plexiform Layer

The internal plexiform layer is immediately subjacent to the mitral cell layer and represents a thin neuropil zone containing a few short axon cells. The main elements composing the internal plexiform layer are numerous axons and axon collaterals of mitral and tufted cells and peripheral dendrites of granule cells. These dendrites are the target for axons of external tufted cells that constitute the intrabulbar associational system (Schoenfeld et al., 1985; Liu and Shipley, 1994). Some axons terminating in the internal plexiform layer originate in neurons of the basal forebrain and brainstem. 6.

Granule Cell Layer

Granule cells are the most numerous neurons in the olfactory bulb, and their number in the rat is said to range from 1000,000 to 3000,000 (Meisami and Safari, 1981; Struble

and Walters, 1982). Rounded or fusiform somata (long axis 6–10 m) of granule cells are densely packed in the granule cell layer and constitute aggregates containing three to five cells apiece. In these aggregates, gap junctions couple granule cells, so the activity of neighboring neurons may be synchronized (Reyher et al., 1991). Granule cells lack axons and have peripheral and deep dendritic processes (Price and Powell, 1970a). Each cell gives rise to one relatively thick peripheral dendrite that ramifies and terminates in the EPL, extending over an area 50–200 m in diameter. One to four fine deep dendrites (length 50–100 m) terminate within the granule cell layer. All parts of the granule cell, including a cell body, are endowed with appendages called spines. Peripheral and basal dendrites have approximately two spines per 10 m of dendritic length. Deep to the mitral cell layer, spines always have a postsynaptic location. In the EPL spines participate in reciprocal synapses between granule and mitral/tufted cells and are therefore presynaptic as well as postsynaptic structures. These spines are also referred to as gemmules to emphasize their special position in the synaptic contacts (Rall et al., 1966; Price and Powell, 1970a). Three types of granule cells have been distinguished in hamsters (Schneider and Macrides, 1978), rabbits (Mori and Kishi, 1982), and mice (Greer, 1987). Type I granule cells (GI in Fig. 2C) have intermediately positioned somata and peripheral dendrites terminating at all levels of the EPL. Type II and type III granule cells (GII and GIII in Fig. 2C) have their cell bodies in the deep and superficial parts of the granule cell layer, respectively. Peripheral dendrites of deep (GII) and superficial granule cells (GIII) terminate, correspondingly, at the deep and superficial levels of the EPL. Only deep and superficial granule cells have been recognized in rats (Orona et al., 1983). These data suggest that there is a segregation of local microcircuits in the EPL, i.e., granule cells of different types are connected to different types of principal cells. Type II and type III granule cells are likely connected to mitral cells and tufted cells, respectively; type I granule cells may receive signals from both classes of principal neurons. Short axon cells are relatively numerous in the granule cell layer. Several types of short axon cells (Fig. 2C) were identified by the Golgi impregnation technique (Ramón y Cajal, 1911; Price and Powell, 1970d; Pinching and Powell, 1971a; Schneider and Macrides, 1978) and immunostaining for calcium-binding protein parvalbumin (Kosaka et al., 1994), and two of them, Golgi cells and Blanes’ cells, had their somata in the granule cell layer. These neurons are intermediate in size between granule and mitral cells (about 15 m in the rat) and have three to eight dendrites mainly restricted to the layer. In Blanes’ cells, only distal parts of the dendrites possess spines; den-

Anatomy and Neurochemistry of the Olfactory Bulb

drites of Golgi cells are almost completely spineless. Axons of Blanes’ cells and Golgi cells terminate within the granule cell layer and, occasionally, in the internal plexiform layer and in the deep of the EPL. B.

Convergence Ratios

In the rabbit, each olfactory bulb receives ~50,000,000 olfactory axons and contains ~2000 glomeruli, 50,000 mitral cells, and 100,000 tufted cells (Allison and Warwick, 1949). Simple calculations suggest that the convergence ratios for olfactory axons are very high in this species: 25,000 onto a single glomerulus, 1000 onto each mitral cell, and 500 onto each tufted cell. The number of PG cells is approximately 20 times higher than that of mitral cells. Rough estimates suggest that, in rabbits, a single glomerulus is composed of 25,000 branching olfactory axons and dividing dendrites of 25 mitral cells, 50 tufted cells, and 500 PG cells. The ratio of granule cells to mitral cells is ~50–100:1. In adult rats, the number of OSNs has been estimated at 20,000,000 (Paternostro and Meisami, 1996a,b), whereas the number of glomeruli is about 2400 (Meisami and Sendera, 1993). Thus, the convergence of olfactory axons onto glomeruli may be about 8000:1 in this species. There are ~6000,000 sensory neurons (Moran et al., 1982) and ~8000 glomeruli (Meisami et al., 1998) in adult humans. This yields a convergence ratio of 750 olfactory axons per glomerulus, or one order of magnitude less than that in rats. C.

Cell Death and Cell Generation

Cell death is inherent in the developing brain, and the olfactory bulb is not an exception to the rule. Studies in the rat indicated that cell death in the layers of mitral and granule cells occurs within 10–15 postnatal days and that early (on postnatal day one) external naris closure prolongs this time period up to 60 days (Fiske and Brunjes, 2001). This demonstrates how sensory input may regulate cell numbers in the bulb. Compensatory reorganization in the bulb following cell death has been investigated in the mutant mouse strain pcd (Purkinje cell degeneration), in which mitral, but not tufted, cells degenerate during early adulthood (Greer, 1987; Greer and Halász, 1987). In these mice, granule cells, denervated from mitral cells, formed new dendrodendritic synapses with tufted cells. The olfactory bulb is one of the few brain structures receiving a supply of newly generated cells throughout adult life (Altman, 1969; Luskin, 1993, 1998; Lois et al., 1996; Doetsch et al., 1997). Neuronal precursors are generated from stem cells in the subventricular zone lining the lateral

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ventricles. Progenitor cells travel via the rostral migratory stream into the bulb, invade the glomerular and granule cell layers, and become PG cells and granule cells. As shown in mice, disconnection of the olfactory bulb from the rest of the brain does not prevent proliferation and differentiation of progenitor cells, but redirects them to the anterior olfactory nucleus and frontal cortex (Jankovski et al., 1998). There is electrophysiological evidence that newly generated PG cells and granule cells in rats establish synaptic connections with existing cells of the bulb neuronal network (O. Belluzzi and J. LoTurco, manuscript in preparation). In mutant mice exhibiting deficits in the migration of bulbar cell precursors, the width of the granule cell layer was significantly reduced and odor discrimination was impaired (Gheusi et al., 2000). The results of this study suggest that new granule cells are not simply added to the bulb, but replace dying neurons, and that granule cell activity is important for olfactory discrimination. We can conclude that processes of cell death and cell generation coexist in the olfactory bulb, resulting in continual remodeling of local synaptic connections. D.

Synaptic Connections

Signal analysis in the olfactory bulb is carried out at two anatomical levels using intrinsic cells specific to each level. Input processing occurs in the glomerular layer on the basis of connections between olfactory axons, principal neurons, and PG cells. Output control results from interactions between principal neurons and granule cells in the EPL. Centrifugal influences may modulate activity in local microcircuits at both anatomical levels. A number of studies have investigated synaptic actions underlying sensory processing in the olfactory bulb (reviewed by Shipley and Ennis, 1996; Shepherd and Greer, 1998) (see Chapter 9). 1.

Input Processing

Within glomeruli, terminals of olfactory axons containing round vesicles make Gray type 1 chemical synapses (with asymmetric membrane thickenings) (see Gray, 1969) onto dendrites of mitral/tufted and PG cells (Pinching and Powell, 1971a,b,c; White, 1973). The dendrites of principal and PG cells are interconnected with one another by synapses of opposite directions. Principal cell dendrites containing round synaptic vesicles make type 1 synapses, whereas dendrites of PG cells containing pleomorphic vesicles establish Gray type 2 synapses (with symmetric membrane thickenings). Serial reconstructions show that 25–40% of these dendrodendritic synapses are arranged as reciprocal pairs. Intraglomerular connections form the

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synaptic triad, in which the olfactory axon (input element) makes excitatory synapses onto principal cell dendrites (output elements) and dendrites of PG cells (intrinsic elements). Within this triad, incoming impulses reach PG cells directly from olfactory axons and indirectly via excitatory synapses from principal cell dendrites. Intraglomerular microcircuits function to process information about a given odorant and to generate a pattern of activity associated with the quality and intensity of a stimulus. Neighboring glomeruli are connected mainly by axons of PG cells that make type 2 synapses with apical dendrites of mitral/tufted cells and somata and dendrites of PG cells. In addition, PG cell axons synapse onto dendrites, somata, and initial axon segments of short axon cells. Axons of the latter cells containing pleomorphic synaptic vesicles establish type 2, presumably inhibitory, synapses with PG cells. Tufted cell axon collaterals that form type 1 contacts onto tufted cell apical dendrites also provide connections between glomeruli. Both inhibitory and excitatory actions between glomeruli may occur (Shepherd and Greer, 1998). Interglomerular microcircuits serve to recruit adjacent glomeruli responsive to a given odorant and to enhance the contrast between glomeruli processing information about dissimilar odors. Spatial clustering of glomeruli of a similar specificity may be due to the lateral inhibition in the glomerular layer and inhibitory actions of granule cells in the deeper layers of the bulb. Intraglomerular and interglomerular microcircuits may be influenced by centrifugal inputs to the olfactory bulb. Centrifugal axons establish synapses with PG cell dendrites, somata and dendrites of short axon cells, and occasionally with dendrites of principal cells (Pinching and Powell, 1972). Most of these contacts are restricted to extraglomerular neuropil, but axons from the raphe nuclei terminate both outside and within glomeruli. 2. Output Control Contacts between basal dendrites of principal neurons and gemmules (spines) of granule cell peripheral dendrites are the predominant type of synaptic connections in the EPL. About 80% of these contacts are organized as reciprocal pairs, in which the synapse from principal to granule cell is type 1, whereas the synapse from granule to principal cell is type 2 (Rall et al., 1966; Price and Powell, 1970b; Jackowski et al., 1978). Electrophysiological data provide strong evidence that the mitral/tufted-to-granule synapse is excitatory and the granule-to-mitral/tufted synapse is inhibitory (see Mori, 1987). The latter synapse is the sole output of the granule cell. Modeling of information processing in granule cell dendritic spines suggests that subsets of spines may function as complex and independent

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units that mediate localized inhibition onto restricted groups of mitral/tufted cell basal dendrites (Woolf et al., 1991a,b). There are about 50–100 granule cells for one mitral cell, and each granule cell has at least 50 gemmules reciprocally connected to mitral/tufted cell dendrites. Such numerous synaptic contacts provide powerful interactions between principal and granule cells. While it has been generally believed that only granule cells have reciprocal synapse with principal cells, recent studies showed that dendrites of mitral/tufted cells in the EPL also participate in reciprocal synapses with processes of some short axon cells (Toida et al., 1994, 1996). Synaptic connections in the EPL form the triad that includes the principal cell apical dendrite (input element), the soma and basal dendrites of the principal cell (output elements), and the granule cell gemmule (intrinsic element). This microcircuit is the basis for output control in the olfactory bulb. Different types of principal neurons connect to different types of granule cells and have differing projection sites. This suggests that parallel pathways exist in the olfactory bulb to convey information about specific characteristics of odor stimuli Dendrodendritic synapses provide the substrate for self-inhibition within each pathway and lateral inhibition in parallel pathways. Granule cells in the EPL also participate in axodendritic type 1 synapses made by centrifugal axon terminals that contain round synaptic vesicles and establish synapses on granule cell gemmules (Price and Powell, 1970b,c). Granule cells are much more numerous than centrifugal axons, but the terminals of such axons make multiple synapses onto several gemmules that are clustered around them (Price, 1968; Price and Powell, 1970b,c). Therefore, each granule cell peripheral dendrite has at least one synaptic contact with a centrifugal axon (Fig. 3A), sometimes very close to a reciprocal synapse (Fig. 3B). In addition, type 2 synapses, formed by terminals with pleomorphic vesicles, are present on the shafts of granule cell peripheral dendrites, but are never observed on gemmules (Price and Powell, 1970b). Axons that make these synapses may originate in GABAergic brain neurons. A centrifugal axon terminal is an input element in the synaptic triad that also involves a granule cell gemmule and principal cell dendrite. Central modulation of intrabulbar processing and output formation are mediated by granule cells and may be realized exclusively through a dendrodendritic synapse between principal and granule cells. This synapse is the site of a convergence of intrabulbar signals and inputs from olfactory and nonolfactory brain structures. Therefore, it is characterized as a “multifunctional synapse” (Shepherd and Greer, 1998), and granule cells are considered the “final common path” for both intrinsic signals and modulatory extrinsic influences (Price and Powell, 1970c).

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II.

Figure 3 Reconstruction of centrifugal axon terminals making synaptic contacts onto granule cell dendritic gemmules (spines) in the external plexiform layer. (A) One centrifugal axon terminal (c) establishes synapses with gemmules (g) of five granule cell peripheral dendrites (p) a short distance from the reciprocal synapse (r) between the gemmule and mitral cell dendrite (m). (B) Termination of a centrifugal fiber (c) on the granule cell gemmule, in close proximity to the reciprocal synapse. (From Price and Powell, 1970c.)

The entire surface of granule cells in the internal plexiform and granule cell layers participate in contacts with axons, which derive from both intrabulbar and central sources (Price and Powell, 1970b,c). One group of axon terminals contains round vesicles and makes type 1 synapses commonly placed on spines of the dendrites and perikarya of granule cells. Another group of terminals contains pleomorphic vesicles and makes type 2 synapses mainly onto the dendritic shafts and somata of granule cells. The axon terminals of both groups also form contacts with somata and dendrites of short axon cells. Intrabulbar axons, which form type 1 synapses with granule and short axon cells, are the axon collaterals of principal neurons (Price and Powell, 1970b). Short axon cells are thought to be the source of intrabulbar axons that make type 2 synapses on granule cells (Price and Powell, 1970b). Results of electrophysiological experiments suggest that short axon cells exert inhibitory influences on granule cells (Mori, 1987). Centrifugal axon terminals that contain round synaptic vesicles and make type 1 contacts with dendrites and somata of granule and short axon cells have been described (Price, 1968; Price and Powell, 1970b,c). Axons of GABAergic brain neurons presumably establish type 2 synapses on granule and short axon cells. Centrifugal axons are likely to make synaptic contacts with different types of granule cells, thus modulating bulbar output signals generated by different types of principal neurons.

ODOR CODING IN THE OLFACTORY BULB

There is a body of evidence suggesting that olfactory glomeruli are structural and functional units for odor coding in the olfactory bulb (Kauer et al., 1991; Shepherd, 1991; Kauer and Cinelli, 1993; Mori and Yoshihara, 1995; Shepherd and Greer, 1998; Mori et al., 1999, 2000; Xu et al., 2000). The idea that a glomerulus is a functional unit and different glomeruli may receive information about different odors goes back to early anatomical (Clark and Warwick, 1946; Allison and Warwick, 1949; Clark, 1951, 1957) and electrophysiological investigations (Adrian, 1950, 1951) of the olfactory system. Further studies in the field have demonstrated that (1) each glomerulus receives projections from OSNs located in many regions of the olfactory epithelium, (2) a single glomerulus is activated by different odorants, and (3) each odorant activates many glomeruli. Methods used in these studies included horseradish peroxidase tracing (e.g., Jastreboff et al., 1984; Astic and Saucier, 1986) and monoclonal antibody immunolabeling of the primary olfactory projection (Schwob and Gottlieb, 1986, 1988; Carr et al., 1994; Mori and Yoshihara, 1995; Ring et al., 1997; Nagao et al., 2000); behavioral testing (e.g., Slotnick et al., 1987; Lu and Slotnick, 1999; Rubin and Katz, 2001); analysis of odor-induced changes in c-fos expression (Onoda, 1992; Guthrie et al., 1993; Sallaz and Jourdan, 1993; Guthrie and Gall, 1995) and [14C]2-deoxyglucose uptake in the olfactory bulb (Sharp et al., 1975; Stewart et al., 1979; Jourdan et al., 1980; Lancet et al., 1982; Royet et al., 1987; Sallaz and Jourdan, 1993; Johnson and Leon, 1996, 2000a,b; Johnson et al., 1998, 1999); volatage-sensitive dye (Kauer, 1991; Kauer et al., 1991; Kauer and Cinelli, 1993; Cinelli et al., 1995) and intrinsic signal imaging of odor-evoked neuronal activity in the bulb (Rubin and Katz, 1999, 2001; Uchida et al., 2000; Belluscio and Katz, 2001; Meister and Bonhoeffer, 2001); and electrophysiological recordings of sensory and bulbar neuron responses to odorants (e.g., Leveteau and MacLeod, 1966; Getchell, 1974; Getchell and Getchell, 1974; Kauer and Moulton, 1974; Getchell and Shepherd, 1978; Mori and Yoshihara, 1995; Yokoi et al., 1995; Kashiwadani et al., 1999). In 1991, a large multigene family encoding up to 1000 putative olfactory receptor (OR) proteins in rat OSNs was discovered (Buck and Axel, 1991). Homologous multigene families were then identified in some other vertebrates and in humans (see Lancet and Ben-Arie, 1993; Ressler et al., 1994a; Sullivan et al., 1995; Buck, 1996; Mombaerts, 1999; Glusman et al., 2000, 2001) (see Chapter 4). This discovery, confirmed at the functional level (Zhao et al., 1998), provided a vital clue to understanding mechanisms

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of odor coding in the olfactory system. In situ hybridization studies with specific gene probes have shown that (1) each OSN may express only one OR, (2) OSNs expressing the same OR are randomly scattered within one of four spatial zones of the olfactory epithelium, and (3) OR mRNAs are present in olfactory axons. The latter finding allowed the investigation of the projection pattern of OSNs expressing different ORs. Studies of OR gene-labeled olfactory axons in the olfactory bulb have demonstrated that (1) OSNs expressing a given OR project to two individual glomeruli located in the dorsomedial and ventrolateral portions of the bulb, (2) the position of these glomeruli is bilaterally symmetric and constant in different animals within a species, and (3) there is a clear correspondence between the number of genes and the number of glomeruli identified with each probe (Resssler et al., 1994a,b; Vassar et al., 1994; Buck, 1996; Mombaerts et al., 1996). These findings suggested that each glomerulus receives input from OSNs expressing a given OR and that information, broadly distributed in the olfactory epithelium, “is transformed in the bulb into a highly organized and spatially stereotyped information map, which is, in essence, a map of information provided by different ORs” (Buck, 1996). The OR protein appears to be an important, but not the sole, determinant in establishing such sensory map and maintaining its constancy throughout life (Singer et al., 1995; Mombaerts et al., 1996; Wang et al., 1998; Gogos et al., 2000). The results of functional and molecular studies indicated that a single odorant might activate a number of different ORs. This suggested that different ORs recognize different structural features of the odor molecule and map them onto distinct groups of glomeruli. According to this suggestion, a unique combination, or ensemble, of activated glomeruli would encode each odorous chemical. Systematic studies using 2-deoxyglucose autoradiography provided strong evidence that different odorants are represented by distinct spatial activity patterns in the glomerular layer and that modules of activity within these spatial patterns correlate with specific structural features of odor molecules (Johnson et al., 1998, 1999; Johnson and Leon, 2000a,b). These results “are consistent with a combinatorial mechanism of olfactory coding wherein unitary responses of olfactory receptors to odorant features would produce spatial patterns of bulbar activity that are characteristic for a given odorant” (Johnson et al., 1988). This mechanism allows for the discrimination of very subtle structural features that distinguish geometric isomers (Johnson and Leon, 2000b) and enantiomers (Rubin and Katz, 2001). Moreover, it has been found that an increase in the odorant concentration results in stimulation of additional glomeruli, located

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at a large distance from the glomeruli activated at lower concentrations (Johnson and Leon, 2000a). The gathering of voluminous data has led to a concept of “odor maps” in the olfactory bulb and allowed formulation of basic principles underlying their organization at the molecular, cellular, and systems level (Xu et al., 2000). This concept considers olfactory glomeruli as structural and functional modules that extend to the deep of the bulb, involving principal and intrinsic cells associated with the activated glomerulus. Such multicellular modules in the bulb are similar to columns and barrels in the cortex. Thus, it appears that glomerular modules synthesize, piece by piece, molecular information about odorant features, providing the basis for the recognition of odor quality and intensity, and the discrimination between odors. Perceptual reconstitution of odors occurs at higher levels of the olfactory system, giving birth to the sensation of smell.

III.

CENTRIFUGAL INNERVATION

The central nervous system controls and adjusts incoming flow and processing of afferent signals via centrifugal projections to various levels of sensory pathways (Hagbarth, 1960). The olfactory bulb is unique among primary sensory centers in receiving extraordinary dense centrifugal, or bulbopetal, inputs (Ramón y Cajal, 1911; Ottoson and Shepherd, 1967; Macrides and Davis, 1983; Kratskin, 1987; Halász, 1990; Shipley and Ennis, 1996). Two major groups of axons project to the bulb from brain. One group is comprised of the afferent fibers that arise from the primary olfactory cortex, whereas the other group is comprised of axons originating in nonolfactory structures of the basal forebrain and brainstem (Fig. 4). A.

Projections from the Primary Olfactory Cortex

The most prominent projection to the olfactory bulb originates in the anterior olfactory nucleus (AON), a structure that contains about 54% of bulbopetal neurons in the mouse brain (Carson, 1984a). All parts of the AON, except the pars externa, project to both olfactory bulbs; neurons of the pars externa send their axons exclusively to the contralateral bulb (Haberly and Price, 1978b; Davis and Macrides, 1981; Luskin and Price, 1983; Schoenfeld and Macrides, 1984). In the AON of rats, approximately 50% of bulbopetal neurons have bilateral projections with different branches of the same axon (Valverde, 1965). Afferents from the AON predominantly terminate in the granule cell layer and, to a lesser extent, in the internal plexiform layer and glomerular layer. Connections between the olfactory bulb and the AON pars externa are

Anatomy and Neurochemistry of the Olfactory Bulb

topographically organized; distinct sectors of the pars externa receive inputs from restricted areas of the ipsilateral bulb and project to corresponding areas in the contralateral bulb (Schoenfeld and Macrides, 1984). The dorsal peduncular cortex and ventral taenia tecta contain neurons projecting to the granule cell layer, internal plexiform layer, and glomerular layer of the ipsilateral olfactory bulb. The piriform cortex is another substantial source of projections to the olfactory bulb. In mice, this structure contains about 36% of bulbopetal brain neurons. Cells projecting to the bulb are located in layers IIb and III of the piriform cortex, with the highest density in its rostral

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part, and their axons terminate in the granule cell layer (Haberly and Price, 1978a). In addition, the piriform cortex is the source of a pathway reaching the olfactory bulb after a synaptic relay in the AON (Haberly and Price, 1978a; Davis and Macrides, 1981). Projections from the AON and piriform cortex are found in the olfactory bulb of rats at the time of birth (Schwob and Price, 1984). Relatively numerous bulbopetal cells (2.4% in the mouse brain) are found in the nucleus of the lateral olfactory tract. Axons arising from this nucleus project to the ipsilateral and contralateral olfactory bulbs (DeOlmos et al., 1978; Carson, 1984a), and terminate in the deep part of the granule cell layer (Davis and Macrides, 1981; Luskin and Price, 1983). Neurons projecting to the ipsilateral bulb are also located in the entorhinal cortex, anterior and posterolateral cortical amygdaloid nuclei, and in the periamygdaloid area (DeOlmos et al., 1978; Shipley and Adamek, 1984). B.

Figure 4 Sources of centrifugal projections to the olfactory bulb: 1, olfactory bulb; 2, anterior olfactory nucleus; 3, dorsal peduncular cortex; 4, ventral taenia tecta or anterior hippocampal rudiment; 5, nucleus of the vertical limb of the diagonal band; 6, nucleus of the horizontal limb of the diagonal band; 7, primary olfactory cortex; 8, lateral preoptic area; 9, lateral hypothalamus; 10, nucleus of the lateral olfactory tract; 11, posterolateral cortical nucleus of the amygdala; 12, raphe nuclei; 13, locus coeruleus. Broken lines show the amygdaloid nuclei and subdivisions of the anterior olfactory nucleus. Pathways of centrifugal fibers are shaded. (Adapted from DeOlmos et al., 1978.)

Projections from the Basal Forebrain and Brainstem

The nucleus of the horizontal limb of the diagonal band (NHDB) is the major source of bulbopetal axons arising from nonolfactory brain structures (Price, 1969; Price and Powell, 1970e; Macrides and Davis, 1983; Shipley and Ennis, 1996). The NHDB is a component of the basal forebrain system that innervates neocortex and hippocampus and plays an important role in learning and memory. There are two compartments in the NHDB of rats; the medial compartment contains small-to-medium-sized cells, whereas the lateral part is composed of large neurons and is often referred to as the magnocellular preoptic nucleus (Záborszky, et al., 1986). Both parts of the NHDB contribute 3.5% of bulbopetal cells found in the mouse brain (Carson, 1984a). Fibers from the NHDB reach the ipsilateral bulb within the medial forebrain bundle (DeOlmos et al., 1978; Macrides et al., 1981) and lateral olfactory tract (Price, 1969; Price and Powell, 1970e) and have restricted, nonoverlapping projection areas (Luskin and Price, 1982). Brain lesions and tract-tracing studies indicate that NHDB neurons project to the glomerular layer, granule cell layer, and EPL (Price, 1968; Price and Powell, 1970c; Godfrey et al., 1980a,b; Macrides et al., 1981). Local injections of biotin dextran amine into the medial and lateral compartments of the rat NHDB give rise to terminal arborizations in the glomerular layer and granule cell layer, respectively (Kratskin and Yu, 1996a). A few bulbopetal neurons (about 0.2% in the mouse brain) have been found in the ventral part of the nucleus of the vertical limb of the diagonal band, substantia innomi-

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nata, and the ventral pallidum (DeOlmos et al., 1978; Carson, 1984a; Záborszky, et al., 1986). A small number of bulbopetal cells are also located in the lateral and dorsomedial hypothalamic areas and in the subthalamic zona incerta (Carson, 1984a; Shipley and Adamek, 1984). Axons from these brain sources project to the ipsilateral olfactory bulb. The dorsal and median raphe nuclei are the source of the bilateral projection to the olfactory bulb. These unpaired midbrain nuclei contribute 0.5% (about 400 neurons) of the total number of bulbopetal cells in mice (Carson, 1984a). Almost 1300 raphe neurons project to the rat olfactory bulb (McLean and Shipley, 1987a). Tracttracing studies have shown that axons of raphe neurons pass within the medial forebrain bundle. These axons largely terminate around and within glomeruli and, to a lesser extent, in the deeper layers of the bulb (Bobillier et al., 1979; McLean and Shipley, 1987a). The locus coeruleus, the paired pontine nucleus, also projects to both olfactory bulbs. This nucleus contains about 0.4% of mouse bulbopetal cells (Carson, 1984a). In the rat, up to 40% of the 1600 neurons in the locus coeruleus send axons to the bulb (Shipley et al., 1985). Fibers from the locus coeruleus run in the medial forebrain bundle and terminate on different parts of granule cells in the granule cell layer and internal plexiform layer (Macrides et al., 1981; Halász, 1990). C.

General Characteristics

The characteristic features of centrifugal innervation of the mammalian olfactory bulb are as follows: (1) multiple afferent fibers to the bulb originate in both olfactory and nonolfactory brain structures; (2) there is no clear correspondence between the position of a bulbopetal neuron in the brain and the location of its terminal field in the bulb; (3) the AON and piriform cortex contain the vast majority (about 90% in the mouse) of bulbopetal neurons; (4) centrifugal axons largely project to the ipsilateral olfactory bulb, but the locus coeruleus, nucleus of the lateral olfactory tract, raphe nuclei, and the AON (except for the pars externa) have bilateral projections; (5) axons from the AON pars externa exclusively project to the contralateral bulb, and this projection is the only one that is topographically organized; (6) centrifugal axons mostly terminate on intrinsic neurons; and (7) fibers from all brain sources contact different parts of granule cells, whereas projections from the AON and nonolfactory brain structures also reach interneurons in the glomerular layer. Extensive centrifugal innervation of the primary olfactory center is observed across vertebrate species and has a conservative structural and functional organization

(Macrides and Davis, 1983; Kratskin, 1987, 1989; Halász, 1990). In bony fishes, amphibians, and reptiles, the olfactory bulb receives afferent fibers from the olfactorecipient regions of the telencephalon and nonolfactory structures of the forebrain and brainstem (Prasada Rao and Finger, 1984; Kratskin, 1987; Belekhova et al., 1995; DuchampViret and Duchamp, 1997; Lanuza and Halpern, 1998). Ultrastructural observations in the frog show that many centrifugal axons terminate on deep dendrites of granule cells (I. Kratskin, J. P. Rio, N. Kenigfest and J. Repérant, manuscript in preparation). D.

Functional Implications

Centrifugal inputs to the olfactory bulb are likely tonic in character (e.g., Paolini and McKenzie, 1997b) and may effectively influence bulbar processing by modulating the activity of local interneurons (see Linster and Gervais, 1996; Linster and Hasselmo, 1997). Reciprocal connections between the bulb and secondary olfactory centers from multiple feedback loops, which may serve to coordinate signal processing and self-regulation in the olfactory system. Inputs from the basal forebrain and brainstem likely exert modulatory influences on the bulbar neuronal network and provide interactions between olfactory and other sensory systems. Bulb output signals are transmitted directly to specific cortical zones, thus avoiding reticular modulation at the thalamic level of sensory processing. Projections from the raphe nuclei, which are part of the ascending reticular system, may allow for the reticular control over the olfactory input to brain. The projection from the lateral hypothalamus, a structure that receives inputs from the secondary olfactory centers, complete a complex path that may influence, for example, the organization of feeding behavior. The lateral hypothalamic area projects to the NHDB, and “olfactory information does reach the nucleus of origin of the olfactory centrifugal fibres, but only after convergence of these different pathways upon the hypothalamus and interaction with midbrain and hypothalamic influences” (Price and Powell, 1970f ). Inputs to the olfactory bulb from the NHDB, whose neuron activity may be modulated by the bulbar output (Paolini and McKenzie, 1997a; Linster and Hasselmo, 2000), are believed to play an important role in olfaction (Paolini and McKenzie, 1993, 1996). The function of the olfactory bulb is likely not confined to the sense of smell. It is possible that, at least in nonhuman mammals, the bulbs perform not only sensory functions, but are also directly involved in nonspecific, limbic-related mechanisms of arousal and forebrain excitation (Herrick, 1933; Cain, 1974; Wenzel, 1974; Shepherd et al., 1981). Diverse

Anatomy and Neurochemistry of the Olfactory Bulb

behavioral changes, alterations in learning and memory, and impairments of brain transmitter systems are observed in bulbectomized rats (e.g., Kelly et al., 1997; Yamamoto et al., 1997), suggesting a complex function of the olfactory bulbs. This may be one of the reasons why the olfactory bulb has an enormously rich supply of centrifugal fibers, a feature that distinguishes it from other primary sensory centers.

IV. NEUROTRANSMITTERS AND MODULATORS The olfactory bulb “appears to be a veritable cornucopia of putative transmitters and neuroactive peptides” (Macrides and Davis, 1983), “for the number and variety of which it rivals all other regions of the brain” (Halász and Shepherd, 1983). The presence of some transmitters in the bulb is entirely associated with centrifugal axons, thus contributing to its neurochemical diversity. The transmitters and modulators proposed for bulb neurons and afferent fibers are shown in Figure 5. A.

Olfactory Axons

1.

Transmitter Glutamate

Glutamate (Glu) is enriched in axon terminals of OSNs in the olfactory bulb (Sassoè-Pognetto et al., 1993; Didier et al., 1994). Electrophysiological studies provided evidence that Glu is a transmitter at excitatory synapses between olfactory axons and dendrites of mitral / tufted and PG cells within the glomeruli (Berkowicz et al., 1994; Bardoni et al., 1996; Ennis et al., 1996; AroniadouAnderjaska et al., 1997, 1999a). Two types of ionotropic Glu receptors (GluRs) mediate postsynaptic responses evoked in mitral cells by stimulation of olfactory axons. The early fast response is due to activation of the AMPA type GluRs, whereas GluRs of the NMDA type mediate the late long-lasting excitation. Prolonged NMDA GluR-mediated postsynaptic activity appears to facilitate synaptic integration and plasticity and, thus, may play an important role in olfactory processing and memory (AroniadouAnderjaska et al., 1997; Ennis et al., 1998). Subunits of various GluRs are expressed in the olfactory bulb, and some of them are located on dendrites within glomeruli (Trombley and Shepherd, 1993; Giustetto et al., 1997; Shepherd and Greer, 1998; Montague and Greer, 1999). 2.

Modulation of Glutamate Release

Several types of metabotropic Glu receptors (mGluRs) are present in different regions of the bulb, including glomeru-

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lar neuropil (see Trombley and Shepherd, 1993; Shepherd and Greer, 1998). Some mGluRs are involved in postsynaptic effects of Glu, whereas those located on terminals of olfactory axons (Kinzie et al., 1997) are “autoreceptors” that may regulate presynaptic release of the transmitter. GABAB receptors, detected on terminals of olfactory axons (Bonino et al., 1999), also represent autoreceptors, and these definitely modulate transmission at the primary olfactory synapse (Potapov, 1985; Nickell et al., 1994; Keller et al., 1998; Aroniadou-Anderjaska et al., 2000). GABA released from interneurons in the glomerular region triggers both tonic and stimulus-evoked inhibition of Glu release, thus reducing postsynaptic responses. Even a single impulse coming from OSNs can evoke sufficient GABA release to activate presynaptic GABAB receptors (Aroniadou-Anderjaska et al., 2000). This regulatory mechanism may serve to shape activity patterns in glomerular modules. D2 dopamine (DA) receptors that are located on terminals of olfactory axons (Nickell et al., 1991; Koster et al., 1999) provide another possibility for regulating Glu release (Hsia et al., 1999; Berkowicz and Trombley, 2000). A decrease in presynaptic Glu release, induced by D2 receptor activation, may be due to intraglomerular actions of various bulb neurons (see below). Thus, different autoreceptors may modulate the efficacy of sensory input to the olfactory bulb. 3.

Taurine

The amino acid taurine (Taur) is produced by OSNs (Kratskin and Hao, 2001), and terminals of olfactory axons co-localize Glu and Taur (Didier et al., 1994; Kratskin and Yu, 1996b). Taur is abundant in the brain and is known to cause neuronal inhibition (see Puopolo et al., 1998; Kratskin et al., 2000). Several factors are involved in regulating Taur release (Oja and Saransaari, 2000). Olfactory axons, in particular, may release Taur via the gaseous second messenger nitric oxide, which is present in OSN terminals within the bulb (Broillet and Firestein, 1996), as well as upon depolarization and activation of presynaptic mGluRs. Application of Taur to a slice preparation of rat olfactory bulb produces strong inhibition of mitral and tufted cells (Puopolo et al., 1998). This action is due to direct GABAA receptor activation and does not involve glycine receptors, which are expressed by bulb neurons (Trombley and Shepherd, 1993, 1994; Trombley et al., 1999). Further experiments have shown that Taur does not influence the membrane potential of PG cells (O. Belluzzi, M. Puopolo, and I. Kratskin, manuscript in preparation). Such a difference in the effects of Taur may be related to the differential subunit composition of

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Figure 5 Neurotransmitters and modulators in the olfactory bulb: ACh, acetylcholine; Carn, carnosine; CCK, cholecystokinin; DA, dopamine; Enk, enkephalin; Glu, glutamate; 5-HT, serotonin; LHRH, luteinizing hormone releasing hormone; NE, norepinephrine; OMP, olfactory marker protein; SOM, somatostatin; SP, substance P; Taur, taurine. Small arrows show the direction of synaptic transmission; solid arrows indicate centrifugal projections to the bulb. (Adapted from Halász and Shepherd, 1983.)

GABAA receptors from mitral/tufted and PG cells (see Laurie et al., 1992; Persohn et al., 1992; Fritschy and Mohler, 1995), implying specific molecular structure requirements of GABAA receptors for Taur sensitivity. In these studies, Taur significantly reduced GluR-mediated excitatory currents evoked in external tufted cells by olfactory axon stimulation. The GABAB receptor antagonist CGP55845A blocked this effect (which could not be ascribed to GABAA receptor activation), suggesting that Taur acts on presynaptic GABAB receptors and decreases Glu release. It is possible that one function of Taur in the olfactory bulb is to moderate the excitability of principal cells at both pre-and postsynaptic levels.

4.

Olfactory Marker Protein, Carnosine, Zinc

OSNs express a specific protein called olfactory marker protein (OMP) and the dipeptide carnosine (Margolis et al., 1986). OMP gene deletion in mice alters the ability of OSNs to generate the electro-olfactogram, suggesting that the neural activity directed toward the bulb is also decreased (Buiakova et al., 1996). Carnosine and Glu are co-localized in olfactory axon terminals (Sassoè-Pognetto et al., 1993), which also contain high levels of zinc (see Trombley and Shepherd, 1993). Bath application of carnosine increases membrane conductance in cultured neurons of the bulb (Kanaki et al., 1997). Furthermore,

Anatomy and Neurochemistry of the Olfactory Bulb

carnosine reduces inhibitory actions of zinc on NMDA and GABA receptor-mediated currents and synaptic transmission (Trombley et al., 1998). These observations suggest that carnosine released from olfactory axons may modulate the excitability of bulb neurons.

B.

Principal Cells

1.

Mitral Cells

Mitral cells and internal tufted cells contain Glu (e.g., Ottersen and Storm-Mathisen, 1984; Liu et al., 1989), and a high density of GluRs is found in the EPL (Cotman et al., 1987), where principal neurons make numerous synaptic contacts. Electrophysiological and pharmacological analyses provide evidence that Glu is a transmitter in mitral / tufted-to-granule dendrodendritic synapses and suggest that granule cell excitation is mediated predominantly by NMDA GluRs (Isaacson and Strowbridge, 1998; Schoppa et al., 1998; Chen et al., 2000; Halabisky et al., 2000; Christie et al., 2001; Isaacson, 2001). Calcium influx through NMDA GluRs may trigger directly GABA release from granule cell dendrites. It has been shown that, under particular conditions, AMPA GluRs can mediate synaptic actions of mitral cells (Isaacson, 2001). Indeed, a recent immunocytochemical study localized NMDA and AMPA GluRs at postsynaptic sites on granule cell dendritic spines, and revealed the spatial organization of these receptors (Sassoè-Pognetto and Ottersen, 2000). Various GABAA receptor subunits are found in the glomerular layer and EPL, where mitral cells have numerous synaptic contacts (Pirker et al., 2000). Several groups of investigators have shown that Glu released from both apical and basal dendrites of mitral/tufted cells causes activation of the same and neighboring principal cell dendrites (AroniadouAnderjaska et al., 1999a,b; Isaacson, 1999; Friedman and Strowbridge, 2000; Salin et al., 2001). The NMDA type autoreceptors were found to mediate self-excitation of principal cells at dendritic sites. No synaptic contacts between dendrites of mitral/tufted cells have been observed in mammals, so the process of self-excitation is thought to be nonsynaptic in nature. It may be noted, however, that an ultrastructural study of the salamander olfactory bulb revealed type 1 synapses between dendrites of principal cells in the glomerular layer and EPL (Allen and Hamilton, 2000). In addition to ionotropic GluRs, mitral cells express mGluRs, and these are located on dendrites within glomeruli (van den Pol, 1995; Kinzie et al., 1997). Activation of mGluRs results in suppression of Glu release from mitral cell terminals (Schoppa and Westbrook, 1997). These findings suggest that both GluRs

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and mGluRs are involved in regulating transmitter release from mitral cell endings. 2.

Tufted Cells

Dopamine (DA) is a putative transmitter in external and middle tufted cells. Several studies have shown that many tufted cells exhibit immunoreactivities for the enzymes of DA synthesis tyrosine hydroxylase (TH) and dopadecarboxylase (see Davis and Macrides, 1983; Halász, 1990). In the olfactory bulb of hamsters, more than 80% of TH-containing neurons are external tufted cells. THimmunostained external tufted cells are also present in the human olfactory bulb (Smith et al., 1991). Type D1 and D2 receptors for DA are expressed in the glomerular layer, EPL, and the mitral cell layer (Coronas et al., 1997). External naris closure in rats produces a rapid and long-lasting decrease in the TH activity and expression of TH mRNA in the olfactory bulb (Cho et al., 1996), as well as a reduction in the DA content (Philipot et al., 1998). However, high-frequency stimulation of the olfactory nerve in rats whose external nares have been closed results in a partial and temporary increase in the DA levels. These findings demonstrate that sensory input regulates DA production in the olfactory bulb. Recent in vitro experiments have confirmed the notion that sensorydependent TH expression occurs in bulb neurons and have shown that NMDA GluRs may mediate the influence of sensory input (Puche and Shipley, 1999). Substance P is found in external tufted cells of the hamster (Burd et al., 1982), and mRNA encoding substance P is expressed in external tufted cells of rats (Warden and Young, 1988). In rats, external tufted cells, which constitute the intrabulbar associational system, are immunoreactive for cholecystokinin (Liu and Shipley, 1994). C.

Intrinsic Neurons

1.

Periglomerular Cells

GABA and DA are the most likely transmitters of PG cells (Mugnaini et al., 1984; Halász, 1990; Kosaka et al., 1995; Toida et al., 2000). Studies in rats have shown the coexistence of TH and the GABA-synthesizing enzyme glutamic acid decarboxylase (GAD) or TH and GABA in PG cells (Kosaka et al., 1985; Gall et al., 1987). Indeed, 30–70% of immunoreactive cells produce both GABA and DA. Many PG cells contain enkephalin and substance P (Davis et al., 1982; Halász, 1990). Co-localization of enkephalin with GABA and/or TH (Kosaka et al., 1995) and coexistence of substance P with GABA and DA (Kosaka et al., 1988) have been observed in PG cells. It is generally believed that PG cells are largely inhibitory in nature. It has been

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shown that inhibitory synaptic interactions between neighboring PG cells are mediated by GABAA receptors (Puopolo and Belluzzi, 1998b). However, somata and dendrites of PG cells may accumulate high concentrations of C1 (Siklos et al., 1995), thus providing the basis for excitatory actions of GABA observed in the glomeruli (Rhoades and Freeman, 1990). The lack of odor stimulation (without denervation of the bulb) results in an alteration in DA, but not GABA, production in the PG cells. This suggests differential regulation of the transmitterspecific phenotype of PG cells by OSN activity–dependent factors (Baker, 1990). On the other hand, the density of synapses, made by olfactory axons, is greater on TH than on GABA-immunostained cell processes; therefore, GABA-containing PG cells may be less sensitive to the lack of sensory input (Bartolomei and Greer, 1993). DA, GABA, and substance P were observed in presumed PG cells of the human olfactory bulb (Ohm et al., 1990; Smith et al., 1991, 1993). 2.

Granule Cells

Granule cells are the most neurochemically homogeneous intrinsic neurons, and GABA most fully satisfies the traditional criteria for transmitter identification, clearly being a transmitter of granule cells (Halász and Shepherd, 1983; Halász, 1990). The highest levels of GABA, the greatest activity of its metabolic enzymes, GAD and GABAaminotransferase, as well as release of GABA and its specific uptake, are found in the EPL. The sites of dendrodendritic synapses between granule and mitral/tufted cells have the highest level of 3H-GABA binding and very high GAD activity. Nearly all somata and dendrites of granule cells display immunoreactivities for GAD and GABA. GAD-immunostained granule cells are also observed in the bulb of humans (Ohm et al., 1990). Many granule cells in the rat and hamster contain enkephalin; granule cell dendrites immunostained for enkephalin are found in the EPL (Davis et al., 1982; Matsutani et al., 1988). Accordingly, a high level of -receptor binding sites is observed in the EPL (McLean et al., 1986). In the rat granule cell layer, 95% of enkephalin-stained cell bodies are immunoreactive for GAD, whereas only 11% of GAD-immunostained somata show immunoreactivity for enkephalin. This suggests that enkephalin-containing neurons represent a subpopulation of GABAergic granule cells (Kosaka et al., 1987). Granule cells express NMDA and AMPA GluRs (Sassoè-Pognetto and Ottersen, 2000) and a specific type of mGluRs (van den Pol, 1995). Striking results have been recently obtained using a combination of whole-cell recordings in a slice preparation of rat olfactory bulb and electron microscopic immunocytochemistry (Didier et al.,

2001). These results suggest that there are granule cells that contain Glu in synaptic vesicles and exert NMDA GluR-mediated postsynaptic excitation of mitral cells in the EPL. Whether the same granule cell may release both GABA and Glu is unknown. 3.

Short Axon Cells

Short axon cells likely use GABA and DA as neurotransmitters (Halász, 1990). The coexistence of these substances has been reported in the superficial short axon cells of rats (Gall et al., 1987). In rats and hamsters, some short axon cells are immunopositive for enkephalin and somatostatin (Davis et al., 1982). Presumptive short axon cells containing somatostatin, GABA, and DA have been described in the bulb of humans (Ohm et al., 1988, 1990; Smith et al., 1993). D.

Centrifugal Fibers

1.

Serotonin

The only source of serotonin (5-HT) in the bulb is the projection from the raphe nuclei (McLean and Shipley, 1987a; Araneda et al., 1989). Some bulbopetal raphe neurons co-localize 5-HT and galanin or somatostatin (Araneda et al., 1999). The density of 5-HT fibers in the glomerular layer is two to three times that of any other layer; depletion of 5-HT from axons in the bulb produces shrinkage of glomeruli (Moriizumi et al., 1994). 5-HT-containing fibers are present in the glomerular region of the human bulb (Smith et al., 1993). During rat postnatal development, the rate of 5-HT fiber arborization exceeds the growth rate of glomeruli (McLean and Shipley, 1987b). 5HT receptor subtypes 1A and 2A are dominant in the bulb (Wright et al., 1995). While immunostaining localized 2A receptors to PG and granule cells (Morilak et al., 1993), in situ hybridization studies showed expression of specific mRNA in mitral and tufted cells (Pompeiano et al., 1994; McLean et al., 1995). Surprisingly, no 5-HT receptors were found in the glomeruli. An involvement of 5-HT in olfactory learning (McLean et al., 1993) and odor discrimination in rats (Moriizumi et al., 1994) has been shown. 2.

Norepinephrine

Norepinephrine (NE) is also not intrinsic to the olfactory bulb. NE-containing fibers arise from the locus coeruleus and terminate on dendrites and somata of granule cells in the internal plexiform and granule cell layers (Macrides et al., 1981; Font et al., 1982; McLean et al., 1989; Halász, 1990). Postnatal maturation of this input (McLean and

Anatomy and Neurochemistry of the Olfactory Bulb

Shipley, 1991) correlates with the development of NE influences on the interaction between granule and mitral cells (Wilson and Leon, 1988). Both  and  NE receptors are present the olfactory bulb (Woo and Leon, 1995; Shipley and Ennis, 1996). Locus coeruleus lesion with 6-hydroxydopamine reduced the NE content of the bulb (Sullivan et al., 1993) and increased the density of 1 and 2 receptors (Woo et al., 1995). About 20% of bulbopetal neurons in the locus coeruleus co-localize NE and neuropeptide Y (Bouna et al., 1994). Only inhibitory actions of NE on mitral cells were found in vivo (Salmoiraghi et al., 1964; McLennan, 1971), whereas NE-evoked mitral cell excitation was observed in isolated turtle olfactory bulb (Jahr and Nicoll, 1982). In bulb cell culture, NE suppressed granule-to-mitral cell inhibition (Trombley and Shepherd, 1992) due to 2 receptor–mediated reduction of mitral cell Ca2 currents (Trombley, 1992). Activation of the locus coeruleus (Jiang et al., 1996) and application of NE to a bulb slice preparation (Ciombor et al., 1999) increased rat mitral cell responses to perithreshold olfactory nerve stimulation. This suggests that one function of NE in the bulb is to enhance detection of weak odor stimuli. Both sectioning the olfactory peduncle and  antagonist injection decreased the number of c-fos–containing granule cells in odor-specific areas, suggesting NE-mediated centrifugal influences on c-fos expression in the bulb (Sallaz and Jourdan, 1996). Behavioral studies showed NE modulatory influences on formation of specific odor memories (Royet et al., 1983; Sullivan et al., 1993, 2000). 3. GABA The observation of decreased levels of GABA in the bulb following its central deafferentation (Godfrey et al., 1980a), the finding by electron microscopy of 3H-GABA–labeled terminals in the granule cell layer (which did not resemble intrinsic neuron terminals) (Halász, 1990), and early pharmacological experiments (McLennan, 1971) have pointed to GABA as a transmitter in centrifugal axons. This was definitively established when GABAergic bulbopetal neurons were found in the rat NHDB (Záborszky et al., 1986). About 30% of bulbopetal cells in this structure are GABAergic; most of them are located caudally in the lateral NHDB compartment. A few such cells are also observed in the ventral pallidum, anterior amygdaloid area, piriform cortex, and nucleus of the lateral olfactory tract. GABAergic centrifugal innervation is likely not specific to the mammalian bulb, as GABA-stained bulbopetal cells are found in the amphibian brain as well (Kratskin et al., 1991, 1992, 1997). A field potential study suggested that continuous electrical stimulation (at 10 Hz) of the lateral NHDB in

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rats causes inhibition of mitral cells and that this effect might be due to granule cell activation (Nickell and Shipley, 1988b). Intracellular recordings, however, demonstrated inhibition of granule cells and facilitation of mitral cells following single- or brief multiple-pulse stimulation of the lateral NHDB (Kunze et al., 1991, 1992a,b). These observations were consistent with the activation of an inhibitory input to granule cells. A subsequent study using extracellular recordings showed that suppression of tonic neuronal activity in the lateral NHDB by the long-lasting GABA agonist muscimol results in sustained facilitation of presumed tufted cells in the EPL and does not influence mitral cell firing (Paolini and McKenzie, 1997b). Changes in unit activity, observed in the granule cell layer, suggested complex interactions between granule and tufted cells. Further studies are needed to elucidate the influence of the GABAergic input on neurons of the olfactory bulb. 4.

Acetylcholine

There is evidence that acetylcholine (ACh) is a transmitter of bulbopetal neurons. Bulbar cells do not express mRNA encoding choline acetyltransferase (ChAT), the enzyme of ACh synthesis (Oh et al., 1992), and central deafferentation of the olfactory bulb almost completely decreases its ChAT activity (Godfrey et al., 1980b). While several studies have shown no bulb neurons immunopositive for ChAT (see Kratskin, 1989; Halász, 1990), some authors have observed ChAT-stained cells in the rat olfactory bulb (Ojima et al., 1988; Phelps et al., 1992). However, the number of these cells was very small, suggesting that ACh fibers in the bulb mostly originate from extrinsic sources. The main source of cholinergic afferents to the olfactory bulb is the NHDB (Carson, 1984b; Záborszky et al., 1986). In the rat NHDB, up to 20% of bulbopetal neurons are cholinergic, and most of them are located rostrally in the medial compartment of the NHDB. Some cholinergic cells in the NHDB give rise to divergent projections to the bulb and hippocampus (Okoyama et al., 1987). Presumably cholinergic bulbopetal neurons are located in the NHDB of frogs (Kratskin and Ragimova, 1985). Cholinergic fibers terminate in the glomerular layer, EPL, and, to a lesser extent, in the internal plexiform and granule cell layers. Ultrastructural studies have shown that ChAT-stained terminals make synapses mainly with dendrites/somata of intrinsic neurons, i.e., PG cells, short axon cells, and granule cells, and also with external tufted cells (Le Jeune and Jourdan, 1993; Kasa et al., 1995). Importantly, many of these contacts were of the symmetric type, which is generally associated with inhibitory synaptic actions. Cholinoceptive cells have been studied by light and electron microscopic histochemistry of the ACh-

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degrading enzyme acetylcholinesterase (AChE). These studies suggest that likely cholinoceptive neurons are short axon cells across the bulb and TH-stained tufted cells; a few PG cells, but not granule cells, are also stained for AChE (e.g., Nickell and Shipley, 1988a; Le Jeune and Jourdan, 1994). It is possible, however, that cholinoceptive bulb cells have no AChE or that its activity is present in neurons that do not receive cholinergic input (Kasa et al., 1996). ChAT-stained fibers, but not cell bodies, are found in the human olfactory bulb; such fibers form a dense plexus around glomeruli (S. Arnold, personal communication, 1999). The possibility that alterations in cholinergic innervation of the bulb may be one of the causes of olfactory dysfunction in Alzheimer’s disease is discussed (e.g., Kasa et al., 1997; Durand et al., 1998). Both muscarinic and nicotinic ACh receptors (mAChRs and nAChRs) are expressed in the olfactory bulb (see Shipley and Ennis, 1996). The EPL has the highest concentration of mAChRs in the brain (Rotter et al., 1979). Intermediate levels of type 1 and 2 mAChRs are observed deep to the EPL, whereas the glomerular layer shows the lowest density of mAChRs. In contrast, nAChRs are concentrated in superficial layers, including the glomerular layer (Le Jeune et al., 1995). At the ultrastructural level, type 1 and 2 mAChRs are found on dopaminergic and GABAergic cells that receive input from olfactory axons, on granule cell gemmules in the EPL, and somata and dendrites of deep short axon cells (Crespo et al., 2000). In general, the laminar distribution of AChRs correlates with that of ChAT-stained terminals and AChE-stained cells, suggesting that different AChR types may mediate cholinergic actions at different levels of odor processing. It is also possible that excitatory and inhibitory effects of ACh on bulb neurons are mediated by nAChRs and mAChRs, respectively (Castillo et al., 1999). Atypical glomeruli receive a dense supply of cholinergic axons but fail to express any AChRs (Zheng et al., 1987; Le Jeune and Jourdan, 1993; Le Jeune et al., 1995). In development, cholinergic input and nAChRs appear much earlier than mAChRs (Le Jeune et al., 1996). An increase in the number of mAChRs (within the first 4 weeks of life) likely coincides with granule cell maturation. Electrical stimulation of the NHDB reduces the bulb field potential evoked by activation of the anterior commissure, suggesting that bulbopetal input from the NHDB regulates transmission of olfactory information between hemispheres (Nickell and Shipley, 1993). Iontophoresis of ACh produces both facilitation and depression of spontaneously active neurons in different layers of the rat olfactory bulb (Ravel et al., 1990). Local infusion of ACh, nicotine, and the irreversible AChE inhibitor soman increases the basal NE content of the

Kratskin and Belluzzi

bulb, whereas a muscarinic agonist has an opposite action (El-Etri et al., 1999). This suggests that release of NE from centrifugal fibers is differentially regulated through different AChRs. There is a possibility that presynaptic nAChRs facilitate Glu-mediated synaptic transmission in the bulb (Girod et al., 2000). 5.

Other Neuroactive Substances

Centrifugal fibers terminating in different regions of the olfactory bulb may contain excitatory amino acids, such as Glu and aspartate, DA, and various peptides. In particular, atypical glomeruli in rats receive a dense supply of axons displaying immunoreactivity for luteinizing hormone–releasing hormone (Zheng et al., 1988). Other peptides found in bulbopetal fibers include substance P, enkephalin, somatostatin, neuropeptide Y, oxytocin, vasopressin, and cholecystokinin. The sources of these projections remain unknown. Most likely, bulbopetal neurons co-localize peptides and “classical” transmitters, but the significance of such coexistence is not understood. ACKNOWLEDGMENTS The authors wish to acknowledge the support of the National Institutes of Health (grant DC04083; I. K.) and MURST-Cofin (O. B.). REFERENCES Adrian, E. D. (1950). The electrical activity of the mammalian olfactory bulb. EEG Clin. Neurophysiol. 2:377–388. Adrian, E. D. (1951). Olfactory discrimination. Ann. Psychol. 50:107–113. Allen, D. M., and Hamilton, K. A. (2000). Ultrastructural identification of synapses between mitral/tufted cell dendrites. Brain Res. 860:170–173. Allison, A. C., and Warwick, T. T. (1949). Quantitative observations on the olfactory system of the rabbit. Brain 72:186–196. Altman, J. (1969). Autoradiographic and histological studies of postnatal neurogenesis. IV. Cell proliferation and migration in the anterior forebrain, with special reference to persisting neurogenesis in the olfactory bulb. J. Comp. Neurol. 137:433–458. Araneda, S., Magoul, R., and Calas, A. (1989). Tracing specific transmitter pathways in the rat CNS: combination of [3H] serotonin retrograde labeling with immunocytochemical detection of endogenous transmitters. J. Neurosci. Meth. 30:211–218. Araneda, S., Gysling, K., and Calas, A. (1999). Raphe serotonergic neurons projecting to the olfactory bulb contain galanin or

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8 Central Olfactory Structures Thomas A. Cleland and Christiane Linster Cornell University, Ithaca, New York, U.S.A.

In this chapter we review central olfactory structures (Fig. 1), with an emphasis on those receiving direct inputs from the olfactory bulb, generally referred to as secondary olfactory structures (Fig. 2) (see Chapter 7). We will first provide an overview of these secondary olfactory structures, then describe their common organizational principles, provide detailed descriptions of the incoming and outgoing projections of each structure, and finally discuss evidence regarding their putative olfactory functions.

I.

including the bullfrog (Kemali and Guglielmotti, 1987; Northcutt and Royce, 1975; Scalia et al., 1991) and snake (Halpern, 1976). The major secondary olfactory structures described in mammals and discussed in this chapter include (listed roughly rostrocaudally): the anterior olfactory nucleus; a group of rostromedial cortices including the ventral tenia tecta, anterior hippocampal continuation, and indusium griseum; the olfactory tubercle; the anterior and posterior piriform cortices and endopiriform nucleus; the periamygdaloid cortex and anterior cortical nucleus of the amygdala; and the lateral entorhinal cortex. Despite a basic conservation of bulbar projection patterns among vertebrates, and particularly among mammals, there are nonetheless important species differences. Such differences may provide insight into the multiple mechanisms by which different species employ olfactory information to solve similar adaptive problems and ultimately contribute to an understanding of the respective roles played by the diverse central structures receiving olfactory information. In general, the canonical features of the secondary olfactory projections described in this review are derived primarily from studies in rat, and secondarily from studies in mouse and primates; however, data from other species are also reviewed to emphasize specific points and highlight the commonalities and diversity of the vertebrate radiation. All secondary olfactory structures are paired, except for interhemispheric commissures; there is no evidence of asymmetry in the anatomy or function of any of these areas. The axons of mitral cells and a subset of tufted cells emerge from the olfactory bulb, forming the olfactory peduncle; this path is also the route of the rostral migratory

OVERVIEW OF SECONDARY OLFACTORY STRUCTURES

Secondary olfactory structures include all areas of the brain to which mitral and tufted cell axons from the olfactory bulb (OB) directly project. This term is synonymous with the common term primary olfactory cortices (de Olmos et al., 1978) (see also Haberly, 2001; Halasz, 1990; Price, 1973, 1987; Shipley, 1995); however, the latter term is not used in this chapter, as recognition of the olfactory bulb as a cortical structure has rendered it ambiguous. The centripetal projection patterns of bulbar mitral and tufted neurons have been described in several mammalian species, among them being the rat (Price, 1973), opossum (Meyer, 1981; Scalia and Winans, 1975; ShammahLagnado and Negrao, 1981), monkey (Turner and Mishkin, 1978; Turner et al., 1978), hamster (Davis et al., 1978), rabbit (Broadwell, 1975a), tree shrew (Skeen and Hall, 1977), and hedgehog (Radtke-Schuller and Kunzle, 2000), as well as in several nonmammalian vertebrates 165

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stream, along which new presumptive olfactory bulb neurons migrate throughout life from progenitor cells in the subventricular zone (Jankovski et al., 1998; Meisami and Hamedi, 1986). Within the olfactory peduncle, and immediately caudal to the olfactory bulb, lies the anterior olfactory nucleus (AON), actually a cortical structure incorporating several morphologically diverse subdivisions with characteristic projection patterns. The AON is predominantly two-layered (with superficial plexiform and deep cellular layers) but gradually assumes a trilaminar form near its caudal extreme adjacent to the anterior commissure (Halasz, 1990; Valverde et al., 1989). Among

Figure 1 Overview of olfactory structures. Each of the panels shows a sagittal section through a rat brain at different lateral locations from medial to lateral. (A) Sagittal section at 0.4 mm lateral from bregma, showing rostromedial olfactory cortices and the olfactory tubercle. OB: olfactory bulb; AOM, AOP: medial and posterior anterior olfactory nucleus; VTT: ventral tenia tecta; AHC: anterior hippocampal continuation (also known as dorsal tenia tecta); Tu: olfactory tubercle; DP: dorsal peduncular cortex; IG: indusium griseum. CA1, CA3, DG: hippocampus; MO: medial orbitofrontal cortex; IL: infralimbic cortex; Cg1 and Cg2: cingulate cortex; ac: anterior commissure; MTN: medial thalamic nuclei; DA and AH: dorsal and anterior hypothalamic areas; VMH: ventromedial hypothalamic nucleus. (B) Sagittal section at 2.4 mm lateral from bregma. AOL: lateral anterior olfactory nucleus; lo: lateral olfactory tract; Pir: piriform cortex; VO: ventral orbitofrontal cortex; DEn: dorsal endopiriform nucleus; aca: anterior part of the anterior commissure; HDB: nucleus of the horizontal limb of the diagonal band; LOT: nucleus of the lateral olfactory tract; STh: subthalamic nuclei; VT: ventral thalamic nuclei. (C). Sagittal section at 3.9 mm lateral from bregma. LO: lateral orbitofrontal cortex; DEn and VEn: dorsal and ventral endopiriform nucleus; Pir: piriform cortex; lo: lateral olfactory tract; CxA: periamygdaloid cortex (also known as the cortex-amygdala transition zone); ACo: anterior cortical amygdaloid nucleus; PMCo: posteriomedial cortical amygdaloid nucleus: LEnt: lateral entorhinal cortex; CA1, CA2, CA3, DG: hippocampus. (Adapted from Paxinos and Watson, 1986.)

Figure 2 Schematic depiction of canonical secondary olfactory projections. Pathways depicted represent the most commonly reported connections and are neither exhaustive nor universal. (A) Projections from the olfactory bulb to secondary olfactory structures. Directionality is implicit. (B) Projections from secondary olfactory structures to the olfactory bulb. Directionality is implicit. (C) Associative connections among secondary olfactory structures and output projections to prominent tertiary olfactory structures. Directionality denoted by arrows. OB: olfactory bulb; AON: anterior olfactory nucleus; aPC and pPC: anterior and posterior piriform cortex; PC: piriform cortex (anterior and posterior); EC: entorhinal cortex; Tu: olfactory tubercle; PAC: periamygdaloid cortex; ACo: anterior cortical amygdaloid nucleus; VTT: ventral tenia tecta; AHC: anterior hippocampal continuation; IG: indusium griseum; IC: insular cortex; OC: orbitofrontal cortex; HT: hypothalamus; Th: thalamus.

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other possible functions, the AON (via the anterior commissure) mediates interhemispheric communication between the olfactory bulbs in mammals. Dorsomedial to the AON, and medial to the lateral olfactory tract, lie several secondary olfactory structures collectively termed the rostromedial olfactory cortices: the indusium griseum (also known as the dorsal hippocampal continuation or the supracallosal gyrus), the anterior hippocampal continuation (also known as the dorsal tenia tecta), and the ventral tenia tecta, which medially adjoins the caudal AON (Carmichael et al., 1994; Haberly and Price, 1978a,b; Kier et al., 1995; Luskin and Price, 1983b; Shipley and Adamek, 1984; Wyss and Sripanidkulchai, 1983). The olfactory tubercle is also sometimes included in this group (Shipley, 1995) (see below), as is the dorsomedial peduncular cortex (Haberly, 1998; Haberly and Price, 1977; Halasz, 1990). The anterior hippocampal continuation and indusium griseum are structurally comparable to, and often considered part of, the hippocampal formation (Adamek et al., 1984; Wyss and Sripanidkulchai, 1983); they probably derive from the medial (limbic) pallium, as does the ventral tenia tecta (Kier et al., 1995). Just caudal to the olfactory peduncle, and medial to the lateral olfactory tract and cortices, lies the distinctive olfactory tubercle. The olfactory tubercle, like the amygdaloid complex and the basal ganglia, is derived from the striatal subpallium (Butler and Hodos, 1996; Heimer and Wilson, 1975), though in architecture it varies between cortical (predominant in the lateral tubercle, adjacent to the lateral olfactory tract) and striatal (predominant in the medial tubercle) organization (Heimer and Wilson, 1975; Millhouse and Heimer, 1984). The olfactory tubercle caudally adjoins the rostromedial olfactory cortices and has been grouped together with them by some authors, differentiating them collectively from the anterior olfactory nucleus and from the continuous lateral olfactory cortices (Haberly, 2001; Shipley, 1995). However, it may ultimately be preferable to consider the olfactory tubercle (a part of the ventral striatum, with its subpallial and diencephalic projections and its lack of associative connections with other olfactory cortices) separately from the rostromedial cortices (with their probable medial pallial derivation and interconnectivity with the hippocampal formation). Laterally, bulbar mitral and tufted cell axons exit the peduncular region following the lateral olfactory tract (LOT). The LOT is heavily myelinated, though it also contains numerous unmyelinated fibers which, at least in cat, may outnumber the myelinated axons (Price and Sprich, 1975; Willey et al., 1983). Collaterals from these axons enter the anterior and posterior piriform cortices

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(defined in Haberly, 1985, 1998, 2001) and the lateral entorhinal cortex, derivatives of the lateral pallium, as well as the transitional periamygdaloid cortex and the anterior cortical nucleus of the corticomedial division of the amygdaloid complex. The piriform cortex is a three-layered allocortex (Haberly and Price, 1978a), incorporating a superficial plexiform layer and two cell body layers, that has been extensively studied in the context of olfactory function (Haberly, 1985, 1998) (see Chapter 9). Deep to the piriform cortex lies the endopiriform nucleus, which some scholars regard as layer IV of piriform cortex, either alone or in conjunction with the deep portion of layer III (Tseng and Haberly, 1989; Valverde, 1965). Within the amygdaloid complex, bulbar collaterals from the LOT innervate the anterior cortical nucleus and periamygdaloid cortex; the latter adjoins and is sometimes also considered part of the piriform cortex (Paxinos and Watson, 1986). While these amygdaloid structures exhibit trilaminar structures similar to that of piriform cortex, their layers II and III are somewhat underdeveloped in comparison (Krettek and Price, 1978). The lateral entorhinal cortex is the most caudal target of olfactory bulb axons (Davis et al., 1978; Heimer, 1968; Price, 1973; Scalia and Winans, 1975). Entorhinal cortex, which includes medial, lateral, and intermediate divisions, has six layers as opposed to the three (or four) layers observed in piriform cortex; entorhinal cortex has thus been considered a transitional cortex between olfactory allocortices and the isocortex. The piriform, entorhinal, and periamygdaloid cortices are often collectively termed the lateral olfactory cortices.

II.

ORGANIZATIONAL PRINCIPLES

A.

Projections from the Olfactory Bulb

The diverse cortices receiving direct input from the OB exhibit some fundamental similarities in their architectures. Each, for example, consists of a superficial plexiform layer (layer I) and one or more deeply located cell layers. Afferent inputs from the OB and associational inputs from other regions project to layer I, where they synapse with pyramidal cell dendrites as well as with local interneurons. In the lateral cortices, bulbar afferents are sharply restricted to the most superficial portion of the layer, layer Ia, while associational projections from other regions arborize within the deeper portion of this plexiform layer, termed layer Ib (see below) (Price, 1973). In the anterior hippocampal continuation and indusium griseum, in contrast, bulbar afferents and associational projections mix within layer I (Wyss and Sripanidkulchai, 1983). The AON is organized similarly to the lateral

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cortices, although in the rhesus monkey, a microsmatic primate, afferents from the olfactory bulb to the AON terminate throughout that structure rather than being restricted to layer I (Turner et al., 1978). Deeper layers in secondary olfactory cortices are all cell body layers—one in the AON, two in the piriform cortex (or three if the endopiriform nucleus is included), four in the indusium griseum and anterior hippocampal continuation—except for the six-layered entorhinal cortex, which in the nomenclature of Amaral and Witter (1995) includes four cellular layers and one additional plexiform layer (the lamina dissecans). Note that while the entorhinal cortex is commonly regarded as six-layered, these layers are dissimilar to the six layers of mammalian isocortex. Bulbar projections to the rostromedial cortices emerge from the superficial plexiform layer of the AON, which is continuous with that of the ventral tenia tecta, and extend dorsally along the midline within the superficial plexiform layers of the ventral tenia tecta, anterior hippocampal continuation, and indusium griseum (Adamek et al., 1984; Shipley and Adamek, 1984). Bulbar afferents to the lateral cortices, olfactory tubercle, and amygdaloid complex in mammals project caudally via the LOT and the superficial plexiform layers of these cortices. In the LOT, both the number and the diameter of bulbar axons decrease as the projections extend further caudally (Price and Sprich, 1975); furthermore, the density of innervation of secondary olfactory structures by LOT axon collaterals parallels the developmental sequence of innervation, being greatest near the lateral olfactory tract and sparsest in the medial olfactory tubercle (Schwob and Price, 1984). While both mitral and tufted cells project to the AON and to the rostral piriform cortex and olfactory tubercle (Haberly and Price, 1977; Schoenfeld and Macrides, 1984), the bulbar projection to more caudal lateral olfactory cortices becomes progressively dominated by mitral cells (Haberly and Price, 1977). Other than the short-latency interbulbar projection via the pars externa of the AON (Haberly and Price, 1978b), no clear topographic organization of bulbar projections to secondary olfactory structures is in evidence: the limited topographical regularity immediately caudal to the olfactory bulb is lost before the LOT emerges from the olfactory peduncle (Price and Sprich, 1975), small regions of the olfactory bulb project to large secondary areas while small areas within olfactory cortex receive projections from widely distributed areas in the olfactory bulb (Haberly and Price, 1977), and individual mitral and tufted cells innervate diverse secondary regions (Luskin and Price, 1982; Scott, 1981) (see Scott et al., 1980). Indeed, in rabbit, individual mitral cells project collaterals into multiple secondary olfactory structures— typically arborizing in both the AON and the anterior

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piriform cortex, with one quarter of the neurons studied additionally projecting into the olfactory tubercle (Ojima et al., 1984) (note that these three structures are to date the only ones in which individual mitral cell projections have been traced). However, within each of the innervated structures, the locations of each neuron’s multiple dense terminal arborizations are highly localized, exhibiting a patchy distribution (Ojima et al., 1984). Consequently, while bulbar projections are clearly diverse, they are also likely to be highly organized. Furthermore, the collateral architecture ensures that multiple, widely spaced secondary olfactory structures could receive nearly identical input patterns from the same set of activated mitral cells (Ojima et al., 1984). The LOT in mammals is considered to contain all of the bulbar axons projecting caudally to the lateral olfactory cortices and olfactory tubercle. However, medial and lateral subdivisions are apparent within the rabbit LOT which reflect the distinct medial and lateral tracts observed in some other vertebrates. In rabbit, one type of mitral cell axon collateral courses through the LOT and terminates within the lateral olfactory cortices and the lateral, more cortically organized portion of olfactory tubercle. A second type of axon collateral branches from the main axon within the olfactory bulb, travels through the ventromedial olfactory peduncle, remaining medial to the LOT, and innervates the medial, more striatally organized portion of the olfactory tubercle. While evolutionary divergence renders it challenging to compare olfactory systems in specific detail, there are many conserved characters in nonmammalian tetrapods that can shed light on the probable plesiomorphic organization of these projections. In the garter snake, Thamnophis sirtalis, bulbar projections are clearly segregated into three tracts: a lateral olfactory tract that projects to lateral (piriform) cortex and rostral amygdala, an intermediate olfactory tract that projects to the olfactory tubercle, and a medial tract that projects ipsilaterally to the dorsomedial retrobulbar formation (Lanuza and Halpern, 1998). These three projections are comparable to the lateral and medial portions of the rabbit LOT and the projection to the mammalian rostromedial cortices, respectively. In bullfrogs (Rana spp.), two distinct tracts are observed: one lateral tract corresponding to the LOT (in that it projects to lateral pallium, dorsal striatum including the cortical amygdaloid nucleus, and a ventral portion of dorsal pallium, as does the mammalian LOT) and one medial tract projecting to the medial pallium (corresponding to the mammalian rostromedial cortices) as well as to multiple septal nuclei (Northcutt and Royce, 1975; Scalia et al., 1991). Interestingly, in rats, transection of either the medial or lateral portions of the olfactory peduncle disrupted normal performance in a two-choice

Central Olfactory Structures

behavioral test, whereas the medial pathway was required in order to mediate normal olfactory arousal in isocortex during slow-wave sleep (Gervais and Pager, 1982). However, unimpaired olfactory task performance can be maintained even in rats with more severe LOT lesions (Slotnick and Berman, 1980; Slotnick and Risser, 1990; Slotnick and Schoonover, 1993), suggesting that a greater understanding of the respective contributions of different secondary olfactory structures to the performance of subtly different behavioral tasks may be necessary. Many more species-specific deviations from the canonical bulbar projection have been described. For example, in the lesser hedgehog tenrec (Echinops telfairi), reciprocal connections have been observed between the olfactory bulb and frontal isocortex, rendering those areas (sulcal and orbitofrontal cortices) secondary olfactory structures in this species (Radtke-Schuller and Kunzle, 2000), unlike most studied species to date in which orbitofrontal cortex is a tertiary recipient of olfactory information (Barbas, 1993; Carmichael et al., 1994). Similarly, in mice, the olfactory bulb directly projects to the insular cortex, an isocortical structure that also typically receives only tertiary olfactory input (Shipley and Adamek, 1984; Shipley and Geinisman, 1984). In both macaque monkeys (Macaca spp.) and hedgehog (Erinaceus europaeus), a direct projection from the olfactory bulb to the nucleus of the horizontal limb of the diagonal band of Broca has been described, which has not been described in most species studied (Carmichael et al., 1994; De Carlos et al., 1989), and a direct projection from the OB to the supraoptic nucleus of the hypothalamus has been reported in rats (Smithson et al., 1989). Finally, in the lemur (Microcebus murinus), in addition to the canonical projections, bulbar fibers also directly and bilaterally innervate the hippocampus proper (usually considered a tertiary projection area) as well as the septum, caudate-putamen, and, via the medial forebrain bundle, several hypothalamic nuclei and two mesencephalic modulatory centers (the locus coeruleus and the raphe nuclei) (Mestre et al., 1992). B.

Associational Connections Within and Among Secondary Olfactory Structures

Extensive connections, termed associational fibers, project between secondary olfactory cortices; their axons arborize in layer I as do the bulbar afferents, although in the lateral cortices they are sharply segregated from the afferents, projecting into the deeper portion of that layer (layer Ib) and also into the two superficial cell body layers (layers II and III) (Luskin and Price, 1983a). These associational connections have been grouped into two classes: local (or intrinsic; short connections between neurons in different layers of a

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given cortical structure) and associative (connections between different cortices) (Shipley, 1995). In piriform cortex, local connections are mediated by a variety of excitatory and inhibitory interneurons as well as pyramidal cell collaterals (Haberly, 1998). Associative connections among olfactory cortical structures are extensive and exhibit a degree of laminar and regional organization. Most intercortical projections among secondary olfactory structures can be classified into one of two fiber systems according to their laminar pattern of termination (Luskin and Price, 1983a,b). The first of these fiber systems, termed the layer Ib fiber system, includes projections from the AON and the piriform and entorhinal cortices, which terminate in layers Ib and often layer III; the projections from each of these different structures are typically concentrated at different characteristic levels within layer Ib. The second fiber system, termed the layer II–deep Ib fiber system, originates from the dorsal peduncular cortex, ventral tenia tecta, and periamygdaloid cortex, and terminates in layer II. Projections from the anterior cortical nucleus of the amygdaloid complex arborize throughout layers Ia–Ib. A second system of classification is apparent based on the origins of these projections: projections from layer II pyramidal cells tend to project to more caudal sites, whereas pyramidal cells in layer III target more rostral sites. Layer II cells of anterior piriform cortex also send commissural projections contralaterally, though these are limited in number and distribution compared to ipsilateral projections (Haberly and Price, 1978a,b; Luskin and Price, 1983a,b). Interestingly, the olfactory tubercle is the only secondary olfactory structure that does not give rise to associational projections (Haberly and Price, 1978a). C.

Feedback Projections to the Olfactory Bulb

Excepting the olfactory tubercle and indusium griseum, all of the secondary olfactory structures described herein send direct feedback projections to the olfactory bulb (Carmichael et al., 1994; Davis and Macrides, 1981; Davis et al., 1978; de Olmos et al., 1978; Haberly and Price, 1978a,b; Luskin and Price, 1983b; Shipley and Adamek, 1984; Wyss and Sripanidkulchai, 1983). Among the secondary structures served by the LOT, corticobulbar feedback projections are heavier from rostral areas (AON and anterior piriform cortex) than from posterior piriform cortex and other caudal areas (Shipley and Adamek, 1984) and arise mainly from layer II and III pyramidal cells. Most of these feedback projections are thought to terminate on granule cells in the olfactory bulb, though some extend into the glomerular layer. Most feedback projections are ipsilateral, with the notable exception of those originating in the AON, which project bilaterally or

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contralaterally. Although the functional roles of these projections are not known, it is interesting to note that in rabbit, there are complex changes in olfactory bulb dynamical activity when the feedback projections are blocked with a cooling probe (Gray and Skinner, 1988). These changes in dynamic activities may be at the basis of experimental results showing that isolation of the olfactory bulb from secondary olfactory structures impairs the formation of epileptiform activity in the olfactory bulb of rabbits, suggesting a dependency on more central structures for the induction and maintenance of epileptiform activity in the olfactory bulb (Gray et al., 1987). D.

Projections to Tertiary Olfactory Structures

Olfactory information is also distributed from secondary olfactory structures to several other regions of the brain, including orbitofrontal cortex, insular cortex, the mediodorsal, submedial, and anterior nuclei of the thalamus, the hypothalamus, the amygdaloid complex and the hippocampus (Barbas, 1993; Carmichael et al., 1994; Cavada and Reinoso-Suarez, 1985; Cavada et al., 2000; Datiche and Cattarelli, 1996b, Krettek and Price, 1977a; Luskin and Price, 1983b; Price, 1985; Price and Slotnick, 1983; Price et al., 1991; Reep and Winans, 1982; Smithson et al., 1989; Takagi, 1986). Generally, corticocortical projections from secondary olfactory structures originate in more superficially located cell layers (typically layer II), while corticodiencephalic projections originate from deeper layers [e.g., endopiriform nucleus, polymorphic (medial, striatal) zone of the olfactory tubercle, deep cells within periamygdaloid and entorhinal cortices] (Price, 1985; Price and Slotnick, 1983). Different secondary olfactory structures projecting to common tertiary structures typically project to discrete subregions; for example, olfactory projections to the thalamus include both highly convergent projections from the lateral olfactory cortices to the mediodorsal and submedial thalamic nuclei (Price, 1987; Price and Slotnick, 1983), as well as projections from the indusium griseum and anterior hippocampal continuation to the anterior thalamic nuclei (Wyss and Sripanidkulchai, 1983). Tertiary olfacto-hypothalamic projections arise from the anterior olfactory nucleus, the piriform cortex, the olfactory tubercle and the amygdaloid nuclei (Price et al., 1991). Notably, in rat, the piriform cortex provides input by way of the mediodorsal thalamic nucleus to the same prefrontal areas to which it projects directly (Ray et al., 1992); in contrast, no corticothalamic projections from the piriform cortex have been observed in cat or rabbit (Motokizawa et al., 1988), though the olfactory tubercle, amygdala, and

insular cortex do project to the mediodorsal thalamic nucleus in those species. E.

Neuromodulatory Inputs

Neuromodulatory inputs to the secondary olfactory structures described herein arise from four main sources: the nucleus of the diagonal band (acetylcholine, GABA), the dorsal and medial raphe nuclei (serotonin), the locus coeruleus (norepinephrine), and the substantia nigraventral tegmental area (dopamine). Both cholinergic and GABAergic neurons from the diagonal band (emerging primarily from the horizontal limb) project to the olfactory bulb and secondary olfactory structures (Gaykema et al., 1990; Haberly and Price, 1978a; Zaborszky et al., 1986). The known cellular and synaptic effects of cholinergic modulation in piriform cortex are detailed in Chapter 9 (see also Linster and Hasselmo, 2001), and a number of researchers have demonstrated the importance of cholinergic modulatory inputs for olfactory learning and discrimination (De Rosa and Hasselmo, 2000; Doty et al., 1999; Hunter and Murray, 1989; Linster et al., 2001; Paolini and McKenzie, 1993; Roman et al., 1993). The OB and secondary olfactory structures also receive noradrenergic, serotonergic, and dopaminergic modulatory inputs (Datiche and Cattarelli, 1996a; Datiche et al., 1995; Fallon and Moore, 1978; Fallon et al., 1978; Jones et al., 1977; Moore et al., 1978). While the physiological effects of these other neuromodulators have been studied in olfactory cortex (Gellman and Aghajanian, 1993, 1994; Marek and Aghajanian, 1994, 1995; Sheldon and Aghajanian, 1990, 1991) (reviewed in Hasselmo, 1995), to our knowledge only norepinephrine, along with acetylcholine, has been related to olfactory learning and memory (reviewed in Sullivan and Wilson, 1994; Sullivan et al., 1992). F.

Chemoarchitecture

Although the transmitters used by associational and output fibers in secondary olfactory structures have not been definitively established, there is strong evidence, derived from a variety of experimental techniques, that glutamate is the principal excitatory neurotransmitter for both afferent and associative fiber systems (Carnes et al., 1990; Fuller and Price, 1988; Fuller et al., 1987; Godfrey et al., 1980; Hoffman and Haberly, 1993; Jung et al., 1990; Ray et al., 1992); aspartate has also been suggested as an excitatory neurotransmitter in these structures. Excitatory EPSCs are mediated via both AMPA- and NMDA-type glutamate receptors; glutamate also acts on metabotropic receptors in the piriform cortex. GABA is believed to be the predominant inhibitory neurotransmitter in the

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secondary olfactory cortices (Haberly, 1985), acting on postsynaptic GABAA receptors as well as metabotropic GABAB receptors on both pre- and postsynaptic membranes. In addition, several neuropeptides have been identified in neurons of the olfactory bulb and secondary olfactory structures (reviewed in Shipley, 1995).

III. CONNECTIVITY OF SECONDARY OLFACTORY STRUCTURES A.

Anterior Olfactory Nucleus

The AON, a subset of which has also been termed anterior olfactory cortex (Haberly, 2001), is a laminated structure embedded within the olfactory peduncle. The AON has been divided into several subregions with distinct architectures and connectivities (described by Broadwell, 1975b, Davis and Macrides, 1981, de Olmos et al., 1978, Haberly and Price, 1978b, Shipley, 1995, Shipley and Adamek, 1984). It is predominantly two-layered, consisting of a superficial plexiform layer containing incoming projection fibers and the apical dendrites of its intrinsic neurons, and a tightly packed cell body layer (Haberly and Price, 1978b), but gradually assumes a trilaminar form near its caudal extreme adjacent to the anterior commissure (Halasz, 1990; Valverde et al., 1989). The AON receives projections from olfactory bulb mitral and tufted cells in its superficial plexiform layer, layer Ia (Scott et al., 1985); bulbar afferents also course along this superficial layer into the superficial plexiform layers of the adjoining ventral tenia tecta (medially) and anterior piriform cortex (laterally) (Shipley and Adamek, 1984), the latter forming the lateral olfactory tract. In 3-week-old cats, a study of neuropeptide Y–immunoreactive neurons observed in the OB and olfactory peduncle revealed a dense contralateral projection through the anterior commissure, which was dramatically reduced after the first 4 postnatal months. In contrast, ipsilaterally projecting neurons of this type are not substantially reduced in the adult (Sanides-Kohlrausch and Wahle, 1990). Such dense contralateral projections during development could conceivably aid in correlating the odotopic projections of the two olfactory bulbs with one another. In neonatal rats unilaterally trained on odors (before the development of a functional olfactory anterior commissure), the learned odor preference was stored unilaterally, as evidenced by the animals’ exhibition of a preference for the odor only when presented to the trained (ipsilateral) side. Older rat pups, however, exhibited learned preference when the odor was presented to either side, even if they had been trained before the anterior commissure developed; i.e., the development of contralateral

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projections enabled contralateral access to previously obtained, ipsilaterally stored odor preferences. If the anterior commissure was then sectioned in these older rat pups, this acquired contralateral access to learned preference was lost, demonstrating that the odor preference was still maintained unilaterally (Kucharski and Hall, 1987, 1988). These data suggest that some form of odotopic crossattunement between the paired olfactory bulbs and/or anterior olfactory nuclei occurs as the contralateral projections between the bulbs develop, and further that this putative cross-attunement does not require further training after development but can access previously formed, unilateral memory traces. The AON is the major source of feedback connections to the olfactory bulb from any source (Carson, 1984); all subdivisions of the AON project to both the ipsilateral and the contralateral olfactory bulb except for pars externa, which projects only to the contralateral olfactory bulb via the anterior commissure (Broadwell, 1975b; Davis and Macrides, 1981; Haberly and Price, 1978b). The AON also projects to the piriform cortex, olfactory tubercle, ventral tenia tecta, orbitofrontal cortex, and hypothalamus (Barbas, 1993; Luskin and Price, 1983b; Price et al., 1991) and receives projections from several structures including the piriform and entorhinal cortices (Luskin and Price, 1983b; Wyss, 1981), as well as the CA1 region of the hippocampal formation (van Groen and Wyss, 1990). While no clear topographical organization is apparent in most of these projections (e.g., Luskin and Price, 1982; Price and Sprich, 1975), a few are clearly topographical. Bulbar projections to the AON pars externa, and that structure’s projections to the contralateral OB, are both strictly topographical (Schoenfeld and Macrides, 1984; Scott et al., 1985); the pars lateralis may also exhibit some topographical organization (Scott et al., 1985). Finally, the AON receives topographically organized inputs from the ventral tenia tecta (Luskin and Price, 1983b). Direct bulbobulbar contralateral projections that bypass the AON have also been demonstrated in several species including cat, rabbit, caiman, turtle, and fish, but are believed to be absent in others such as rat, mouse, rhesus monkey, hamster, guinea pig, frog (Rana spp.), and some lizards (reviewed in Halasz, 1990; Kemali and Guglielmotti, 1987; Scalia et al., 1991; Shipley and Adamek, 1984; Turner et al., 1978) (see Leveteau et al., 1993). However, in frogs (Rana esculenta), primary olfactory receptor neurons themselves have been shown to project bilaterally and innervate both olfactory bulbs; this contralateral projection is mediated by an interbulbar adhesion distinct from the anterior and habenular commissures (Leveteau et al., 1992).

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Little is known about the functional role of the AON for olfactory processing, save that it mediates most or all bilateral bulbobulbar communication in many species and is thus presumably important for the bilateral comparison of olfactory information. Odor responses recorded in rabbit AON neurons appeared less odor-selective than those recorded in the OB (Boulet et al., 1978). When adult rats were trained on simple discrimination tasks, changes in odor-evoked neural activity as measured by 2-deoxyglucose (2DG) staining were observed in the AON of the trained animals compared to their untrained counterparts; interestingly, no changes in 2DG uptake were observed in the piriform cortex in this experiment (Hamrick et al., 1993). Enhanced c-fos expression has also been observed in the AON of rats that received forward pairing of odors with a foot shock stimulus, demonstrating that odorinduced c-fos expression can be modified through aversive conditioning in the AON as well as in the olfactory bulb (Funk and Amir, 2000). C-fos expression within OB odoractivated regions was also reduced bilaterally when centrifugal afferents were severed by unilateral section of the olfactory peduncle or by application of noradrenergic antagonists within the OB, while 2DG uptake patterns were unaffected (Sallaz and Jourdan, 1993, 1996). These results suggest that the AON is a plastic structure that participates in olfactory learning along with the olfactory bulb and other secondary olfactory structures. B.

Rostromedial Olfactory Structures

The ventral tenia tecta, anterior hippocampal continuation, and indusium griseum receive input from the OB in their small molecular layers (Adamek et al., 1984; de Olmos et al., 1978; Levy et al., 1999; Shipley and Adamek, 1984; Wyss and Sripanidkulchai, 1983); in addition, the latter two cortical structures receive input from the entorhinal cortex. Because the anterior hippocampal continuation and indusium griseum have been considered part of the hippocampal formation, their inputs from the OB have been suggested to provide a more direct olfactory input to the hippocampus proper than that via the entorhinal cortex (Adamek et al., 1984). While there are projections from the ventral tenia tecta and anterior hippocampal continuation to the OB, the indusium griseum does not project back to the OB. The ventral tenia tecta additionally receives projections from, and projects back to, the AON, while the indusium griseum receives additional input from the piriform cortex (Adamek et al., 1984; Luskin and Price, 1983b; Wyss and Sripanidkulchai, 1983). Interestingly, connections from the OB to the ventral tenia tecta are absent in the microsmatic rhesus monkey (Turner and Mishkin, 1978).

C.

Olfactory Tubercle

The olfactory tubercle in mammals is a prominent bulge on the base of the brain just caudal to the olfactory peduncle and medial to the lateral olfactory tract; it receives afferent input from mitral and tufted cells in the OB (de Olmos et al., 1978; Heimer, 1968). The olfactory tubercle exhibits a superficial plexiform layer like the lateral and rostromedial olfactory cortices, but its cellular architecture varies: medially, it resembles other striatopallidal complexes, whereas laterally (adjoining the piriform cortex) it exhibits a trilaminar cortical organization (Heimer and Wilson, 1975; Millhouse and Heimer, 1984). However, the olfactory tubercle differs from the piriform cortex in that it does not send output projections to the OB or to any other secondary olfactory structures (Haberly and Price, 1978a; Luskin and Price, 1983b); the outputs of the olfactory tubercle are directed towards the mediodorsal and submedial nuclei of the thalamus (Price and Slotnick, 1983), the ventral pallidum, and the nucleus accumbens (Heimer and Wilson, 1975; Luskin and Price, 1983b), and, in monkeys, the orbitofrontal cortex (Barbas, 1993). The inputs and projections to and from the olfactory tubercle can vary substantially among species; for example, in many macrosmatic animals (in which the olfactory sense is well developed), the olfactory tubercle receives copious direct bulbar input, and cell bridges exist between the olfactory tubercle and other striatal structures (Butler and Hodos, 1996), whereas in humans and other microsmatic primates, the region of the tubercle receiving afferent input from the OB is greatly reduced (Shipley, 1995). Neurons in the olfactory tubercle of rats can respond to electrical stimulation of the OB, as suggested by their direct bulbar inputs. Both excitatory and inhibitory responses have been observed and were modulated by the application of dopamine (Inokuchi et al., 1987, 1988). D.

Piriform Cortex and Endopiriform Nucleus

Of all secondary olfactory structures, the piriform cortex (also termed the primary olfactory cortex) has been most intensively studied with respect to olfactory function (Haberly, 1985, 1998, 2001) (see Chapter 9). This threelayered allocortex receives abundant afferent input from the OB as well as inputs from other secondary olfactory cortices, excepting the olfactory tubercle (Kowianski et al., 1999; Krettek and Price, 1977a; Luskin and Price, 1983b; Wyss, 1981). Mitral and tufted cells project to the piriform cortex by way of the lateral olfactory tract (LOT) and arborize exclusively in layer Ia. The piriform cortex also receives input from the orbitofrontal and insular cortices, hippocampal formation, basal forebrain, brainstem,

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thalamus, and hypothalamus (Haberly and Price, 1978a; Kowianski et al., 1999) and sends extensive projections back to the OB (Carmichael et al., 1994; de Olmos et al., 1978; Haberly and Price, 1978a,b; Luskin and Price, 1983a). These feedback projections terminate mainly on or near OB granule cells, which are inhibitory to mitral and tufted projection neurons in the bulb. Many projections from the piriform cortex to other regions have also been described (Carmichael et al., 1994; Haberly and Price, 1978a; Kowianski et al., 1999; Price et al., 1991; Takagi, 1986), including projections to many other secondary olfactory structures as well as to the hippocampal formation, orbitofrontal and insular isocortices, the amygdaloid complex, the hypothalamus, and the mediodorsal and submedial nuclei of the thalamus. A more detailed review of the known architecture, connectivity and function of the piriform cortex is provided in Chapter 9 (see also Haberly, 1985, 1998, 2001; Linster and Hasselmo, 2001). Deep to the piriform cortex lies the endopiriform nucleus, a large group of multipolar cells interconnected with the overlying cortex (to the extent that it, either alone or in combination with the deep portion of layer III, is considered layer IV of piriform cortex by some authors) (Tseng and Haberly, 1989; Valverde, 1965). The function of the endopiriform nucleus is unknown; however, studies with animal models suggest that it plays an important role in temporal lobe epileptogenesis (Behan and Haberly, 1999). The input and output connections of the endopiriform nucleus are very similar to those of the piriform cortex (Kowianski et al., 1999), but efferents from the endopiriform nucleus lack the precise laminar order of those from the piriform cortex and form a heavy caudorostral pathway that the piriform cortex lacks (Behan and Haberly, 1999). E. Periamygdaloid Cortex and the Anterior Cortical Nucleus of the Amygdaloid Complex In mammals, axons from the main OB project to the periamygdaloid cortex (considered part of the piriform cortex by some) (Paxinos and Watson, 1986) and the anterior cortical nucleus of the amygdaloid complex. While the accessory olfactory bulb also projects to the amygdaloid complex, its target regions are not shared with those of the main OB (Haberly and Price, 1978a; Krettek and Price, 1978; Luskin and Price, 1983b). These “extended amygdalar” structures exhibit a characteristic trilaminar structure, although layers II and III are somewhat less developed than in the piriform cortex (Krettek and Price, 1978). Olfactory output targets of the periamygdaloid cortex and the anterior cortical nucleus include the piriform cortex, entorhinal cortex, infralimbic area, ventral agranular insular area, and perirhinal area (Kevetter and

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Winans, 1981; Kowianski et al., 1999; Krettek and Price, 1977b; Kunzle and Radtke-Schuller, 2000; Wyss, 1981). A second superficial corticoid structure within the amygdaloid complex, adjoining the anterior cortical nucleus, is the nucleus of the lateral olfactory tract; this structure exhibits a trilaminar structure similar to that of the anterior cortical nucleus, though its interconnectivity with other secondary olfactory structures is less established. Research in rats and in monkeys has shown that, in awake, behaving animals, neurons in the amygdaloid complex respond selectively to olfactory stimulation. In rats, neurons in the basolateral amygdala responded to odors (Cain, 1975; Cain and Bindra, 1972), displayed selective odor responses in an odor discrimination task, and rapidly reversed this selectivity during reversal learning (Schoenbaum et al., 1998). In monkeys, odor selectivity in medial amygdalar neurons could be obtained without training (Tanabe et al., 1975). In PET studies of humans, aversive odors have been shown to activate the amygdala in both hemispheres (Zald and Pardo, 2000). Furthermore, in neonatal rats, lesions of the amygdaloid complex blocked the acquisition of odor preferences in a conditioned odor association paradigm, although this impairment could be overcome by overtraining (Sullivan and Wilson, 1993). In contrast, in adult rats, lesions of either the lateral olfactory tract inputs to the amygdala or of the amygdala itself did not affect simple odor discrimination learning (Slotnick, 1985; Slotnick and Risser, 1990; Sutherland and McDonald, 1990). Lesions of the bed nucleus of the stria terminalis (described in Broadwell, 1975b; Krettek and Price, 1978; Turner and Zimmer, 1984) specifically blocked activation of the hypothalamic paraventricular nucleus (PVN), which regulates adrenocortical secretion by olfactory stimuli while sparing activation of the PVN via other sensory modalities (Mor et al., 1987). F.

Entorhinal Cortex

The lateral portion of the entorhinal cortex is the most caudal projection of olfactory bulb axons (Davis et al., 1978; Heimer, 1968; Price, 1973; Scalia and Winans, 1975). Entorhinal cortex is divided into medial, lateral, and intermediate divisions and is commonly categorized into six [or seven; there is some disagreement between the primate and rat literatures (Amaral and Witter, 1995)] layers as opposed to the three (or four) layers seen in piriform cortex. Entorhinal cortex has been considered transitional between olfactory allocortices and the isocortex, although its six layers do not directly correspond to the six layers of mammalian isocortex. While entorhinal cortex projects back to the OB, and also to other olfactory cortical

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structures including the anterior olfactory nucleus, ventral tenia tecta, indusium griseum, piriform cortex, endopiriform nucleus, olfactory tubercle, and amygdaloid cortices (Kowianski et al., 1999; Luskin and Price, 1983a,b; Wyss, 1981), its strongest projection is to the hippocampal formation. A number of studies have shown that olfactory stimuli can modulate neural activity in the entorhinal cortex of behaving rats (Chabaud et al., 2000; Kay and Freeman, 1998; Mouly et al., 2001). Highly coherent dynamical neural responses from piriform cortex and entorhinal cortex have been evoked by odor application, and these dynamics change in the entorhinal cortex as a function of the behavioral relevance of a given odor stimulus (Chabaud et al., 2000; Kay and Freeman, 1998; Mouly et al., 2001). While these data suggest the functional relevance of entorhinal cortex to olfactory processing, behavioral lesion studies have demonstrated that rats with posterior sections of the LOT (severing bulbar projections to the entorhinal and amygdaloid cortices) are not impaired in odor discrimination tasks (Slotnick and Risser, 1990; Thanos and Slotnick, 1997; Zhang et al., 1998). While these data in turn may superficially suggest that entorhinal cortex is not a crucial structure for olfactory discrimination learning, it is also clear that entorhinal cortex receives olfactory input not only from the OB, but also from many other secondary olfactory structures; that is, posterior LOT lesions may not eliminate the participation of the entorhinal cortex in olfactory stimulus processing. Finally, it has been reported that short-term memory for olfactory stimuli in a delayed-nonmatch-to-sample task in rats can be increased in duration by lateral entorhinal cortex lesions (Ferry et al., 1996; Wirth et al., 1998); these results could of course also be interpreted as a decrease in the rats’ ability to extinguish associations likely to be no longer appropriate due to the passage of time, suggesting that such lesions would impair reversal learning.

IV.

FUNCTIONAL ASPECTS

The anatomy of the olfactory pathways described in this chapter clearly shows that olfactory processing involves a large number of structures, interconnected with each other in complex fashion, and incorporating both feedforward and feedback interactions. As early as in the olfactory bulb, feedback projections from more central brain structures influence neural dynamics and are crucial for olfactory learning and processing. Whole-brain imaging studies in humans have shown that multiple, diverse neural structures become activated during tasks involving olfactory stimulation, and furthermore that the nature of the task strongly

influences which of these structures become most activated. For example, in studies measuring regional cerebral blood flow increases using PET, presentation of single odors increased activity in the piriform, periamygdaloid, orbitofrontal, insular and cingulate cortices as well as in the thalamus, indicating that olfactory stimuli activate diverse regions throughout the human brain. When subjects were tested on olfactory discrimination and memory tasks, additional regions were activated, including the cerebellum (Savic et al., 2000). In a similar PET study, the orbitofrontal cortex was differentially activated when subjects were asked to make judgments about odor presence, familiarity, intensity, hedonicity, or edibility; furthermore, that activation was differentially lateralized depending on the task (Royet et al., 2001). Aversive odor stimuli have been shown to produce regional cerebral blood flow increases in orbitofrontal cortex; highly aversive odor stimuli additionally evoked such increases in the amygdaloid complex (Zald and Pardo, 1997). Finally, in a functional magnetic resonance imaging study differentiating effects of odor stimulation per se from those deriving from motor and other correlates of odor sampling behaviors, Sobel and colleagues (1998) showed that active sniffing, either in the absence or in the presence of an odor, induced activation in the piriform and the medial and posterior orbitofrontal cortices; in contrast, smelling an odor, regardless of sniffing activity, induced activation mainly in the lateral and anterior orbitofrontal cortex (see Chapter 12). These results emphasize a crucial caveat to imaging and other physiological studies: regions in which activity correlates with or is shown to mediate important features of an olfactory task are not necessarily chemosensory in nature. Physiological and behavioral data from nonhuman animals have also offered considerable insight into the functional roles of various secondary olfactory structures in odor acquisition, processing, and memory. Neural responses evoked or modulated by olfactory stimulation have been reported in the anterior olfactory nucleus (anesthetized rabbit) (Boulet et al., 1978), amygdala (awake, behaving rats) (Schoenbaum et al., 1998) (monkeys) (Tanabe et al., 1975), orbitofrontal cortex (awake rats) (Lipton et al., 1999; Ramus and Eichenbaum, 2000, Schoenbaum et al., 1998, 2000) (monkeys) (Tanabe et al., 1975), and in the hypothalamus (awake monkeys) (Karadi et al., 1989; Tazawa et al., 1987). Several experiments have shown that odor-evoked neural activity is modified by experience: in the AON, enhanced c-fos transcription was observed in rats that had been trained to associate a footshock with an odor (Funk and Amir, 2000); similarly, changes in neural activity recorded with [14C]2-deoxyglucose were observed in the AON of rats that had learned a

Central Olfactory Structures

simple odor detection task (Hamrick et al., 1993). Odorevoked single-unit responses in the orbitofrontal cortex and amygdala of awake, behaving rats are modulated by the reward associations that rats learned during the task; in addition, the activity of most neurons in both these structures is also modulated by other task-related events (Lipton et al., 1999; Ramus and Eichenbaum, 2000; Schoenbaum et al., 1998, 2000). In the entorhinal cortex, the learning of olfactory stimuli is accompanied by changes in neural dynamics, as measured by local field potentials (Chabaud et al., 2000; Kay and Freeman, 1998; Mouly et al., 2001). All of these data together demonstrate that an animal’s experience with and expectations about odors can durably alter the odor-evoked response patterns of individual neurons and the overall spatial activity patterns in response to odorants, as well as the dynamics of the interplay of neural populations. While these effects are readily apparent, it remains unclear just what factors are being encoded or what the functional meanings of such changes might be. Behavioral lesion studies can also yield valuable information about the putative contributions of various structures to tasks such as odor detection, identification, discrimination, responsivity, and learning. In a series of behavioral lesion studies, Slotnick and colleagues have shown that deficits in odor detection and learning are related to the extent to which the olfactory bulb is disconnected from the forebrain. For example, transections of only the lateral olfactory tract, the anterior limb of the anterior commissure, or the olfactory tubercle had little effect on performance of a simple odor discrimination task, whereas combined lesions of these structures produced severe impairments (Slotnick and Schoonover, 1992). Interestingly, transections of the lateral olfactory tract, sparing the more medially directed outputs of the olfactory bulb, had little effect on odor retention, suggesting that medial olfactory projections can suffice to perform certain olfactory tasks (Slotnick and Berman, 1980). Lesions of either the lateral or medial portions of the olfactory peduncle impaired rats’ performance in a food odor detection task, and medial lesions specifically impaired mitral cell responsivity to food odor presentation during slow-wave sleep (Gervais and Pager, 1982). More posterior lesions of the lateral olfactory tract, disconnecting the amygdaloid complex and entorhinal cortex from direct olfactory bulb inputs, had no detectable effects on either retention of a previously learned odor detection task or the acquisition of a simple odor discrimination (Slotnick, 1985; Slotnick and Risser, 1990); however, substantial associational projections to the entorhinal cortex from multiple secondary olfactory structures remained intact under this procedure. Thus, the contribution of entorhinal cortex activity to the olfactory task may

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not have been eliminated by the LOT lesions. Indeed, in some types of olfactory memory tasks, lesions of the entorhinal cortex can be interpreted to facilitate olfactory recognition. In an olfactory habituation task, rats with aspirative entorhinal cortex lesions displayed recognition of a previously investigated odor at latencies for which control rats did not (Wirth et al., 1998). Similar results have been obtained using conditioned odor aversion, in which entorhinal cortex lesions lengthened the time window during which an association between the odor stimulus and the subsequent aversive stimulus could be formed (Ferry et al., 1996, 1999). Lesions of the mediodorsal thalamic nucleus, in contrast, impaired both acquisition of an odor discrimination task and its reversal (Slotnick and Risser, 1990). Finally, lesions of the olfactory inputs to the amygdala did not impair performance on olfactory detection and discrimination tasks (Slotnick, 1985); however, Sullivan and Wilson (1993) reported that bilateral amygdala lesions in neonatal rats affected learned odor preferences and that these effects could be reversed by extensive training.

V.

CONCLUSION

Olfactory sensory input pathways diverge immensely after emerging from the relative bottleneck of the olfactory bulb. As reviewed in this chapter, these secondary olfactory projections innervate a broad diversity of structures deriving from several distinct telencephalic pallial and subpallial tissues, as well as diencephalic, midbrain, and brainstem structures. Many of these structures are highly interconnected with one another via associative projections, while some are relatively isolated from other secondary olfactory influences; furthermore, each of them receives characteristic extrinsic and modulatory inputs from other regions of the brain. While these structures and projections are remarkably conserved among vertebrates, there are also numerous species-specific variations that presumably derive from the divergent adaptive needs of each species, both in terms of novel or missing projections and in terms of the relative densities of projection patterns among secondary and tertiary olfactory structures. Lacking specific knowledge of what purposes most of these structures serve, or even of the physiological and adaptive tasks that must be performed by the organism and for which it requires olfactory perceptual information, what framework for analysis is likely to be the most conducive to elucidating an understanding of these structures over time? It may be counterproductive to think of secondary olfactory structures as primarily “olfactory” in nature; in particular, it may be misleading to judge such structures primarily on the basis of the purported odor selectivity of

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individual neurons, or even of ensembles. Aside from the typically unwarranted assumptions about mechanisms that necessarily underlie statistical measures of selectivity, it is unlikely that ever-increasing specificity is the general goal of all secondary processing. Rather, a functional approach is likely to be stronger: for what various purposes might a given organism require olfactory sensory data, and what elements of those data are needed for the organism to respond adaptively? How precise must an olfactory identification be in order to meet the organism’s needs, and what are the probable costs of false-positive errors compared with false negatives? Even if maximally specific odor identification were prerequisite to all decision processes utilizing olfactory information—an unlikely possibility—neural activity based increasingly on contingency and less on the physical characteristics of the stimulus would be expected as the response cascade proceeds beyond primary sensory areas in the brain. In some tissues, studying how the categorization of different olfactory stimuli changes, for example, may be more indicative than measuring how theoretically orthogonal their representations may be. In short, a functional approach to understanding the contributions of secondary olfactory structures might be to hypothesize an information-processing task to which a given structure might contribute, to assess what elements of olfactory sensory information would be required in order for it to fulfill that task, and to predict what cellular and network mechanisms would be useful in order to extract the needed information from the ensemble activity of the mitral/tufted cell projection neurons that innervate it. Differences in species—e.g., microsmatic compared to macrosmatic, predator compared with prey species—along with what is known about the functions and connectivities of different regions within the brain, will likely be useful in hypothesizing about the respective utilities of divergent secondary olfactory structures and the purposes for which each may sample the data provided by the mitral/tufted ensemble representation.

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9 Sensory Physiology of Central Olfactory Pathways Donald A. Wilson and Regina M. Sullivan University of Oklahoma, Norman, Oklahoma, U.S.A.

I.

INTRODUCTION

help clarify this issue (Singer and Shepherd, 1994), it does appear that, similar to other sensory systems, odorant stimuli are broken down into component features, each recognized by a particular receptor, and the problem for the remainder of the olfactory pathway is to reconstruct those features into a perceptual whole. In addition to discriminating pure, isolated stimuli, a problem for all sensory systems is that they must function in the real world. Thus, the visual system is able to recognize a stimulus partially obscured by other objects and the auditory system can interpret speech against a background of other noises. Similarly, the olfactory system is able to recognize garlic in the spaghetti sauce even when the odors from the freshly cut lawn are blowing in the window. On the other hand, that garlic odor is a mixture of many individual molecular components, yet is perceived as a single stimulus. This review will focus on what is currently known about the sensory physiology of central olfactory structures that allows odorant discrimination and odorant mixture analysis and synthesis to occur (see Chapters 7 and 8 for the detailed anatomy of these structures). This review will focus on terrestrial vertebrates, although striking similarities with invertebrates will be noted (Christensen and White, 2000). While excellent work has been done on synaptic physiology of this system, much of which utilized in vitro preparations (see Haberly, 1998; Shepherd and Greer, 1998; Shipley and Ennis, 1996 for reviews), this review will focus on in vivo sensory physiology in vertebrates and response to odorants. The central olfactory system of vertebrates (Fig. 1) includes the main olfactory bulb and the primary olfactory

Central sensory pathways construct representations of the external world based on a combination of spatiotemporal patterns of receptor neuron input and a running average of internal activity patterns. In most sensory systems, the relationship between stimulus energy in the external world and the spatiotemporal pattern of receptor neuron activity appears relatively straightforward. For example, spatial relationships of visual stimuli are maintained by spatial patterns of visual receptor cell activity in the retina and subsequent precise retinotopic projections to visual cortical centers. Similarly, auditory stimulus frequency information is extracted by a spatial gradient of frequency sensitivity along the basilar membrane of the cochlea and subsequent precise tonotopic projections to auditory cortical centers. Lateral inhibition along both the visual and auditory sensory pathways helps to more precisely define the specific visual spatial pattern or auditory frequency of the initiating stimulus. However, how the olfactory system constructs a representation of the external odor world is not so obvious. Simple analytical chemistry does not appear to be sufficient to account for olfactory perception. Molecules that are structurally very similar may be perceptually very different, and vice versa. Furthermore, it is not clear at present which features of olfactory stimuli the olfactory system uses for odorant discrimination (e.g., carbon chain length, presence and location of functional groups, molecular resonant frequency). While further analysis of ligandreceptor interactions at the olfactory receptor sheet should 181

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Figure 1 Basic schematized organization of the vertebrate olfactory system. Circled structures receive direct input from the main olfactory bulb. Note that most areas receiving direct input from the main olfactory bulb project back to the bulb. Modulatory inputs project broadly to all primary olfactory structures, although there is substantial heterogeneity in laminar density of terminations within each area. Abbreviations: OB, main olfactory bulb; AON, anterior olfactory nucleus; Amy, amygdala; PC, piriform cortex; Ent, entorhinal cortex; PFC, prefrontal cortex; Hyp, hypothalamus; DMN, dorsomedial nucleus of the thalamus; LC, locus coeruleus, NE, norepinephrine; HLDB, horizontal limb of the diagonal band of Broca; ACh, acetylcholine; Raphe, raphe nucleus; 5-HT, 5-hydroxytryptamine (serotonin).

cortex (piriform cortex). In mammals, a higher-order olfactory cortex exists, the orbitofrontal/insular cortex. The thalamic relay to the olfactory orbitofrontal cortex is the dorsomedial nucleus of the thalamus, although a direct projection from the piriform cortex to the orbitofrontal cortex also exists (see Chapter 8). While odorant responses have been examined in a number of other central brain regions, such as the amygdala (Cain and Bindra, 1972; Schoenbaum et al., 1999; Tanabe et al., 1975a) and hypothalamus (Karadi et al., 1989; Kogure and Onoda, 1983; Pfaff and Gregory, 1971; Scott and Pfaffmann, 1972), the sensory physiology of the main olfactory bulb, piriform cortex, and orbitofrontal cortex will be emphasized here.

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MAIN OLFACTORY BULB

A.

Glomerular Layer

Olfactory receptor axons synapse onto second-order olfactory neurons within the main olfactory bulb (Fig. 2). As described elsewhere in this volume, single receptor neurons appear to express a single receptor protein. Receptor neurons expressing the same receptor protein, while randomly scattered within one of four zones of the olfactory receptor sheet, converge on two individual glomeruli within the olfactory bulb, one located dorsomedially in the bulb and one more ventrolaterally (Buck, 1996; Mombaerts, 1999). Thus, each of the approximately 2000 glomeruli of the rodent olfactory bulb is believed to each receive relatively homogeneous input from neurons expressing one of the 1000 different receptor proteins. Furthermore, receptor neurons expressing similar or homologous receptor genes (Tsuboi et al., 1999), and with similar odorant receptive fields (Bozza and Kauer, 1998), tend to terminate in neighboring glomeruli, enhancing the possibility of lateral inhibitory interactions between similar molecular features. The high convergence ratio of olfactory receptor neurons to mitral cells within a glomerulus (1000: 1) significantly amplifies sensitivity of the system by reducing odorant response threshold in mitral cells compared to olfactory receptor neurons (Duchamp-Viret et al., 1989) Vertebrate olfactory receptor neurons have relatively broad odorant receptive fields (Duchamp-Viret et al., 1999; Kaluza and Breer, 2000; Malnic et al., 1999; Sato et al., 1994; Sicard and Holley, 1984). Similarly, individual main olfactory bulb glomeruli respond to multiple odorants, although each odorant produces a unique spatial pattern of glomerular activation as determined by 2-deoxyglucose autoradiography (Johnson and Leon, 2000; Johnson et al., 1998, 1999; Jourdan et al., 1980; Stewart et al., 1979), c-fos immunohistochemistry (Guthrie et al., 1993; Sallaz and Jourdan, 1993), and optical imaging (Joerges et al., 1997; Rubin and Katz, 1999; Uchida et al., 2000). A recent optical imaging study of intrinsic signals in the rat revealed that the specific functional group present in an odorant determined the glomerular zone of activation (e.g., anteromedial or dorsolateral), while more subtle features of the odorant molecule (e.g., carbon chain length or branching pattern) determined which glomeruli within that zone would be activated (Uchida et al., 2000). This spatial pattern of glomerular activation is believed to encode the molecular features present in the sampled odorant. However, while individual odorant features may be encoded by individual

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Figure 2 Basic schematized neural connectivity of the main olfactory bulb and piriform cortex. Individual receptors within the olfactory epithelium express one of 1000 different receptor proteins and are randomly scattered within one of four zones, yet receptors expressing the same receptor protein converge onto a small number of exclusive glomeruli (three receptor types are labeled A, B, and C in this example). The receptors are hypothesized to be responsive to individual odorant features, rather than odorant molecules as a whole. Mitral cells receive receptor input from a single glomerulus (and thus convergent receptor input; e.g., A or B) and project to the piriform cortex. Within the olfactory bulb, interglomerular and interoutput neuron lateral inhibition is mediated by juxtaglomerular and granule cells, respectively, heightening contrast between similar odorant features. Neurons in the piriform cortex appear to form a combinatorial array, allowing convergence of multiple odorant features (e.g., AB or ABC) and/or behavioral state/nonolfactory inputs to occur on single neurons. Both the olfactory bulb and piriform cortex receive extensive input from neuromodulatory and nonolfactory inputs.

glomeruli, odorants in a mixture can interact at the receptor level and/or within the glomerular layer to produce odorant mixture specific glomerular activation in both vertebrates (Bell et al., 1987) and invertebrates (Cromarty and Derby, 1998; Derby; et al., 1991; Joerges et al., 1997). Thus, some aspects of odor synthesis may occur even before the first central synapse of the olfactory pathway. Within glomeruli, olfactory receptor axons synapse onto the primary output neurons of the olfactory bulb, mitral cells, as well as onto a second class of output neurons, tufted cells (Fig. 2). Juxtaglomerular cells, a class of olfactory bulb interneurons that mediate interglomerular inhibition, also receive direct olfactory nerve input. Olfactory receptor axons release the excitatory amino acid glutamate from their axon terminals and activate both NMDA and non-NMDA receptors on second-order neuron dendrites (Berkowicz et al., 1994; Ennis et al., 1996).

Many juxtaglomerular cells express both the inhibitory amino acid neurotransmitter GABA and dopamine (Gall et al., 1987; Kosaka et al., 1985). Juxtaglomerular cells respond to odorants with simple depolarizations and bursts of spikes and may directly mirror olfactory nerve input (Onoda and Mori, 1980; Wellis and Scott, 1990). One role of juxtaglomerular cell GABA release may be to presynaptically inhibit glutamate release from olfactory nerve axons (Aroniadou-Anderjaska et al., 2000; Nickell et al., 1994). Mitral/tufted cells also express GABA receptors (Bowery et al., 1987), and thus, juxtaglomerular cell activation could mediate either lateral or feedforward inhibition of these output neurons. In the frog, activation of either GABAB receptors or dopamine D2 receptors in the glomerular/external plexiform layers results in a decrease in mitral cell spontaneous activity with a sparing of odorant-evoked activity (Duchamp-Viret et al., 1997, 2000). These results suggest that one role of inhibition in

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the glomerular layer may be to increase the signal-tonoise ratio of bulb output and thus odor saliency. In the rat, dopamine D2 receptors are located on presynaptic olfactory receptor cell axons (Coronas et al., 1997; Koster et al., 1999; Nickell et al., 1991). Stimulation of dopamine receptors reduces olfactory nerve evoked potentials in olfactory bulb (Gurski and Hamilton, 1996; Hsia et al., 1999; Nowycky et al., 1983), and more specifically activation of D2 receptors in rat reduces glomerular layer odorant-evoked spatial patterns of 2-deoxyglucose uptake (Sallaz and Jourdan, 1992). In contrast, D2 receptor blockade or reduction in olfactory bulb dopamine content enhances and blurs odorant-specific glomerular activation (Guthrie et al., 1990) and increases mitral/tufted cell responsiveness to odorants (Wilson and Sullivan, 1995). In accordance with these physiological results, the D2 receptor agonist quipirole reduces odor detection performance in a dose-dependent manner (Doty and Risser, 1989). Interestingly, systemic injection of the D1 receptor agonist SKF38393 enhances odor-detection performance (Doty et al., 1988). Juxtaglomerular cell dopamine expression is highly odorant experience dependent. Olfactory bulb dopamine levels increase following brief odorant exposure (Coopersmith et al., 1991), while odorant deprivation significantly reduces bulb dopamine content (Brunjes et al., Wilson and Wood, 1992) via an experience-dependent decrease in tyrosine hydroxylase expression (Baker, 1990; Baker et al., 1993; Kosaka et al., 1987; Puche and Shipley, 1999). Given the described effects of dopamine on odorant responses, glomerular layer dopamine may function as a form of experience-dependent volume control—during periods of intense odorant stimulation, dopamine may suppress olfactory nerve input, perhaps to maintain bulb activity within an optimal dynamic range. During periods of weak odorant stimulation, dopamine levels fall to enhance sensitivity of the system. This enhanced sensitivity, however, comes at the price of a decrease in glomerular and mitral/tufted cell odorant discrimination (Guthrie et al., 1990; Wilson and Sullivan, 1995). A strikingly similar dopaminergic mechanism of gain control exists in the vertebrate retina. Dark adaptation leads to changes in dopamine release and a reduction in lateral inhibition in the retina, which increases sensitivity but reduces spatial resolution (Daw et al., 1989). The olfactory bulb glomerular layer thus creates an odorant-specific spatial feature map through precise projection patterns of olfactory receptor axons, while inhibition in the glomerular layer both acts as an experiencedependent gain control and allows sharpening of the odorant-specific spatial patterns.

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B.

Olfactory Bulb Output Neurons

In the rat, mitral cells extend an apical dendrite into a single glomerulus, with each glomerulus innervated by approximately 25 mitral cells (Fig. 2) (Shepherd and Greer, 1998). Mitral cells respond to olfactory nerve input with both a fast AMPA receptor–mediated depolarization and a slower, NMDA receptor–mediated depolarization (Berkowicz et al., 1994; Ennis et al., 1996). Mitral cell responses to odorants are generally more complex than the simple responses described for juxtaglomerular neurons above, reflecting the additional circuit processes affecting these cells. Intracellular recordings of mitral/tufted cell responses to odorants reveal prominent short- and long-latency hyperpolarizations, in addition to the depolarization and evoked spikes presumably mediated by direct glutamatergic excitation from the olfactory nerve (Hamilton and Kauer, 1985, 1989; Wellis et al., 1989). Similar multiphasic membrane potential responses to odorants have been observed with intracellular recordings from invertebrate antennal lobe neurons (Christensen et al., 1998). Low-intensity odorant stimulation within the mitral cell odorant-receptive field evokes a low-amplitude depolarization that may be suprathreshold for spike initiation (Hamilton and Kauer, 1989; Wellis et al., 1989). In salamander, this depolarization is frequently preceded by a brief hyperpolarization (Hamilton and Kauer, 1989). As stimulus intensity increases, the amplitude of the odorantevoked depolarization increases and latency decreases, resulting in a high-frequency burst of spikes. This burst is then followed by a second period of hyperpolarization that can last several hundreds of milliseconds under artificial respiration conditions. As stimulus intensity increases further, the second period of hyperpolarization begins to truncate the evoked spike burst, in some cases leading to a single, short-latency evoked spike followed by hyperpolarization in response to high-intensity odorant. These membrane potential results correspond well with extracellular spike train recordings in a variety of terrestrial species (Chaput and Holley, 1985; Duchamp-Viret and Duchamp, 1997; Harrison and Scott, 1986; Imamura et al., 1992; Kauer, 1974; Mair et al., 1982; Mathews, 1972; Meredith, 1986; Scott, 1977). Thus, in response to a single odorant pulse, a triphasic membrane potential response can be observed in mitral/tufted cells. Odorant intensity appears to be encoded by a rate code and/or a latency code, with responses to high-intensity odorants often consisting of a single spike followed by inhibition. Given the short latency of the initial hyperpolarization, it is assumed to be mediated by juxtaglomerular neurons in a feedforward manner. The initial depolarization is mediated by AMPA and NMDA receptor activation on

Sensory Physiology of Central Olfactory Pathways

apical dendritic tufts of mitral cells. There is also recent anatomical (Allen and Hamilton, 2000) and physiological (Aroniadou-Anderjaska et al., 1999; Friedman and Strowbridge, 2000; Isaacson, 1999) evidence for glutamatergic mitral-mitral cell excitation and/or autoexcitation. These mitral-mitral cell connections could contribute to the synchrony observed in odorant responses of neighboring mitral cells (Buonviso et al., 1992; Kashiwadani et al., 1999; Stopfer et al., 1997), which could also contribute to an intensity code as well as play an important role in odorant quality coding, as discussed below. The late-onset, slow hyperpolarization is mediated by GABAergic granule cell interneurons. Mitral and tufted cells connect with granule cells via dendrodendritic reciprocal synapses along mitral/tufted cell lateral dendrites (Shepherd and Greer, 1998). Glutamate released by mitral/tufted cell dendrites excites AMPA and NMDA receptors on granule cells (Chen et al., 2000; Isaacson and Strowbridge, 1998; Jacobson et al., 1986; Schoppa et al., 1998; Trombley and Westbrook, 1990; Wilson et al., 1996), which in turn release GABA back onto mitral/tufted cell dendrites. Mitral cell lateral dendrites can extend for up to 1 mm around the olfactory bulb, and thus may contact many granule cells. The granule cells are believed to perform lateral inhibitory functions, with GABAergic synapses on distal lateral dendrites, perhaps primarily functioning to reduce backpropagation of spikes along these extended dendrites, rather than directly influencing spike initiation at the initial segment. Direct evidence for such lateral inhibitory actions comes from in vitro studies showing that inhibitory post-synaptic currents (IPSCs) can be evoked in both mitral cells and tufted cells by electrical stimulation of distant glomeruli (Christie et al., 2001). Tufted cells are influenced by a more narrow region of glomerular input (glomerular distances up to 400 ) than mitral cells (glomerular distances up to 800 ), which have much longer lateral dendrites (Christie et al., 2001). This, along with other structural differences (Ezeh et al., 1993; Macrides et al., 1985; Orona et al., 1983, 1984; Scott, 1981), suggests a potential important functional difference between the two principal bulb output neurons, although no detailed comparisons of odorant evoked activity have been made between these two cell types. In addition to the phasic nature of the response within a single odorant pulse, single-unit studies in freely breathing animals demonstrate a strong respiratory cycle modulation of mitral/tufted cell activity (Chalansonnet and Chaput, 1998; Macrides and Chorover, 1972; Ogawa, 1998; Onoda and Mori, 1980; Pager, 1985). Mitral/tufted cell spontaneous activity generally oscillates with the respiratory cycle, with different cells maximally active at different phases of the cycle (inspiration or expiration).

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Odorant stimulation can either enhance the spontaneous patterning of a single cell, or shift cell activity to a different phase of the respiratory cycle (Chalansonnet and Chaput, 1998). The respiratory entrainment of activity during odorant stimulation is stable over a wide range of odorant concentrations, despite potential changes in lateral inhibition discussed above (Chalansonnet and Chaput, 1998). The effects of active sniffing (i.e., an increase in inhalation rate to 5–10 Hz during exploration and arousal) on odorant response patterns has not been thoroughly examined in vertebrates, although it is assumed to modify odorant access to the receptor sheet (Dethier, 1987; Youngentob et al., 1987) and appears to modify granule cell–mediated inhibition in the bulb (Young and Wilson, 1999). More attention has been paid to effects of odorant stimulation frequency in invertebrates (Christensen and Hildebrand, 1988; Gomez et al., 1999; Loudon and Koehl, 2000; Schneider et al., 1998). In sphinx moths stimulated with puffs of odorant at different rates, antennal lobe neuron response patterns varied significantly, with some cells able to make discrete responses to odorant pulses at stimulation frequencies as high as 10 Hz (Christensen and Hildebrand, 1988). Given the ubiquity of active sniffing during exploration across animal species (Dethier, 1987), additional research into the consequences of variations in stimulus frequency on peripheral and central odorant coding seems warranted. For example, olfactory cortical targets of mitral cells must be able to discriminate between a mitral cell weakly excited by an odorant inhaled at normal respiration rates (perhaps evoking a short spike train at 10 Hz), from a mitral cell activated by an intense odorant while sniffing (perhaps evoking a single spike on each inhalation with inhalations occurring at 10 Hz). Of course, in addition to detecting odorants and encoding odorant intensity, mitral/tufted cells encode odorant quality/identity. Odorant quality appears to be encoded by variations in odorant/molecular receptive fields of individual mitral/tufted cells and spatial clustering of cells with similar receptive fields within the olfactory bulb. As with olfactory receptor neurons (Bozza and Kauer, 1998; Malnic et al., 1999; Sato et al., 1994), odorant-receptive fields of mitral/tufted cells are based on responsiveness to molecular features rather than to an odorant as a whole. Odorants within the receptive field of an individual mitral/tufted cell evoke excitatory/suppressive changes in firing rate, generally in phase with the respiratory cycle, as described above. Because of the spatial clustering of cells with similar receptive fields and the lateral inhibitory networks described above, however, mitral/tufted cell receptive fields may be more focused or precise than receptor neurons.

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Individual mitral/tufted cells respond to many odorants (Duchamp-Viret and Duchamp, 1997; Harrison and Scott, 1986; Imamura et al., 1992; Katoh et al., 1993; Kauer, 1974; Mair et al., 1982; Mathews, 1972; Meredith, 1986; Mori et al., 1992). The receptive field appears to include odorants that share a similar molecular feature (carbon chain length or functional group), although blend-or mixture-specific neurons have been identified in the invertebrate antennal lobe (Vickers et al., 1998). Using a homologous alkane odorant series, cross-habituation studies demonstrate that habituation of mitral/tufted cell responses to one odorant within its receptive field significantly suppresses responses to other receptive field odorants (Wilson, 2000b), strongly suggesting that mitral/tufted cell responses to multiple odorants are mediated by a single input. Odorant receptive fields of mitral/tufted cells appear to be organized in a roughly center-surround fashion (Meredith, 1986; Wilson and Leon, 1987). Using a stimulus set of homologous odorants varying in carbon chain length, individual mitral/tufted cells are excited by a range of chain lengths (Imamura et al., 1992; Katoh et al., 1993; Mori et al., 1992) and inhibited by neighboring longer or shorter chain lengths (Yokoi et al., 1995). This inhibitory surround is largely due to granule cell mediated lateral inhibition and can be reduced by GABA receptor antagonists (Yokoi et al., 1995). The excitatory region of the receptive field is believed to be largely dependent on the glomerulus from which that cell receives its afferent input. Thus, just as there are odorant-specific spatial patterns of glomerular activation noted above, there are spatial patterns (or differential spatial responsiveness) of mitral/tufted cell odorant–evoked unit activity (Imamura et al., 1992; Katoh et al., 1993; Kauer and Moulton, 1974; Mori and Yoshihara, 1995; Wilson and Leon, 1988) and local field potential activity (Adrian, 1953; Freeman and Skarda, 1985; Viana DiPrisco and Freeman, 1985). For example, mitral/tufted cells connected to glomeruli in the dorsomedial region of the olfactory bulb have receptive fields that include aliphatic acids and exclude alkanes, while cells in the ventrolateral olfactory bulb have receptive fields that include alkanes and exclude aliphatic acids (Imamura et al., 1993; Katoh et al., 1993; Mori and Yoshihara, 1995). In addition to global variation in odorant receptive field characteristics, local circuit interactions produce more regional variations in odorant receptive fields. Mammalian glomeruli are approximately 100–150  in diameter and include apical dendrites of around 25 mitral cells (Royet et al., 1989; Shepherd and Greer, 1998). Mitral/tufted cells physically near each other, and thus likely to receive input from the same glomerulus (Buonviso et al., 1991a), are

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more likely to respond similarly to odorants, while cells more distant (150 ) are more likely to respond differently (Buonviso and Chaput 1990; Meredith, 1986; Wilson and Leon, 1987). For example, simultaneous recordings from pairs of mitral/tufted cells reveal that if a mitral/tufted cell is excited by amyl acetate, most cells within 100  of that cell will also be excited, while cells 150  will most likely be inhibited or nonresponsive (Buonviso and Chaput 1990). Furthermore, cells stimulated simultaneously with odorant in their receptive fields tend to synchronize their firing (Buonviso et al., 1992; Kashiwadani et al., 1999). Given that individual odors are composed of many features, each of which activates glomeruli at some distance from each other, this synchronization of co-active neurons could be critical for binding of the features into perceptual wholes by higher-order neurons (see below). Granule cell–mediated feedback/lateral inhibition is again implicated in this synchronization (Bressler and Freeman, 1980; Buonviso et al., 1996; Kashiwadani et al., 1999; Rall et al., 1966). Similar observations have been made in the invertebrate olfactory system (Laurent, 1999, Wehr and Laurent, 1996). Desynchronizing antennal lobe output neurons with local infusion of GABA antagonists impairs behavioral odorant discrimination by honey bees (Stopfer et al., 1997). In summary, mitral and tufted cells express odorant receptive fields for molecular features, similar to that described for olfactory receptor neurons. Receptive field characteristics are largely driven by the specific glomerulus from which the cell derives its afferent input, and thus, the specific receptive field expressed by a mitral/tufted cell is largely dependent on that cell’s location in the olfactory bulb. The receptive field appears to include odorants sharing a common molecular feature. Cells near to each other have similar receptive fields and are under lateral inhibitory influences from neighboring glomeruli-output neuron groups. Odorant responses consist of excitatory-inhibitory sequences, which are significantly shaped by both odorant intensity and quality. Respiration parses the response into 100–500 ms long components depending on respiration rate. Within these respiratory cycles, activity is further organized by synchronization of simultaneously firing mitral/tufted cells. C.

Modulation and Nonolfactory Responses

Although mitral/tufted cells in the main olfactory bulb are second-order neurons in the olfactory system, they are already heavily influenced by both current behavioral state and past odorant experience. The olfactory bulb receives massive centrifugal inputs from a variety of olfactory and nonolfactory structures (Shepherd and Greer, 1998).

Sensory Physiology of Central Olfactory Pathways

Centrifugal inputs include acetylcholine (ACh) from the horizontal limb of the diagonal band, norepinephrine from the locus coeruleus, and serotonin from the raphe nucleus, as well as strong feedback from olfactory cortical areas (feedback from olfactory cortical areas constitutes 80% of centrifugal inputs to the bulb) (Haberly, 1998). One of the initial paradigms demonstrating behavioral state modulation of olfactory bulb odorant responsiveness described food-deprivation effects on responses to food odor (Pager et al., 1972). Multiunit and single-unit recordings of mitral/tufted cells in awake rats revealed that responses to food odor or odors associated with food were greater in food-deprived rats than in satiated rats (Pager, 1974, 1983; Pager et al., 1972). Deprivation state had no effect on responses to novel odorants (Pager, 1972, 1983). The enhanced responsiveness to food odor in deprived rats appears to be related to a state-dependent reduction in habituation to the food odor mediated by centrifugal inputs to the bulb (Gervais and Pager, 1983). Lesions of centrifugal input to the bulb (olfactory peduncle cut) eliminate the deprivation-induced modulation of responses to food odor (Pager, 1978). Similar behavioral state or nonolfactory modulation of mitral/tufted cell unit activity (Garcia-Diaz et al., 1985; Jiang et al.,1996; Kay and Laurent, 1999; Nickell and Shipley, 1988; Potter and Chorover, 1976; Scott, 1977; Wilson and Sullivan, 1990) or olfactory local field potentials (Chabaud et al., 2000; Viana DiPrisco and Freeman, 1985) has been demonstrated in other paradigms. Activation of centrifugal inputs to the main olfactory bulb can hyperpolarize mitral cells (e.g., anterior commissure) (Nakashima et al., 1978), enhance mitral/tufted cell spontaneous activity (e.g., norepinephrine) (Wilson and Sullivan, 1991); suppress spontaneous activity (e.g., acetylcholine) (Nickell and Shipley, 1988), or enhance mitral/tufted cell responsiveness to weak afferent input (e.g., norepinephrine) (Jiang et al., 1996). Olfactory bulb output and responsiveness to odorants, therefore, is under constant dynamic regulation by centrifugal inputs responsive to behavioral state and nonolfactory events. Thus, as in other sensory systems, olfactory bulb responses to odorants in behaving animals is a reflection not only of odorant quality and quantity, but also of the context and state of the receiving animal. Finally, mitral/tufted cell odorant–response patterns are modulated not only by current conditions, but also past odorant experience and olfactory learning. As mentioned above, periods of reduced odorant stimulation cause a decrease in glomerular layer dopamine, which, upon subsequent return of odorant input, enhances glomerular and mitral/tufted cell responses to odorant at the expense of odorant discrimination (Guthrie et al., 1990; Wilson and

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Sullivan, 1995). Olfactory associative conditioning also modifies subsequent glomerular (Coopersmith and Leon, 1984; Johnson et al., 1995; Sullivan and Leon, 1986), mitral/tufted cell (Wilson et al., 1987), granule cell (Woo et al., 1996), and local field potential responses (Viana DiPrisco and Freeman, 1985) to the learned odorant. Associative learning during early development enhances odorant-specific focal glomerular 2-deoxyglucose uptake to that odorant (Coopersmith and Leon, 1984; Sullivan and Leon, 1986). Furthermore, mitral/tufted single units near these modified glomeruli display enhanced inhibitory responses selectively to the learned odorant, while cells distant to those glomeruli do not (Wilson and Leon, 1988; Wilson et al., 1987). These changes in olfactory bulb physiology require co-activation of centrifugal noradrenergic input from the locus coeruleus during odorant exposure for induction (Sullivan et al., 1989). The mitral/tufted cell response modification has been hypothesized to be due to learning-induced changes in granule cell–mediated dendrodendritic inhibition (Wilson and Sullivan, 1994). Similar norepinephrine-dependent, learning-induced changes have been observed in odorant-evoked spatiotemporal olfactory bulb local field potentials in adult animals (Viana DiPrisco and Freeman, 1985) and in the accessory olfactory bulb (Brennan and Keverne, 1997) In summary, despite being the first central relay for olfactory information, a variety of nonolfactory signals converge on olfactory bulb neurons to allow dynamic modulation of odorant processing, as well as more permanent odorant memories. In fact, even the first synapse of the olfactory pathway between olfactory receptors and secondorder neurons is capable of experience-dependent plasticity (e.g., LTP) (Ennis et al., 1998) and neuromodulation that can shape spatial and temporal odorant-response patterns.

III.

PIRIFORM CORTEX

A detailed description of the anatomy and synaptic physiology of the piriform is outside the scope of this review, but several excellent reviews exist (Bower, 1991; Haberly, 1998; Lynch, 1986). What follows is a brief introduction to the functional organization of the piriform cortex followed by a description of what is known about the sensory physiology of the piriform cortex. Mitral/tufted cell axons project via the lateral olfactory tract to the olfactory cortex, which is composed of several structures including the anterior olfactory nucleus, a major source of commissural connections in the olfactory system, and the piriform cortex (Fig. 2). While the anterior and posterior regions of the piriform cortex appear to be both structurally (Haberly, 1998; Johnson et al., 2000) and

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functionally (Chabaud et al., 2000; Haberly, 1998; Illig and Haberly, 2000; Litaudon and Cattarelli, 1995; Litaudon et al., 1997a; Mouly et al., 1998; Wilson and Bower, 1992) quite distinct, there are several basic characteristics of piriform cortical functional organization that apply to the entire structure. The piriform cortex is a relatively simple, three-layered cortical structure with pyramidal cell bodies arranged in a tight Layer II and more dispersed in Layer III. Dendrites of both groups of pyramidal cells extend into Layer I, where mitral/tufted cell axons terminate on approximately the most distal half. The proximal half of the dendritic tree receives association and commissural input from other regions of the olfactory cortex. Both the afferent input via the lateral olfactory tract and the commissural/association fibers are glutamatergic, and cortical pyramidal cells express both NMDA and nonNMDA receptor types. GABAergic inhibitory interneurons are located in both Layers I and III. Similar to mitral/tufted cells, piriform cortex neurons display both excitatory and inhibitory responses to odorants (Haberly, 1969; McCollum et al., 1991; Nemitz and Goldberg, 1983; Tanabe, et al., 1975; Wilson, 1998a). Intracellular recordings reveal somewhat more simple odorant-evoked postsynaptic potentials in piriform pyramidal neurons than in mitral cells, although relatively few studies have been reported to date (Nemitz and Goldberg, 1983; Wilson, 1998a, b). In freely breathing rats, odorant stimulation evokes a short-latency large depolarization, in phase with the respiratory cycle (Fig. 3) (Wilson, 1998a). This odorant-evoked depolarization can be suprathreshold for evoking spikes, which can reach instantaneous frequencies of over 100 Hz, but generally are within the range of 50–100 Hz, which corresponds to the odorant-evoked gamma-frequency waves recorded in piriform. The respiratory entrained depolarization is often bounded by periods of hyperpolarization, which accentuate the responses to each inhalation. Despite the remarkable precision and topography of the olfactory nerve input to the olfactory bulb glomerular layer, the mitral/tufted cell projection to the piriform cortex is broadly nontopographic. Projections to the anterior piriform may have some spatial patterning, with individual axons terminating in small clusters rather than being uniformly dispersed (Buonviso et al., 1991b Ojima et al.,), but in general any one region of the olfactory bulb can project to every region of the piriform cortex and any one region of the cortex can receive input from every region of the bulb (Haberly and Price, 1977; Scott et al., 1980). This broadly scattered input from a highly spatially ordered olfactory bulb has led to models of piriform cortex as a combinatorial array, ideally suited to combine odorant molecular features into perceptually whole odors. Thus, co-activation of spatially dispersed mitral/tufted cells

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encoding individual molecular features could in turn activate coincidence detecting pyramidal cells of the piriform cortex, each maximally responsive to a particular combination of features. Broadly dispersive intracortical association fibers further contribute to this associational network (Haberly, 1998; Haberly and Price, 1978; Johnson et al., 2000) If the combinatorial array model of piriform function is correct, then odorant-receptive fields of cortical pyramidal cells might, at least superficially, appear very similar to odorant-receptive fields of mitral/tufted cells, although with the two cell classes responding to odorants for different reasons. That is, as discussed above, a particular odorant may be composed of several features. A mitral cell may respond to that odorant, and similar odorants, because of the presence of a single feature that dominates the receptor input to that mitral cell. A cortical pyramidal neuron, on the other hand, may respond to that odorant, and similar odorants, because of the unique combination of odorant features present (i.e., it responds to the odor(s) as a whole). Odorant responses of piriform cortical single units have been described in several species [frog (Duchamp-Viret et al., 1996), rat (Haberly, 1969), monkey (Tanabe et al., 1975a)] and in both awake (McCollum et al., 1991; Schoenbaum and Eichenbaum, 1995a) and anesthetized preparations (Haberly, 1969; Giachetti and MacLeod, 1975; Nemitz and Goldberg, 1983; Tanabe et al., 1975a; Wilson, 2000). In general, similar to mitral/tufted cells, piriform cortical pyramidal cells have broad odorantreceptive fields (Fig. 3) (Tanabe et al., 1975a; Wilson, 1998a, 2000), although in frog cortex there is also a subpopulation of narrowly tuned cells (Duchamp-Viret et al., 1996). In one of the few direct comparisons of receptive fields between olfactory areas, Tanabe et al. (1975) suggest that piriform cortex single units are somewhat more highly tuned (narrow receptive fields) than mitral cells, with cells in orbitofrontal cortex the most highly tuned—forming a hierarchy of odorant discrimination ability along the primary olfactory pathway (see below). In a more direct test of the combinatorial array hypothesis outlined above, a comparison of odorant crosshabituation between mitral/tufted cells and anterior piriform cortex layer II/III single units was made using a homologous series of alkane odorants. It was hypothesized that if mitral/tufted cells respond to multiple odorants because each of the effective odorants shares a common feature, then habituation to that feature should reduce responsiveness to all odorants by that cell. Piriform cortex cells, however, should show less cross-habituation between similar odorants if cortical cells respond to collections of features, because each odorant would contain a unique feature ensemble. These precise results were

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Figure 3 Examples of odorant-receptive fields (A) and an intracellularly recorded odorant response (B) in anterior piriform cortical neurons. The odorant-receptive fields of piriform cortical neurons are similar to those described for both olfactory receptor neurons and mitral cells, with, for example, responses varying with odorant carbon chain length (A). Receptive fields in piriform cortex are highly dynamic, with rapidly habituating odorant responses (B). (C) In contrast to mitral/tufted cells in the main olfactory bulb, however, this habituation is highly odorant-specific. Responses to odorants differing by only 2–4 carbons in length are unaffected in piriform cortex, while mitral/tufted cells demonstrate more generalized habituation.

obtained in urethane-anesthetized, freely breathing rats (Fig. 3C) (Wilson, 2000). In addition, anterior piriform single units showed minimal cross-habituation between binary odorant mixtures and their components (Wilson, 1998a), further supporting the hypothesis that the piriform cortex synthesizes feature ensembles into perceptual odor wholes.

Given the incredible diversity of potential odorant features and odorant mixtures in the world, however, it is unlikely that the synthesis of feature ensembles in the piriform cortex is based on innate synaptic connections, but rather occurs through olfactory experience-induced synaptic plasticity. Experience-dependent perceptual learning of this sort is used to explain receptive fields in visual

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inferotemporal cortex for complex objects and faces (Gilbert et al., 2001). As a test of the role of experience in cortical feature synthesis, we have recently demonstrated that blockade of piriform cortex cholinergic muscarinic receptors with scopolamine during exposure to novel odorants causes piriform cortex neurons to function as featuredetectors similar to mitral/tufted cells (Wilson, 2001). These results are interpreted as a scopolamine blockade of synaptic plasticity that would normally allow feature ensembles to be synthesized by the cortical neurons. In fact, scopolamine can also prevent behavioral perceptual learning—enhanced olfactory acuity—that occurs after exposure to novel odors (Fletcher and Wilson, 2002). As described above, lateral inhibition forms a critical component of odorant-response patterns in mitral/tufted cells of the olfactory bulb, shaping both the temporal nature of the response as well as emphasizing the spatial nature of the response inherent in olfactory bulb organization. While both feedforward and feedback inhibitory circuits exist in the piriform cortex (Haberly, 1998; Kanter et al., 1996; Kapur et al., 1997; Satou et al., 1982; Scholfield, 1978) and membrane hyperpolarization is expressed in cortical neuron response to odorant (Wilson, 1998a), no investigation of the role of inhibition in cortical odorant responses has yet been carried out. Lateral inhibition functions in most sensory systems to enhance existing spatial response patterns, allowing one cell (or group of cells) to inhibit neighboring cells with similar receptive fields. This can enhance contrast and/or signal-to-noise characteristics of the system. If the piriform cortex truly lacks any spatial organization, then the role of lateral inhibition may be different in this system. Several studies have attempted to detect spatial patterns of evoked activity in the piriform cortex with limited success (Cattarelli, and Cohen, 1989; Cattarelli et al., 1988; Sharp et al., 1977), although some of the difficulty may have been due to the rapid odorant habituation that occurs in the piriform (Wilson, 1998a). Optical imaging of in vivo piriform responses to olfactory bulb electrical stimulation has shown some spatial specificity, with different regions of the bulb activating slightly different regions of anterior piriform, but with diffuse activation of more posterior regions (Litaudon et al., 1997a). Similarly, a more recent study using well-spaced odorant stimuli and c-fos labeling has detected odorant-specific spatial patterns of activated neurons in the anterior piriform, but not in the posterior piriform (lllig and Haberly, 2000). The noted functional difference between the anterior and posterior regions of the piriform cortex has been demonstrated with a variety of techniques including local field potential recordings (Chabaud et al., 1999, 2000; Mouly et al., 1998) and optical imaging (Litaudon and

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Cattarelli, 1995; Litaudon et al., 1997a). The anterior piriform may be further functionally divided into dorsal and ventral regions (Haberly, 1998). These functional distinctions presumably arise from the significant variation in such anatomical features as dominance of lateral olfactory tract input over association fiber input (greatest in the ventral region of the anterior piriform and least in the posterior piriform) and some differences in cell populations (Haberly, 1998) and modulatory inputs (e.g., ACh) (Lysakowski et al., 1987). No studies to date have examined differences in odorant-receptive fields between anterior and posterior piriform neurons, although there is some evidence that posterior piriform responses to odorants may be more plastic than anterior responses (Chabaud et al., 2000; Litaudon et al., 1997b; Mouly et al., 1998). Synaptic plasticity can be evoked in both afferent and association fiber synapses (Jung et al., 1990; Kanter and Haberly, 1990; Roman et al., 1987; Stripling and Patneau, 1999; Wilson, 1998b), although some evidence suggests that association fiber synapses may be under more modulatory control than LOT afferent synapses (Hasselmo and Bower, 1992; Hasselmo et al., 1997; Stripling and Patneau, 1999; Tang and Hasselmo, 1994). Together with the dominance of LOT input and potential spatial patterns of odorant evoked activity in the anterior piriform, these results suggest that the anterior regions of piriform may be more involved in odorant discrimination and the posterior piriform more involved in odorant memory and odorant associations (Hasselmo and Barkai, 1995; Litaudon et al., 1997b). Finally, single units in the anterior piriform cortex of the rat appear to express spatial receptive fields in addition to odorant receptive fields. Single units in the anterior piriform cortex can be driven by odorants unilaterally presented to either the ipsilateral or contralateral naris. Different cells express preferred stimulation sites, with some cells responsive only to ipsilateral stimulation, some only to contralateral stimulation, some equally responsive to both, and some requiring bilateral stimulation (Wilson, 1997). The convergence of ipsilateral and contralateral inputs in piriform cortex may be involved in maintaining bilateral access to odorant memories (Kucharski and Hall, 1987), response amplification (Bennett, 1968; Klimek et al., 1998), or even stimulus localization (Wilson and Sullivan, 1999). Imaging work in humans suggests that the two nares may have somewhat different odorant-tuning characteristics (Sobel et al., 1999) and that cortical odorant processing is lateralized (Zatorre et al., 1992). Thus, commissural pathways in both humans and rats may play a critical role in central odorant processing, the precise nature of which is yet to be described.

Sensory Physiology of Central Olfactory Pathways

A.

Modulation and Nonolfactory Responses

Odorant responses of anterior piriform cortex neurons are extremely dynamic, capable of showing marked habituation within a few inhalations of an odorant in anesthetized rats (Wilson, 1998a). As described above, this habituation is highly odorant specific, thus the cortex can filter out background or currently nonsignificant stimuli, while maintaining responsiveness to novel odorants. In awake rats in an odorant-conditioning task, piriform cortex single units also show a decrease in responsiveness to repeated odorants (McCollum et al., 1991). This rapid, experiencedependent, odorant-specific change in cortical receptive fields is similar to that reported in other sensory systems (Edeline, 1999) and may contribute to odorant identification and memory. Similar experience-dependent, odorantspecific decreases in odorant responses of single units in the orbitofrontal cortex of primates have also been observed, as described below. In the auditory system, both experience-dependent, stimulus-specific decreases and increases can be observed within receptive fields of cortical neurons, following habituation (Condon and Weinberger, 1991) and associative learning (Weinberger, 1998), respectively. While no direct studies of learning-induced changes in piriform cortex single-unit odorant-receptive fields have been reported, work in two other paradigms suggest that such associative changes can occur. Rats can learn to discriminate between “artificial” odorants induced by focal electrical stimulation of different regions of the olfactory bulb (Mouly et al., 1985). Evoked responses in the piriform cortex to these artificial odorants are enhanced as the animal learns this discrimination (Litaudon et al. 1997; Roman et al., 1987). Learning to discriminate real odorants in a similar discrimination paradigm enhances 2-deoxyglucose uptake in the anterior olfactory nucleus in response to the learned odorant (Hamrick et al., 1993). While no learning associated 2-deoxyglucose uptake changes were detected in the piriform cortex in this study, any changes may have been masked by the rapid cortical habituation described above. As in the olfactory bulb, piriform cortex odorant responses can be influenced by behavioral state. The hunger modulation of food odor responses observed in the main olfactory bulb also occurs in local field potential responses to food odor in the piriform cortex, although largely in the posterior piriform and not in the anterior piriform (Chabaud et al., 2000). Similar to the olfactory bulb multiunit responses, these hunger-induced changes in cortical responsiveness are specific to food odor (Chabaud et al., 2000). Activity in the piriform cortex is also modulated by a variety of nonolfactory events, as determined by both

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single-unit (Schoenbaum and Eichenbaum, 1995a) and local field potential recordings (Kay and Freeman, 1998). Analysis of oscillatory local field potentials suggest that during odorant sampling 12–35 Hz -frequency oscillations travel from rostral (olfactory bulb) to caudal regions (entorhinal cortex) (Kay and Freeman, 1998; Chapman et al., 1998). However, in an odorant-conditioning task prior to odorant sampling, these oscillations travel in the reverse direction (Kay and Freeman, 1998). Single-unit recordings in freely moving rats performing an odorant discrimination task similarly show changes in cortical activity during many stages of the odorant-discrimination task in addition to the odorant-sampling period itself, including during preparation for odorant sampling and during receipt of a water reward (Schoenbaum and Eichenbaum, 1995a). Furthermore, piriform odorant responses can be affected by the current learned hedonic valence of that odorant (Schoenbaum and Eichenbaum, 1995a). This is in contrast to the learned changes in olfactory bulb mitral/tufted cell single-unit responses described above. Learned changes in olfactory bulb responses are specific to learned odorants but do not encode learned hedonic valence, i.e., learned aversive odorants and learned appetitive odorants are encoded similarly by the olfactory bulb (Sullivan and Wilson, 1991). Much of this experience- or state-dependent modulation of cortical odorant responses is dependent on centrifugal inputs to the piriform cortex from neuromodulatory centers such as the horizontal limb of the diagonal band (ACh) and locus coeruleus (norepinephrine). Cholinergic modulation of piriform cortex function has received the most attention at both the experimental physiological and neural computation levels. ACh input to the olfactory system plays an important role in behavioral odorant memory. Blockade of ACh muscarinic receptors impairs both associative and nonassociative odorant memory (DeRosa and Hasselmo, 2000; Hunter and Murray, 1989; Ravel et al., 1994). ACh also has a variety of specific effects on piriform physiology (Barkai and Hasselmo, 1994; Hasselmo and Bower, 1992; Linster et al., 1999; Zimmer et al., 1999). In vitro physiology has demonstrated that muscarinic receptor agonists reduce piriform cortex pyramidal cell firing adaptation (i.e., increase duration of bursts evoked by depolarization) (Barkai and Hasselmo, 1994; Tseng and Haberly, 1989), selectively suppress association fiber synaptic activation of pyramidal cells (with minimal effect on LOT afferent synapses) (Hasselmo and Bower, 1992), and enhance associative synaptic plasticity in the piriform cortex (Hasselmo and Barkai, 1995). The muscarinic receptor–mediated suppression of association fibers has been replicated in vivo by stimulation of the horizontal limb of the diagonal band to evoke ACh release in piriform

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(Linster et al., 1999; Rosin et al., 1999). Further in vivo work has demonstrated that electrical stimulation of the horizontal limb of the diagonal band increases excitability of piriform cortex single units via a cholinergic muscarinic mechanism (Zimmer et al., 1999). These physiological effects of ACh on piriform function have led to the hypothesis that ACh reduces interference between similar patterns of odorant input, thus enhancing odorant discrimination and recognition of previously learned odorants (Hasselmo, 1995); norepinephrine may have similar effects in the piriform cortex (Bouret et al., 2000; Hasselmo and Bower, 1992). In support of this model, recent work has demonstrated that the ACh muscarinic receptor antagonist scopolamine applied to the piriform cortical surface or systemically injected reduces odorant discrimination by piriform single units as demonstrated by enhanced cross-habituation (Wilson, 2001). Given that stimulation of the olfactory bulb and piriform cortex activates neurons in the horizontal limb of the diagonal band (Linster and Hasselmo, 2000), odorant stimulation itself can regulate ACh feedback to the cortex and thus modify subsequent coding and plasticity. In summary, the anterior piriform cortex may serve to synthesize odorant feature input from the olfactory bulb into perceptual odor wholes. Odorant discrimination within the piriform cortex is enhanced compared to mitral/tufted cells of the olfactory bulb and olfactory receptors. Extensive association connections within the cortex contribute to and reinforce odorant synthesis as well as allow associative memory to tie odorants and odorant-related experiences together. Experience can produce highly specific changes in cortical odorant-receptive fields, with association fibers and the posterior piriform cortex playing a prominent role in these memory functions. Behavioral state and past experience can shape both piriform odorant responsiveness and general cortical activity through extensive centrifugal inputs to the cortex. Finally, the piriform cortex is a major source of centrifugal input to the olfactory bulb; thus, as in thalamocortical sensory systems, the cortex can directly influence its own input via descending control of activity in more peripheral structures.

IV.

ORBITOFRONTAL CORTEX

The major neocortical area processing olfactory information is the orbitofrontal region of the prefrontal cortex, which in rats includes the insular cortex. Neuroanatomical studies have demonstrated that the piriform cortex projects directly to the orbitofrontal cortex (Johnson et al., 2000; Krettek and price, 1977; Price et al., 1991), as well as to the dorsomedial nucleus of the thalamus, which in turn

projects to the orbitofrontal cortex (Krettek and Price, 1977; Price and Slotnick, 1983). Electrophysiological (Cinelli et al., 1987) and anatomical (Shipley and Geinesman, 1984) evidence suggests there may also be a direct projection from the olfactory bulb to the orbitofrontal/insular cortex in rats. Orbitofrontal cortex efferents form feedback loops with primary olfactory structures including the piriform cortex and dorsomedial nucleus of the thalamus (Price et al., 1991). In both rats and primates, lesions of either the orbitofrontal cortex or the dorsomedial nucleus of the thalamus impair odorantdiscrimination learning (Eichenbaum et al., 1980; McBride and Slotnick, 1997; Staubli et al., 1987; Tanabe et al., 1975b; Zatorre and Jones-Gotman, 1991). Single units in the orbitofrontal cortex respond to, and can discriminate between, odorants in both rodents (Onoda et al., 1984; Schoenbaum and Eichenbaum, 1995a) and primates (Rolls and Baylis, 1994; Tanabe et al., 1975a). Odorant discrimination by single units in the orbitofrontal cortex (as measured by receptive field size) is improved over that observed in the olfactory bulb and piriform cortex (Onoda et al., 1984; Tanabe et al., 1975a; Yarita et al., 1980), and odorant discrimination by ensembles of orbitofrontal neurons is improved over single units (Rolls et al., 1996b; Schoenbaum and Eichenbaum, 1995b). The orbitofrontal cortex also receives inputs from several sensory systems in addition to the olfactory system, including the gustatory, visual, and somatosensory systems (Carmichael and Price, 1995; Cavada et al., 2000), as well as spatial location information (Lipton et al., 1999). In fact, these diverse inputs can converge on single neurons, leading to single cells that respond to olfactory, gustatory, visual, or somatosensory stimuli alone or in combination (Rolls and Baylis, 1994; Rolls et al., 1999). Similar to that described for the piriform cortex, orbitofrontal neurons respond to many phases of odorant discrimination behavior, including during preodorant sampling behavior, odorant sampling, and postodorant reward consummation (Schoenbaum and Eichenbaum, 1995a). While behavioral state and previous olfactory experience shape odorant responses in both the olfactory bulb and piriform cortex, as discussed above, this state-dependent, associative nature of odorant processing appears highly refined in the orbitofrontal cortex. Thus, for example, the response of most odorant-sensitive orbitofrontal cortex neurons to odorants is dependent on taste reward associations of the particular odorant (Critchley and Rolls, 1996a; Rolls et al., 1996b). That is, if the odorant is associated with a pleasant sweet taste, the response to that odorant may be greater than if the odorant is associated with an unpleasant salt taste, or vice versa. These differential responses require repeated experience to emerge and thus represent a learned,

Sensory Physiology of Central Olfactory Pathways

cross-modal association (Rolls et al., 1996a). Other learned cross-modal associations that influence primate orbitofrontal single-unit responses to odorants include vision (Rolls and Baylis, 1994) and somatosensation (Rolls et al., 1999). Cross-modal associations with odorants have been described in both the rat and primate orbitofrontal cortex (Lipton et al., 1999; Rolls et al., 1996a). As in both the main olfactory bulb and piriform cortex, behavioral state also influences orbitofrontal cortical responses to odorants. Orbitofrontal cortex single-unit responses to food odors (or associated food gustatory, visual or somatosensory stimuli) are modulated by hunger (Critchley and Rolls, 1996b). Feeding to satiety selectively reduces orbitofrontal responsiveness to the odor of that food (Critchley and Rolls, 1996b). In humans, eating a single food to satiety selectively reduces pleasant ratings of that food odor, although simple exposure to a food odor for a comparable duration has a similar effect (Rolls and Rolls, 1997). Similarly to the single-unit work in monkeys, odorant pleasantness influences activity in the human orbitofrontal cortex as determined by PET studies (Royet et al., 2000), and feeding to satiety reduces orbitofrontal cortex activation in response to that food odor in humans as determined by fMRI (O’Doherty et al., 2000). These results suggest that odorant coding in the orbitofrontal cortex is similar to that for other sensory stimuli processed by prefrontal cortex, namely that responses to stimuli reflect not only the sensory qualities of that stimulus, but also the current and past context of the stimulus, including sensory and hedonic associations and biological significance (Goldman-Rakic, 1987; Kolb, 1984; Rolls, 2001; Schoenbaum and Eichenbaum 1995a). Orbitofrontal cortex neurons in turn provide descending feedback to the piriform cortex and olfactory bulb (Cinelli et al., 1987; Haberly, 1998), which can modulate subsequent peripheral processing as described above for piriform cortex.

V.

GENERAL PRINCIPLES

We can now return to the original problem stated in the introduction that all sensory systems must function in the real world (Fig. 2). Specifically, the olfactory system must be able to recognize garlic in the spaghetti sauce in the presence of odor from a freshly cut lawn and to recognize that odor as the perceptual entity of “garlic,” despite it being a mixture of many individual molecular components. Our current understanding of olfactory system sensory physiology outlined above, as well as extensive theoretical and computer modeling work (Freeman, 1981; Haberly, 1985; Hasselmo et al., 1990; Lynch, 1986; Mori

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and Yoshihara, 1995; Rolls, 2000; Wilson and Shepherd, 1995), leads to the following description of hypothetical events that may allow this remarkable feat. Odorant molecules are broken into informational features by binding with specific receptors in the nose. Interactions between molecules and/or between features may occur at the receptor level, resulting in unique receptor output for some feature combinations (Cromarty and Derby, 1998; Derby et al., 1991). The contrast between features is then sharpened through precise projections to the main olfactory bulb glomerular layer and glomerular layer inhibition. Again, some feature mixing may occur at the glomerular layer (Joerges et al., 1997; Vickers et al., 1998). Thus, the spatial pattern of activity within the olfactory bulb glomerular layer represents the collection of molecular features, including an initial processing of some feature combinations present in the odorants sampled. Mitral/tufted cells then project this alphabet of features into the piriform cortex. Based on the current behavioral state (hunger?) and past experiences (memory of past food odors), the representation of some features by mitral/tufted cells will be selectively enhanced over others through olfactory bulb centrifugal modulation. The piriform cortex takes the mitral/tufted cell input and furthers the process of combining the features into perceptually whole odors initiated in the periphery. This is performed by the combinatorial anatomy of the piriform and past experience with specific combinations of features. That is, features that have been associated together in the past will, due to experience-dependent synaptic plasticity within the piriform cortex, be more effective at driving coincidence detecting piriform cortical neurons. Thus, rather than random association of odorant features within the piriform cortex, past experience will allow some combinations of features to be more easily combined and salient. This role of the piriform cortex in odorant feature synthesis is suggested by behavioral data showing that animals with piriform cortex lesions have difficulty learning odorant discriminations of complex odorant mixtures, but not of more simple odorants (Staubli et al., 1987). Furthermore, piriform cortex neurons can discriminate between odorant mixtures and their components, suggesting a synthesis of odorant features (Wilson, 1998a). The reassembly of molecular features based on past associative experience within the piriform cortex allows extraction and synthesis of perceptual odor wholes from the collection of molecular features (i.e., the stimuli garlic and grass odor are present), in a conceptually similar way to the synthesis of simple visual features into complex visual objects in higher-order visual cortices (Logothetis and Sheinberg, 1996). In addition, the dynamic receptive fields and enhanced odorant discrimination of the piriform cortex

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allows selective filtering of background or currently less relevant odorants. Again, specific activity patterns will be enhanced depending on the behavioral state of the animal. It is hypothesized that within the piriform cortex, identification of the sensory stimulus (what odor is it?) is largely complete. It should be noted, however, that piriform cortex lesions produce little effect on well-learned odorant discrimination behavior, although they may impair learning to discriminate novel odorants (Slotnick and Schoonover, 1992; Staubli et al., 1987; Zhang et al., 1998) In addition to association of odorant molecular features (sensory processing—this is garlic), the piriform cortex and orbitofrontal cortex combine to allow association of odorants with sensory context, memory, and hedonic reactions (perceptual processing—I see food previously associated with garlic, I have eaten and enjoy garlic, I am hungry for garlic). Through descending connections this perception can influence subsequent peripheral sensory processing by the bulb and anterior piriform cortex. Efferent connections of the piriform and orbitofrontal cortices can then shape behavior appropriate for the given stimulus and current internal state. Something smells good, let’s eat.

ACKNOWLEDGMENTS The authors wish to acknowledge the support of grants from NIDCD (DAW), NSF (DAW), and NICHD (RMS).

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201 Wilson, M., and Shepherd, G. M. (1995). Olfactory cortex. In The Handbook of Brain Theory and Neural Networks M. A. Arbib (Ed.), MIT Press, Cambridge, MA, pp. 669–673. Woo, C. C., Oshita, M. H., and Leon, M. (1996). A learned odor decreases the number of Fos-immunopositive granule cells in the olfactory bulb of young rats. Brain Res. 716:149–156. Yokoi, M., Mori, K., and Nakanishi, S. (1995). Refinement of odor molecular tuning by dendrodendritic synaptic inhibition in the olfactory bulb. Proc. Natl. Acad. Sci. 92:3371–3375. Young, T. A., and Wilson, D. A. (1999). Frequency dependent modulation of inhibition in the rat olfactory bulb. Neurosci. Lett., 276:65–67. Youngentob, S. L., Mozell, M. M., Sheehe, P. R., and Hornung, D. E. (1987). A quantitative analysis of sniffing strategies in rats performing odor detection tasks. Physiol. Behav. 41: 59–69. Zatorre, R. J., and Jones-Gotman, M. (1991). Human olfactory discrimination after unilateral frontal or temporal lobectomy. Brain. 114:71–84. Zatorre, R. J., Jones-Gotman, M., Evans, A. C., and Meyer, E. (1992). Functional localization and lateralization of human olfactory cortex. Nature 360:339–340. Zhang, Y., Burk, J. A., Glode, B. M., and Mair, R. G. (1998). Effects of thalamic and olfactory cortical lesions on continuous olfactory delayed nonmatching-to-sample and olfactory discrimination in rats (Rattus norvegicus). Behav. Neurosci. 112:39–53. Zimmer, L. A., Ennis, M., and Shipley, M. T. (1999). Diagonal band stimulation increases piriform cortex neuronal excitability in vivo. NeuroReport 10:2101–2105.

10 Psychophysical Measurement of Human Olfactory Function, Including Odorant Mixture Assessment Richard L. Doty University of Pennsylvania, Philadelphia, Pennsylvania, U.S.A.

David G. Laing University of Western Sydney, Sydney, Australia

I.

mathematical concepts developed in the mid-nineteenth century by Weber (1834) and Fechner (1860) and by Thurstone, Stevens, and others in the twentieth century (e.g., Anderson, 1970; Stevens, 1961; Thurstone, 1927a,b). Tests derived from these traditions include absolute detection thresholds (the lowest odorant concentration that can be perceived), differential thresholds (the smallest difference in concentration of a given chemical that can be perceived), and various indices of suprathreshold sensation magnitude. Most were developed within the theoretical framework of establishing mathematical rules or laws that govern the build-up of suprathreshold sensation relative to stimulus intensity. To achieve these ends, well-defined stimuli (e.g., single chemicals of known chemical purity) were usually employed, allowing for straightforward stimulus specification. Other trends, however, resulted in the development or application of tests more useful in applied settings. For example, eighteenth- and nineteenth- century physicians simply presented familiar odorants to patients to see if they could be identified, usually without insight into prior psychophysical developments. In the twentieth century relatively sophisticated procedures were developed within the food industry (e.g., the forced-choice triangle test), where the need exists for quantifying the discriminability or

INTRODUCTION

As can be gleaned from other chapters in this volume, the perception of an odorant depends upon the activation of a subset of ~1000 olfactory receptor types distributed, in the human, across ~6,000,000 receptor cells. Each receptor cell commonly carries only one type of receptor, and the relative distribution of the receptor types among the ~6,000,000 receptor cells is unknown. Since most odorous substances found in nature are comprised of more than one chemical, a typical stimulus simultaneously activates overlapping subsets or arrays of many olfactory receptor cells. From these arrays the nervous system extracts a unitary sensation for a given stimulus, although, for some substances, a few major “notes” can be discerned, as is well known to wine and beer connoisseurs. Hence, from one perspective olfaction is largely a synthetic sensory system, synthesizing a distinct individual sensory sensation from a complex set of chemicals, many of which have an individual odor. From another perspective, however, it is an analytical sensory system, capable of extracting from hundreds of potential sensations a few dominant qualities. During the last two centuries, numerous tests have been devised to assess the function of this system. Historically, many of these tests have been modeled on procedural and 203

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acceptability of various product formulations in relation to perceived qualitative attributes. Unlike the academic psychophysical traditions, and akin to the clinical traditions, the stimuli were multicomponent or chemically complex. Although quantitative, the metrics employed in these paradigms were more operational and rarely linked to simple physicochemical properties such as odorant concentration. The present chapter has two major goals. The first is to provide the reader with an up-to-date overview of the quantitative methods available for assessing the sense of smell, regardless of the historical traditions that led to their development. Emphasis is placed on the relative utility of various approaches for achieving this end. The second goal is to examine elements of odor mixture perception, including how well individual components can be discerned. An understanding of odor mixture processing is of value in elucidating how the olfactory system works, as its neural architecture seems to be designed to filter or collapse complex arrays of chemical information into distinct, interpretable, and manageable percepts. Although many of the examples described in this chapter come from clinical studies, the tenants of the chapter are broadly applicable to settings outside the clinic, including industrial and regulatory ones.

II.

STIMULUS CONTROL AND PRESENTATION

In some chemosensory paradigms, extremely accurate stimulus specification is required, and elaborate olfactometers and other devices for presenting known concentrations of odorants in specific quantities for various durations have been devised (for review, see Prah et al., 1995). This is particularly true for devices employed in event-related potential research (see Chapter 11). In other paradigms, including those related to assessing olfactory function in patients, it is not necessary to know the exact number of molecules that enter the nose to make the test valid. The key issue in the latter case is that the odorants are presented in a reliable manner and that norms are available to establish whether a patient’s responses are normal or abnormal. Thus, accurate clinical assessment of chemosensory function can be made using surprisingly simple stimulus presentation equipment. Devices used to present odorants to humans include (1) the draw tube olfactometer of Zwaardemaker (1925, 1927), (2) glass sniff bottles (Cheesman and Townsend, 1956; Doty et al., 1986; Nordin et al., 1998), (3) odorized glass rods, wooden sticks, felt-tipped pens, alcohol pads, or strips of blotter paper (Davidson & Murphy, 1997; Hummel et al., 1997; Semb, 1968; Toyota et al., 1978),

(4) plastic squeeze bottles (Amoore and Ollman, 1983; Cain et al., 1988; Doty, 2000; Guadagni et al., 1963), (5) air-dilution olfactometers (Cheesman and Kirkby, 1959; Doty et al., 1988b; Kobal and Plattig, 1978; Lorig et al., 1999; Punter, 1983; Walker et al., 1990; Wenzel, 1948), (6) microencapsulated “scratch and sniff” odorized strips (Doty, 1995; Doty et al., 1984a; Richman et al., 1992), and (7) bottles from which blasts of saturated air are presented (Elsberg and Levy, 1935) (Fig. 1). In environmental control studies, mobile units containing olfactometers, odor exposure chambers, analytical equipment, and subject waiting rooms have been employed (e.g., Berglund et al., 1984; Springer, 1974) (Fig. 2). In addition to these approaches to the presentation of stimuli, intravenous administration of odorants has been employed to produce chemosensory sensations. This has been used primarily by Japanese otolaryngologists in an attempt to determine whether the olfactory receptors are working when nasal congestion or blockage eliminates or mitigates airflow to the receptor region. The assumption underlying this technique is that the stimulus makes its way to the olfactory receptors via the bloodstream. Most commonly thiamine propyldisulfide (Alinamin) is injected into the median cubital vein, and recordings of the duration and latency of the onset of a garlic-like sensation experienced by the patient are made (see Takagi, 1989, for review). Although this procedure may be of value in some cases, there is some controversy regarding its physiological basis (i.e., whether the stimulus reaches the receptors via diffusion from nasal capillaries, from lung air, or both) (see Maruniak et al., 1983). Furthermore, such testing is invasive, highly variable, not readily adaptable to a forced-choice paradigm, and lacks normative referents.

III. PSYCHOPHYSICAL TEST PROCEDURES Today, any procedure that provides a quantitative measure of sensory function and requires a verbal or conscious overt response on the part of the examinee is generally considered to be a psychophysical procedure. In this section, the basic psychophysical paradigms available for measuring olfactory function are discussed and examples of their application are provided. The interested reader is referred to other sources for more detailed information about psychophysical methods, including their mathematical foundations (Ekman and Sjöberg, 1965; Gescheider, 1988; Guilford, 1954; Köster, 1975; Marks, 1974; Stevens, 1961; Tanner and Swets, 1954).

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Figure 1 Procedures for presenting odorants to subjects for assessment. (A) Early draw-tube olfactometer of Zwaardemaker. In this apparatus, an outer tube, made of rubber or another odorous material, slides along a calibrated inner tube, one end of which is inserted into the subject’s nostril. When the odorized tube is slid toward the subject, less of its internal surface is exposed to the inspired airstream, resulting in a weaker olfactory sensation. (B) Sniff bottle. (C) Perfumer’s strip. (D) Squeeze bottle. (E) Blast injection device. The experimenter injects a given volume of odor into the bottle and releases the pressure by squeezing a clamp on the tube leading to the nostril, producing a stimulus pulse. (F) Microencapsulated “scratch-and-sniff ” test. (G) Sniff ports on a rotating table connected to one of the University of Pennsylvania’s dynamic air-dilution olfactometers.

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Figure 2 (Top) Odor evaluation room of mobile odor evaluation laboratory designed to evaluate responses of panel members to diesel exhaust. (Bottom) Schematic of mobile odor evaluation laboratory. (From Springer, 1974.)

A.

Detection and Recognition Threshold Tests

A popular means for assessing chemosensory function is to establish, operationally, a measure of the lowest concentration of a stimulus that can be detected. A qualitative odor sensation (e.g., “banana-like”) is rarely perceived at very low odorant concentrations, where only the faint presence of something is noted. The absolute or detection threshold is the lowest odorant concentration where such a presence is reliably detected, whereas the recognition threshold is the lowest concentration where odor quality is reliably discerned. In modern olfactory detection threshold testing, a subject is asked to indicate, on a given trial, which of two or more stimuli (e.g., a low concentration odorant and one or more nonodorous blanks) smells strongest, rather than to report whether an odor is perceived or not. Recognition thresholds are obtained in a similar manner, but the requirement is to report which one has the target quality. Such “forced-choice” procedures are less susceptible than non-forced-choice procedures to

contamination by response biases (i.e., the conservatism or liberalism in reporting the presence of an odor under uncertain conditions). In addition, they are typically more reliable and produce lower threshold values (Blackwell, 1953; Doty et al., 1995). Two types of threshold procedures that have received the most clinical use are the ascending method of limits (AML) and the single staircase (SS) procedures. In the AML procedure, odorants are presented sequentially from low to high concentrations and the point of transition between detection and no detection is estimated. Forcedchoice responses are required on each trial. In the SS method (a variant of the method of limits technique) (see Cornsweet, 1962), the concentration of the stimulus is increased following trials in which a subject fails to detect the stimulus and decreased following trials where correct detection occurs. In both these procedures, the direction of initial stimulus presentation is made from weak to strong in an effort to reduce adaptation effects of prior stimulation (see Pangborn et al., 1964). An example of a clinical application of the AML procedure is provided by Cain (1982a) who used 60-mL glass sniff bottles to present either water (diluent) or odorant (n-butanol dissolved in water) to 43 patients with various degrees of olfactory dysfunction. Four repeated ascending series were presented to each side of the nose in a twoalternative, forced-choice format. This test, which took approximately half an hour per patient to administer, demonstrated that the olfactory dysfunction in these cases was typically bilateral. An example of the clinical use of a SS procedure comes from a study that demonstrates loss of olfactory function in early Alzheimer’s disease (Doty et al., 1987). In this experiment, a trial consisted of the presentation of two 100-mL glass sniff bottles to the patient in rapid succession. One bottle contained 20 mL of a given concentration of phenyl ethyl alcohol dissolved in USP-grade light mineral oil, whereas the other contained mineral oil alone. The patient was asked to report which of the two bottles in a pair produced the strongest sensation. The first trial was presented at a -6.50 log (liquid volume/volume) concentration step. If a miss occurred on any trial before five were correctly completed at that concentration, the process was repeated at 1 log concentration step higher. When five consecutive correct trials occurred at a given concentration level, the staircase was “reversed” and the next pair of trials was presented at a 0.5 log concentration step lower. From this point on, only one or two trials were presented at each step (i.e., if the first trial was missed, the second was not given and the staircase was moved to the next higher 0.5 log step concentration). When correct performance occurred on both trials, the concentration of the next trial was given at

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0.5 log unit step lower. The average of the last four of seven staircase reversal points served as the threshold estimate. Examples of individual data obtained using the SS procedure are shown in Figure 3. In general, threshold values are relative and dependent upon such factors as the method of stimulus dilution, volume of inhalation, species of molecule, type of psychophysical task, and number of trials presented (Pierce et al., 1996). A number of investigators have been struck with the fact that threshold measures often exhibit considerable intra- and intersubject variability. For example, in one study of 60 subjects, intersubject variation as great as 5 log units was reported (Brown et al., 1968). In another, in which a non– forced-choice ascending threshold procedure was used (the Japanese “T&T Olfactometer”), variation on the order of 16 log units was present among groups of 430 –1000 young

subjects (Yoshida, 1984). More recently, Stevens et al. (1988) obtained 60 threshold values over the course of 30 days from three subjects (20 for butanol, 20 for pyridine, and 20 for -phenylethylmethylethylcarbinol). These investigators found that the within-subject variability across test days was as great as the between-subject variability on a given test day, suggesting to these authors that the large individual differences observed in threshold values are not a reflection of big differences among stable threshold values of subjects but reflect large day-to-day fluctuations in the test measures. Unfortunately, much of this fluctuation likely reflects the use of the single ascending detection threshold technique, in which the apparent limen is traversed only once. Clearly, test procedures with more trials, such as the SS procedure, produce less variable measures and, when employed, do not exhibit as marked day-to-day fluctuations.

Figure 3 Data illustrating single-staircase detection threshold determinations. Each plus (+) indicates a correct detection when an odorant versus a blank is presented. Each zero (0) indicates an incorrect report of an odorant. Threshold value (T; vol/vol in light USP grade mineral oil) is calculated as the mean of the last four of seven staircase reversals. Although the geometric mean is the correct measure, the arithmetic mean usually provides a close approximation. The o’s and d’s on the abcissa indicate the counterbalancing order of the presentation sequences for each trial and are read downward (o-odorant presented first, then diluent; d-diluent presented first, then odorant). In the first reversal point (where five correct sets of pairs occur at the same concentration), the fifth order sequence is determined by the first o or d of the subsequent column of four order sequences. (From Doty, 1991a.)

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Difference Threshold Tests

In classical psychophysics, the smallest amount by which a stimulus must be changed to make it perceptibly stronger or weaker is termed a “just noticeable difference,” or JND. This value is also called a difference or differential threshold (in contrast to an absolute threshold, as described above). The size of the increment in odorant concentration ( I) required to produce a JND increases as the comparison concentration (I) increases, with the ratio approximating a constant; i.e., I/I
K (Weber’s law) (Weber, 1834). K is a rough index of the sensory system’s sensitivity (i.e., the smaller the K value, the more sensitive the system is to fine changes in stimulation). However, numerous studies suggest that K is not a constant, being influenced by the size of I, particularly at the extremes of the sensory continuum (Doty, 1991a). An example of a brief clinical test used to establish a difference threshold is described by Eichenbaum et al. (1983). In this test, 10 binary dilutions (in water) of acetone, ethanol, almond extract, and lemon extract were presented. Initially, the highest and lowest concentrations of a given odorant were presented and the subject was required to choose the stronger stimulus. Successively stronger stimuli were then paired with the strongest stimulus until, on the last of the 10 trials, the two samples were identical. Eichenbaum operationally defined the difference threshold as the lowest concentration for which discrimination up to and including the dilution was effortless. C.

Signal Detection Tests

Signal detection theory (SDT) differs fundamentally from the approach of sensory measurement inherent in classical threshold theory. Thus, SDT rejects the notion of a threshold (whether absolute or differential) and focuses on (1) noise and signal plus noise as the milieu of the detection situation and (2) the influences of subject expectancies and rewards on the detection decision. Signal detection procedures provide both a measure of sensory sensitivity and the subject’s response criterion or bias (Tanner and Swets, 1954). In effect, the response criterion is the internal rule used by a subject in deciding whether or not to report detecting a stimulus (e.g., the liberalism or conservatism in reporting a sensation under uncertain circumstances). For example, two subjects may experience the same subtle degree of sensation from a very weak stimulus. One, however, may report that no sensation was perceived (e.g., perhaps because of lack of self-confidence), whereas the other may report the presence of the sensation. In both cases, the stimulus was perceived to the same degree. However, the two subjects had different criteria for reporting its presence. In

Figure 4 Hypothetical distributions of signal plus noise (SN) and noise alone (N) plotted on the same axes. When the strength of the perceived signal increases, the SN distribution moves to the right, increasing d’, the measure of the distance between the two distributions in standard deviation units (z-scores). (From Doty, 1976.)

a traditional non–forced-choice detection threshold paradigm, the investigator would conclude that these two subjects differed in sensitivity to the stimulus, when, in fact, they only differed in regards to their response biases. SDT assumes that a stimulus is imbedded within a background of noise. Noise can arise from a variety of sources and can be conceptualized at a number of levels (e.g., variations in attention, stimulus fidelity, neural firing unrelated to the stimulus, fluctuations in distracting physiological processes). In most cases noise is assumed to be normally distributed (as is done here to simplify discussion). Whenever a signal is added to the “noise” (N) distribution, a “signal plus noise” (SN) distribution results. Both the N and SN distributions can be placed on the same set of axes, as shown in Figure 4. The measure of the subject’s sensitivity is the distance between the means of these distributions. The concept of the response criterion is illustrated for a hypothetical subject in Figure 5 (Doty, 1991a). On any given trial, a low-concentration odorant (SN) or a blank stimulus (N) is presented, and the subject’s task is to report whether or not an odor was presented. Reports of “yes” are represented by the areas under the N and SN curves to the right of the vertical line depicting the subject’s response criterion, whereas reports of “no” are indicated by the areas to the left of this line. In case 1, the subject exhibits a very liberal criterion, reporting the presence of an odor on the majority of the SN trials () and on half of the N trials (). Thus, although correct detection of the odorant occurred nearly all of the time (), many false alarms () were present. Perhaps in this instance the subject was rewarded for reporting the detection of an odor and not admonished for making false alarms. In case 2, the subject chose a less liberal response criterion. Although fewer correct detections of the odor were made (), fewer false alarms were also made (). In case 3, the observer chose a very conservative response criterion, making few false alarms but similarly making fewer correct detections. This

Psychophysical Measurement of Human Olfactory Function

Figure 5 Hypothetical examples of how the response criterion can vary when perceptual sensitivity (d) remains constant. In case 1, a liberal criterion was chosen in which a relatively large number of false positives occurred [i.e., α, the reports of the presence of odor when the blank (N) is presented]. In cases 2 and 3 more conservative criteria were chosen, decreasing both the number of false positives (α) and hits (β). Traditional threshold measures confound the influences of perceptual sensitivity and the setting of the response criterion. (From Doty, 1976. Copyright © 1976, Academic Press.)

would tend to result, for example, when a subject is penalized for making false positives and given few rewards for successful detection of the odor. In all three of these hypothetical cases, the sensitivity (i.e., d) was equivalent, as indicated by the constant distance between the N and SN distributions. In a typical olfactory experiment employing SDT, the subject is presented with a large number of trials of a single low concentration of odorant interspersed with blank trials (Doty et al, 1981; Semb, 1968). Even though the number of blank and odorant trials need not be equivalent, this is commonly the case. The proportion or percent of the total odor trials (S) on which a subject reports detecting an odor (the hit rate) is calculated, as is the percent of blank trials (N) on which an odor is reported (the false alarm rate). The parametric sensitivity measure, d, can then be computed by converting the proportions to normal distribution standard deviation values (z-scores) via a normal probability table; d equals the z-score for hits minus the z-score for false alarms. A more convenient procedure for

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determining d for any combination of hit and false-alarm proportions is to use the table provided by Elliot (1964). In addition, nonparametric signal detection measures are also available (Brown, 1974; Frey and Colliver, 1973; Grier, 1971; Hodos, 1970; but see Macmillan and Creelman, 1996), as are methods for testing the parametric assumptions of traditional signal detection analysis (Gescheider, 1976; Green and Swets, 1966). The classical parametric measure of response bias is termed . Not to be confused with the  in Figure 5,  represents the ratio, at the criterion point, of the ordinate of the SN distribution to that of the N distribution. This value can be easily calculated from the hit and false-alarm rates by use of ordinate values from the normal curve, as discussed by Gescheider (1976). Despite the fact that hundreds of trials have traditionally been used in signal detection studies, some chemical senses studies have employed far fewer trials, largely out of practicality considerations. For example, Potter and Butters (1980) and Eichenbaum et al. (1983) computed d using only 30 test trials. Even though such estimates are somewhat unstable (because a test’s reliability is a function of its length), they may be less so than typically assumed, and there is at least some empirical rationale for the use of abbreviated signal detection tests. Thus, O’Mahony et al. (1979b), in a study of gustatory sensitivity to sodium chloride, found that Brown’s (1974) nonparametric R index fell, after 40 trials, within 5% of the values obtained after 200 trials in slightly over half the subjects tested. However, an analogous olfactory study has not been performed, and ideally all of the subjects should evidence such response stability. For these reasons it is prudent to use as many trials as possible in signal detection tasks. D.

Suprathreshold Scaling Procedures

A number of psychological attributes can be assigned to odors, including strength, pleasantness, and quality. Although the first of these attributes changes in a systematic way with stimulus concentration, odorant pleasantness or unpleasantness is more variable and idiosyncratic (see Doty, 1975). In regard to odor quality, only in rare instances is it dramatically altered by changes in suprathreshold odorant concentration. Since the perceived intensity of an odorant is a function of its concentration, ratings or other measures of perceived intensity have been used to evaluate olfactory function. Because the intensity of a stimulus is related to the number of neurons that are recruited and the frequency at which they fire, such measures may relate to the extent of neural damage present in the afferent pathway (Drake et al., 1969). However, suprathreshold rating or scaling methods appear to be less

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sensitive to olfactory dysfunction than a number of other tests (e.g., detection threshold tests and tests of odor identification), although they have the advantage of being relatively brief, easy to administer, and less susceptible than threshold tests to subtle stimulus contamination. Negative findings, however, must be conservatively interpreted, as in some cases suprathreshold rating scales have completely missed major changes observed by other methods (e.g., the influences of age on olfactory function) (see Rovee et al., 1975). Despite the fact that olfactory psychophysicists and psychometricians have sought to develop psychological scales with ruler-like properties (i.e., the so-called ratio scale, where distances along the scale have ratio properties and a true zero point is present), the degree to which this is possible is debatable. Judgments of the intensity of odors must be viewed as relative, as they are markedly influenced by both subject idiosyncrasies and contextual factors (e.g., a moderately intense odor is reported to be more intense when presented with weak comparison stimuli than with strong comparison stimuli) (Eyman et al., 1975; Helson, 1964). Fortunately, for the purposes of clinical testing, neither the exact form of the underlying psychological scale nor the influences of stimulus context need to be of great concern to the examiner, as long as the test procedures are standardized and it can be demonstrated that the responses on the scaling tasks are reliable and differentiate among persons with differing degrees of olfactory function. Rating scales can be used to estimate the relative amount of a psychological attribute perceived by a subject. In chemosensory assessment, two types are popular: category scales, where the relative amount of a sensation is signified by indicating which of a series of discrete categories best describes the sensation, and line scales (also termed visual analog or graphic scales), where the subject or patient indicates the strength of the sensation by placing a mark along a line that has descriptors (termed anchors) located at its extremes (e.g., very weak–very strong). Recently, scales have been developed in which logarithmic elements have been incorporated into their design in an effort to overcome ceiling effects and to more closely mimic ratio-like properties of magnitude estimation (see below) (e.g., Green et al., 1996; Neely et al., 1992). The reader is referred elsewhere to discussions of the properties of rating scales, including the influence of category number on their psychometric properties (Anderson, 1970; Doty, 1991b; Guilford, 1954). Intensity matching procedures have also been applied in the clinical and other applied settings, with cross-modal matching procedures (e.g., magnitude estimation) being the most popular. In cross-modal matching, the relative

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magnitude of each member of a stimulus set is estimated by using some other sensory modality or cognitive domain. A key difference between this procedure and rating scale procedures is that the ratio relations among the intensities of the different stimuli are defined, and the subject’s responses are not confined to categories or a short response line. Continua commonly used in the cross-modal matching task termed magnitude estimation include number (e.g., assigning numbers proportionate to an odor’s intensity) and distance (e.g., pulling a tape measure a distance proportional to an odor’s intensity) (Berglund et al., 1971; Stevens, 1961). When intensities of sensations from two or more modalities are judged on a single common scale, the procedure is termed the method of magnitude matching. Magnitude estimation and magnitude matching are among the most commonly used cross-modal matching procedures and are discussed in more detail below. In the typical magnitude estimation paradigm, the subject assigns numbers relative to the magnitude of the sensations. For example, if the number 60 is used to indicate the intensity of one concentration of an odorant, a concentration that smells four times as intense would be assigned the number 240. If another concentration is perceived to be half as strong as the initial stimulus, it would be assigned the value 30. The examinee can assign any range of numbers to the stimuli, as long as they reflect the relative magnitudes of the perceived intensities. In some cases, a standard for which a number has been preassigned (often the middle stimulus of the series) is presented to the subject in an effort to make his or her responses more reliable. In other cases, the individual is free to choose any number system he or she wishes, as long as the numbers are made proportional to the magnitude of the attribute (the “free modulus method”). For example, one subject may choose to assign the first stimulus the number 250, whereas another may choose to assign this same stimulus the number 5. If a second stimulus is perceived to be 10 times stronger than the first by each of these individuals, the first one would assign the number 2500, whereas the second one would assign the number 50. The important point is that the absolute values of the numbers are not important; only the ratios between them are relevant. To obtain an index of suprathreshold function, magnitude estimation data are most commonly plotted on log-log coordinates (log magnitude estimates on the ordinate and log odorant concentrations on the abscissa) and the best line of fit determined using linear regression. The resulting function, log P
n log  log k, where P
perceived intensity, k
the Y intercept,
stimulus concentration, and n
the slope, can be represented in its exponential form as a power function, P
k n, where the exponent n is the slope of the function on the log-log plot. In olfaction,

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Figure 6 Relationship between perceived magnitude of three types of stimuli, as measured by magnitude estimation, and stimulus magnitude. Note that the perceived intensity of the example odorant increases in a negatively accelerated fashion, indicating a power function exponent less than 1 (in this case 0.33). (Adapted and modified from Stevens, 1961.)

n varies in magnitude from odor to odor, but is generally less than 1, reflecting a negatively accelerated function on linear-linear coordinates (Fig. 6). As noted elsewhere, various investigators have made modifications in these equations in an attempt to take into account such factors as threshold sensitivity and adaptation (Doty, 1991a; Overbosch, 1986). It is noteworthy that magnitude estimation, perhaps more so than most other sensory procedures, can be biased or influenced in systematic ways by procedural and subject factors (Doty, 1991a; Marks, 1974). The magnitude estimation task is relatively complex in that accurate responses to a stimulus require a good memory for the prior stimulus. If too much time lapses between the presentation of stimuli, the memory of the prior stimulus fades. On the other hand, if the trials are spaced too closely together, adaptation can distort the relationship. Not all subjects consistently provide ratio estimates of stimuli, and a number do not understand the concept of producing ratios (Baird et al, 1970; Moskowitz, 1977). Furthermore, the magnitude of the exponent is dependent on the choice of the stimulus scale (i.e., the units in which the stimulus concentration is expressed), although in olfaction this is probably of minor consequence (Myers, 1982). The degree to which these and other potential shortcomings hinder the use of magnitude estimation procedures in applied settings, such as the clinic, is not known; however,

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it is likely that such problems can be minimized by ensuring that the instructions, test procedures, and test stimuli are carefully standardized and monitored. Comparative assessments of nine-point rating scales, line scales, magnitude estimation scales, and a hybrid of category and line scales suggest that, for untrained or mathematically unsophisticated subjects, category scales and line scales may be superior to magnitude estimation when such factors as variability, reliability, and ease of use are considered (Lawless and Malone, 1986a,b). Since the magnitude estimation function’s intercept and height above the origin depend to a large degree on idiosyncratic differences in the use of numbers and the specific magnitude estimation method employed (e.g., fixed vs. free modulus), only its slope has traditionally been used as an index of sensory function. In an attempt to gain additional information from the function’s ordinate position, investigators have employed the method of cross-modal magnitude matching, which provides, at least theoretically, information about the perceived intensity of stimuli from the absolute position of the magnitude estimation function and corrects, to some degree, for differences among subjects in number usage (for a detailed discussion of this procedure, see Marks et al., 1988). In the most common application of this method, judgments of the intensity of sensations from two modalities (e.g., loudness and odor intensity) are made on a common magnitude estimation scale (Marks et al., 1986). Under the assumption that subjects experience stimuli on one of the continua (i.e., loudness) in a similar manner (an assumption that some question), differences among their loudness ratings would be expected to reflect differences in number usage. The odor intensity continuum can then be adjusted accordingly. Such normalization allows, theoretically, for a direct comparison of scale values across subjects; thus, if the adjusted odor intensity magnitude value for one subject is 10 and for another subject is 20 at the same concentration level, the second subject is presumed to experience twice the odor intensity as the first subject. E.

Quality Discrimination Tests

The most straightforward chemosensory quality discrimination test requires individuals to decide whether two stimuli have the same or different quality. In one scenerio, a series of same-odorant and different-odorant pairs is presented, and the proportion of pairs that are correctly differentiated is taken as the measure of discrimination (O’Mahony, 1979; O’Mahony et al., 1979; Potter and Butters, 1980;). Variants on this theme include picking the “odd” stimulus from a set from which only the “odd” stimulus differs (e.g., the socalled triangle test) (Frijters et al., 1980).

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Another form of discrimination test is based on a procedure called multidimensional scaling (MDS). In one variant of this procedure, ratings are made for all possible pairs of stimuli (or selected subsets of pairs) on a line scale anchored with descriptors like “completely different vs. exactly the same,” and the correlation matrix among these ratings is subjected to an algorithm that places the stimuli in two- or more dimensional space relative to their perceived similarities (e.g., Schiffman et al., 1981). The process is akin to constructing a map of a country from a list of distances available between the cities of that country. Persons with poor discrimination abilities fail to discern differences and similarities among stimuli, as illustrated by multidimensional spaces that have no distinct or reliable groupings. Because of its time-consuming nature and the fact that statistical procedures for comparing one person’s MDS space to another’s (or to a norm) are poorly worked out, MDS has not been used routinely in the clinic. Interestingly, when subjects are asked to rate the similarity of stimuli that are only indicated to them by name (i.e., the odorants, per se, are never presented), stimulus spaces derived by MDS are analogous to those obtained by the actual use of the odorants (Carrasco and Ridout, 1993; Ueno, 1992). This implies that well-defined imagery, or at least conceptual representations, exist for odorous stimuli. Recently, Wise and Cain (2000) used a response latency approach to determine the discriminability of unmixed odors and mixed odors. A clear monotonic relationship was found between latency and accuracy, with latency decreasing with accuracy. In addition, subjects required more time and made more errors in discriminations between binary mixtures and their unmixed components than between the unmixed components. It was concluded that this approach may provide a novel measure of differences in odor quality, since latency provides information about discriminability. F.

Quality Recognition Tests

Two general classes of quality recognition tests can be defined. In the first class, the subject is asked whether each stimulus of a presented set is recognized. Identification is not required. As indicated at the beginning of the chapter, this procedure is relatively crude, despite the fact that it is perhaps the most common means used by neurologists to measure olfactory function (Sumner, 1962). In the second class, a patient is presented with a “target” stimulus and subsequently asked to select the target from a larger set of stimuli. The number of correct responses of a series serves as the test score. A variant on this theme is the stimulus matching task, in which a set of stimuli are provided and the subject is

required to match the stimuli, one by one, to those of a set of identical stimuli. As an example, Abraham and Matha (1983) presented subjects with eight vials that contained four odorants (two vials per odor). The subject’s task was to pair up the equivalent two-vial containers. The number of pairs correctly matched on each of two administrations of the test was used by these authors as the test score. G.

Quality Identification Tests

Among the most popular procedures for assessing taste and smell function are those that require stimulus quality identification. Such tests can be divided into three groups: naming tests, yes/no identification tests, and multiplechoice identification tests. The respective responses required, on a given trial, in these three classes of tests are (1) to provide a name for the stimulus, (2) to signify whether the stimulus smells like an object named by the examiner (e.g., does this smell like a rose?), and (3) to identify the stimulus from a list of names or pictures. Odor naming tests in which no response alternatives are provided have been used clinically (e.g., Gregson et al., 1981) but are of limited value since many normal individuals have difficulty in naming or identifying even familiar odors without cues. Yes/no identification tests are much more useful, since they require a patient to report whether or not each of a set of stimuli smells like a particular substance named by the experimenter. Two trials with each stimulus are usually given, with the correct alternative provided on one trial and an incorrect one on the other (e.g., orange odor is presented and the subject is asked on one trial whether the odor smells like orange and on another trial whether the odor smells like peppermint). Although such a test requires the patient to keep the percept in memory long enough to compare it with the target word (which, of course, must also be recalled from memory), some of its proponents argue that it is less influenced by cognitive and memory demands than multiple-choice identification tests (see below). Since chance performance on this type of test is 50% compared to 25% on a four-alternative multiplechoice identification test, its range of discriminability is lower, and therefore more trials are needed to obtain the same statistical power as the multiple-choice odor identification test. Numerous multiple-choice odor identification tests have been described in the clinical literature (Cain et al., 1983; Doty, 1991b; Doty et al., 1984a; Gregson et al., 1981; Wood and Harkins, 1987; Hummel et al., 1997; Wright, 1987). These tests are conceptually similar and, in the few cases that have been examined, strongly correlated with one another (Cain and Rabin, 1989; Doty et al., 1994; Wright, 1987). The most widely used of these tests

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[the University of Pennsylvania Smell Identification Test (UPSIT), commercially termed the Smell Identification TestTM, Sensonics, Inc., Haddon Heights, NJ] examines the ability of subjects to identify, from sets of four descriptors, each of 40 “scratch and sniff ” odorants (Fig. 1) (Doty, 1995; Doty et al., 1984a,b). The number of correct items out of 40 serves as the test measure; this value is compared to norms and a percentile rank is determined, depending on the age and gender of the subject (Fig. 1F) (Doty, 1995). This test has several unique features, including amenability to self-administration and a means for detecting malingering (see Sec. VI). Furthermore, it is available in English, French, German, and Spanish versions. The popularity of this test is attested to by the fact that hundreds of scientific publications have arisen from its use by investigators from many laboratories and clinics. Several odor identification confusion matrix tests have been described that are applicable to clinical settings (Köster, 1975; Wright, 1987). The test that has been most widely applied is that of Wright’s (1987). In his test, each of 10 suprathreshold stimuli is presented to a patient in counterbalanced order 10 times (100 total trials). The response alternatives are the names of the 10 stimuli: ammonia, chlorine bleach, licorice, mothballs, peppermint, roses, turpentine, vanilla, Vicks vapor rub, and vinegar. No feedback as to the correctness of the subjects’ responses is given. The percentage of responses given to each alternative for each odorant is determined and displayed in a rectangular matrix (stimuli making up rows and response alternatives making up equivalently ordered columns). Responses along the negative diagonal therefore represent correct responses, whereas those that fall away from the diagonal represent “confusions.” The percentage of correct responses is used as the main test measure, although some of its proponents argue that the confusions (off-diagonal responses) may provide meaningful clinical information. The main limitations of Wright’s confusion matrix are (1) its long administration time (approximately 45 min) and (2) the lack of evidence that the off-diagonal responses provide any meaningful clinical information (although such responses may be of value in detecting malingering) (see Kurtz et al., 1999). It would seem that if off-diagonal responses are to be sensitive to aberrations or distortions seen in most clinical cases, more subtle differences in the response alternatives need to be employed within the matrix. Should subtle aberrations be reliably categorized, this approach would have considerable clinical value.

(termed the target or inspection stimulus or stimulus set) and to select, after an interval of time (e.g., 30 sec up to several days), that odorant or set of odorants from foils (distracters). Repeated trials may be performed at one or more retention intervals for each of several stimuli or sets of stimuli. In an effort to minimize the rehearsal of verbal labels reflecting the odor qualities or referents during the delay intervals, the examinee is sometimes asked to perform an unrelated task during the retention period, such as counting backwards by twos or threes. The proportion of trials where correct performance occurs is a typical measure derived from such tests. The results from an odor memory test must be interpreted with caution. Despite attempts to minimize labeling of the inspection odor with a familiar word or item on the part of a subject, such labeling undoubtedly occurs, and, thus, what is being measured across intervals is the memory of the label, not the memory of the odor. In other words, once an individual recognizes an odor as that of an orange, all that has to be remembered over time is the concept “orange,” not the specific smell of the orange. Later, when given stimuli from which to select the earlier perceived odor, the subject simply looks for the smell of an orange (which has been known for much of his or her life). In effect, the odor is not what is being uniquely remembered over the retention interval, only its name or concept. For this reason, investigators have attempted to employ novel, nondescript, and unfamiliar odorants in such tasks. Unfortunately, it is difficult to find target odors and foils that are not readily labeled by subjects as pleasant or unpleasant, fruity or nonfruity, medicine-like or non– medicine-like, etc. In general, both short- and long-term odor recognition is markedly facilitated by verbal encoding (Jehl et al., 1997). Another point that should be stressed about odor memory tests is that the performance across the delay intervals comprises the “memory” component of the task, not the overall test score. Thus, an odor memory test is essentially an odor discrimination test with varying inspection (delay) intervals. If, for example, scores on a nominal odor memory task differ between two groups (as evidenced by a main group effect in an analysis of variance), then a significant interaction term between delay interval and group must be present for such scores to reflect differences in odor memory per se. Without an interaction with delay interval, the difference would reflect discrimination, not memory. That being said, a number of examples of clinical applications of odor memory tests are available from the literature. Unfortunately, convincing evidence for a true odor memory deficit is lacking in most cases. Campbell and Gregson (1972) developed a test of shortterm odor memory in which four odors in a row were

H. Memory Tests In a basic odor recognition memory test, a subject is required to smell an odorant or a small set of odorants

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presented and the patient was asked if the fourth, which was the same as one of the first three, was equivalent to the first, second, or third odorant. No delay interval, per se, was defined between the presentation of the stimuli, but presumably the trials were presented closely after one another. Seven three-odor combinations of 12 inspection stimuli were administered. Patients who had difficulty with this task were subsequently given two-odor combinations. The test score was the number of odors that were consistently recognized by the subject. This test was shown to be sensitive to olfactory deficits due to schizophrenia (Campbell and Gregson, 1972), Kallmann’s syndrome (Gregson and Smith, 1981), and Korsakoff psychosis (Gregson et al., 1981). However, it is debatable whether the scores truly reflect memory processes per se. Jones et al. (1975) presented 20 pairs of odorants at 0- and 30-second delay intervals to 14 alcoholic Korsakoff psychosis patients, 14 alcoholic controls, and 14 nonalcoholic controls. On a given trial, the subject’s task was to report whether the second stimulus was the same as or different from the first. In the 30-second delay interval, the subjects counted backward by threes. Since the Korsakoff psychosis patients performed significantly more poorly than did the control groups at both the 0- and 30-second retention intervals, it is questionable whether odor memory is the trait being influenced in this case. More recently, Jones-Gotman and Zatorre (1993) reported that, in patients having undergone surgical cerebral extirpation for control of epilepsy, odor memory impairment was noted between the controls and two of the eight surgical groups evaluated—namely, those who had received excision from the right temporal or right orbitofrontal cortices. The memory task consisted of eight target odors and eight new foils, and the yesno recognition testing was performed twice after the initial testing—20 minutes later and 24 hours later. Although the authors interpret their findings as evidence of a “right hemisphere predominance in odor memory,” their underlying data do not support the notion that differences in odor memory, per se, were present among the groups. Thus, in the overall analysis, where the test scores at the various delay intervals were evaluated as a function of operative group and delay interval, main effects of both of these factors were noted, but no interaction between them was present. No interactions with delay interval were noted in any subgroup analyses. Hence, this study suggests that odor discrimination is altered by certain cerebral excisions, but not necessarily odor memory.

IV.

TEST RELIABILITY

The utility of an olfactory test reflects the degree to which it is reliable (consistent, dependable, or stable) and valid

(accurately measures what it portends to measure). Related to a test’s validity are its sensitivity (ability to detect abnormalities when present) and specificity (ability to detect abnormalities with a minimum number of false positives). Although a test cannot be valid without being reliable, the reverse is not the case; i.e., a test can be reliable but not valid. Despite the fact that measures of test reliability and validity are available for many medical and psychological tests, this is not the case for most olfactory tests. Indeed, measures of validity (other than a few intercorrelations among different tests) are extremely rare; hence, in this chapter studies of reliability are emphasized (for more discussion on this point, see Schwartz, 1991). The reliability of a test can be determined in several ways. First, the test can be administered on two occasions to each member of a group of subjects and a correlation coefficient computed between the test scores on the two occasions (termed the test-retest reliability coefficient or the coefficient of stability). Second, when parallel forms of a test are available, the two forms can be administered to the same set of subjects and a correlation coefficient computed between the two forms. Third, subsections of some types of tests (e.g., multiple-item odor identification tests) can be correlated with one another to provide an estimate of test stability. The test is viewed, in this case, as consisting of parallel forms, and the resulting coefficient, when based upon the correlation of half of the items with the other half of the items, is termed the split-half reliability coefficient. Since reliability is related to test length, as will be noted below, a statistical correction for test length must be applied to the correlation coefficient obtained in this way to provide the correct reliability coefficient for the full test (Guilford, 1954). The magnitude of a reliability coefficient depends, to a large degree, on the variation of the test scores of the group upon which it is computed. If all members of a group score exactly the same on a test administered on two test occasions, the reliability coefficient will not be able to be computed, even though, in effect, there is a perfect correlation between the test scores on the two occasions. If only a small variation occurs among the subjects, then the reliability coefficient may be spuriously low. Thus, in assessing reliability one must have some understanding of the variation among the test scores. Also, it should be noted that while a high reliability coefficient indicates that a group of individuals scored similarly relative to one another on a test from one test occasion to the other, all of the individual’s test scores still may be lower (or higher) on the second than on the first test occasion. In other words, systematic changes in the test values can occur which are not reflected in the reliability coefficient. In such a case, a high reliability coefficient is misleading, as the overall stability of the test may vary systematically over time.

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Although there is a trend among modern developers of olfactory tests to assess the reliability of their instruments, there is a dearth of information on this point in the vast majority of cases. In general, forced-choice odor identification tests with a relatively large number of items evidence a high degree of reliability (e.g., both the test-retest and split-half r’s of the 40-item UPSIT are consistently above 0.90) (Doty et al., 1984a, 1985, 1987, 1995). Shorter identification tests evidence lower reliability. For example, the test-retest reliability of the 16-item Scandinavian OdorIdentification Test is 0.79 (Nordin et al., 1998) and that of the 12-item self-administered B-SIT is 0.73 (Doty et al., 1989). Recently, the reliability of the identification component of the ‘Sniffin’ Sticks’ test was reported to be 0.73 (Hummel et al., 1997). Since it has been reported that olfactory thresholds vary considerably among individuals and evidence considerable day-to-day fluctuations within the same individuals (Stevens et al., 1988), one might expect their reliability to be suspect. Indeed, reliability coefficients for various threshold tests do vary considerable from study to study, and extremely low reliability coefficients have been noted in some cases (e.g., Heywood and Costanzo, 1986; Punter, 1983). Nonetheless, particularly in cases where repeated estimates of the threshold are obtained, respectable reliability coefficients have been reported. Jones (1955), for example, presented ascending concentrations of n-butanol, safrol, and n-butyric acid in sniff bottles (with a comparision blank for reference) to 24 college students. The series were repeated six times for each subject for each stimulus, and the subjects were required to recognize the substance. Reliability coefficients, based upon intraclass correlations, were 0.82, 0.77, and 0.80, respectively, for the three substances. In a study of 40 subjects, Koelega (1979) reported test-retest reliability coefficients for a four-alternative forced-choice n-amyl acetate threshold test to be 0.65, 0.51, and 0.59 for bilateral, right nostril, and left nostril presentations, respectively. In a study of 32 subjects ranging in age from 22 to 59 years, Cain and Gent (1991) reported left nostril:right nostril correlations of 0.68, 0.96, 0.86, and 0.83, respectively, for detection thresholds from single ascending series presentations of butanol, phenyl ethyl methyl ethyl carbinol (PEMEC), isoamyl butyrate, and pyridine. Doty et al. (1995) found test-retest reliability coefficients for detection thresholds of the six odorants contained within the non–forced choice T&T olfactometer test series (skatole, isovaleric acid, -undecalactone, -phenyl ethanol, cyclotene) to range from 0.56 to 0.71; recognition thresholds coefficients were lower, ranging from 0.22 to 0.45. The reliability of the single staircase forced-choice phenyl ethyl alcohol detection threshold was found to be 0.88, whereas the reliability coefficients for

single ascending series n-butanol and PEMEC detection thresholds were 0.49 and 0.70, respectively. The reliability of the detection threshold component of the Sniffin’ Sticks test has been reported to be 0.61 (Hummel et al., 1997). Doty et al. (1995) concluded that (1) detection threshold values are more reliable than recognition threshold values, (2) thresholds based upon a single series AML procedure are less reliable that thresholds based upon a staircase procedure, (3) reversal location within a staircase series has no influence on reliability, and (4) a clear relationship between reliability and test length (e.g., number of staircase reversals) exists. Importantly, in a related study it was found that the threshold measures tended to load on the same principal component in a principal components analysis as a number of the other test measures evaluated (e.g., the UPSIT, a yes/no odor identification test, and tests of odor discrimination), suggesting that all of these tests measure a common sensory domain (Doty et al., 1994).

V.

OTHER CONSIDERATIONS

A.

Unilateral Versus Bilateral Testing

Most individuals with chemosensory dysfunction evidence the dysfunction bilaterally (Cain and Rabin, 1989). In cases where unilateral losses are present, they are often unnoticed. When time is at a premium, bilateral testing is preferable to unilateral testing since it reflects clinically meaningful deficits. However, there are a number of occasions when unilateral olfactory testing is of considerable value (e.g., in the detection of some types of tumors) (Doty, 1979), and the ideal assessment of a patient includes unilateral, as well as bilateral, testing. Unilateral testing is straightforward. Although it is possible to present a stimulus to one naris and obtain mainly unilateral stimulation, the possibility of the crossing of odorant to the contralateral side within the rear of the nasopharynx upon exhalation cannot be excluded. Thus, it is prudent to close the contralateral naris without distorting the septum [e.g., by using a piece of MicrofoamTM tape (3M Corporation, Minneapolis, MN) cut to fit tightly over the borders of the naris] and have the patient exhale through the mouth after inhaling through the nose (Doty et al., 1992). As in the case when both nares are blocked, this precaution decreases the likelihood for air to enter the blocked nasal chamber via the retronasal route. Furukawa et al. (1988) noted that 7 of 94 patients (7%) they examined, all of whom evidenced no bilateral threshold deficits, evidenced significant unilateral threshold deficits. They reported a similar phenomenon in 6 of 12 patients who had had brain surgery. Of 82 consecutive

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nonanosmic patients presenting to the University of Pennsylvania Smell and Taste Center with chemosensory dysfunction, 14 (i.e., 17%) were observed whose unilateral detection thresholds were discrepant from one another by at least three orders of magnitude (Doty, unpublished). Interestingly, nine of these 14 individuals were anosmic on one side of the nose, even though only three had bilateral detection threshold values that were obviously abnormal. B.

Detection of Malingering

Because considerable compensation can be available in accident cases for alterations in ability to smell, malingering on chemosensory tasks is not uncommon. It is frequently suggested in the medical literature that if a patient cannot readily perceive the vapors from an irritating substance presented to the nose, he or she is malingering (e.g., Griffith, 1976). However, this is not a definitive method for detecting malingering. Thus, individuals who, on other grounds, are believed to be feigning anosmia usually have difficulty in denying experiencing the effects of NH4 or other irritants, particularly since these stimuli often produce eye watering, coughing, and other reflexes that are manifested overtly. Furthermore, there appears to be considerable variability among normal individuals in trigeminal responsiveness to such stimulants. A more valid approach for detecting cheating on the basis of psychophysical testing is to examine response strategies of patients on forced-choice tests, since malingerers often avoid the correct response more often than expected on the basis of chance. This is well illustrated by responses to the UPSIT. Since the UPSIT is a four-alternative forced-choice test, approximately 25% of the test items (i.e., 10) are correctly answered, on average, by an anosmic. The probability of scoring 5 or less on the UPSIT and not having at least some ability to smell is less than 5 in 100. The probability of scoring zero on the UPSIT and having no sense of smell is less than 1 in 100,000. As noted in Chapter 11, electrophysiological measures are now available that distinguish between intranasal stimulation of the olfactory and trigeminal systems. Although such testing is not possible in all persons, it does allow for a determination as to whether gross responses are present in the olfactory system, adding key information as to the likelihood of malingering. C.

Subject Variables

The reader should be aware that numerous factors influence olfactory function in “normal” individuals and that these factors can significantly alter the ability to smell. Among the variables that meaningfully alter the ability to

smell are age, gender, and smoking habits. Of these three factors, age is the most important (for reviews, see Doty, 1991a; Doty and Snow, 1988; Schiffman, 1993). Indeed, over the age of 80 years, nearly three out of four persons exhibit marked olfactory dysfunction; half of those between the ages of 65 and 80 years evidence such dysfunction (Doty et al., 1984b). Age-related declines in olfactory performance are observed for a variety of olfactory tests, including tests of odor detection threshold, identification, discrimination, adaptation, and suprathreshold odor intensity perception (for reviews, see Corso, 1981; Doty, 1990; Murphy, 1986; Schiffman et al., 1979; Weiffenbach, 1984). In addition, age influences the responsiveness of the nasal mucosa to volatile chemicals that produce irritation and other skin sensations (Stevens and Cain, 1986). In general (1) large individual differences are present in the test scores of older individuals, (2) olfactory dysfunction is most evident after the sixth decade of life, and (3) women, on average, evidence age-related declines in odor perception at a later age than do men. The decline in the ability to smell in later life is not inconsequential. Thus, a disproportionate number of older persons die from accidental gas poisoning (Chalke et al., 1958), and many complain that their food has no flavor (Doty et al. 1984b). The latter phenomenon, which can lead to decreased interest in food, may explain some cases of age-related nutritional deficiencies. As documented clinically (e.g., Deems et al., 1991), decreased “taste” perception during deglutition largely reflects the loss of stimulation of the olfactory receptors via the retronasal route (Burdach and Doty, 1987; Mozell et al., 1969). In general, women of all ages outperform men on tests of odor identification, detection, discrimination, and suprathreshold intensity and pleasantness perception (Cain, 1982b; Doty, 1986; Doty et al., 1984a; Koelega and Köster, 1974; Le Magnen, 1952). Such differences are present for a wide variety of odorants, including human breath and bodily secretions (Doty et al., 1975, 1978b, 1982), and are observed as early as such testing can be reliably performed (Doty, 1986). The fact that female babies more readily show a preference for odors from their own mothers than do male babies suggests that such sex differences are present at birth and are either inborn or due to early developmental sexually dimorphic influences (Makin and Porter, 1989) (see Chapter 15). The influence of tobacco smoking on olfactory function is less marked, on average, than that of age or gender (e.g., Doty et al., 1984b). This influence, however, is doserelated and present in both previous and past smokers (Frye et al., 1990). Interestingly, cessation from smoking results in some improvement of olfactory function over time—improvement that is related to the amount of previous smoking and the duration of such cessation.

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Both reversible and irreversible changes in smell function have been observed following exposure to a wide variety of environmental agents, including industrial chemicals and dusts (see Chapter 27). In the most extensive study on this point, the olfactory function of 731 workers at a chemical plant that manufactures acrylates and methacrylates was tested (Schwartz et al., 1989). Decrements in odor identification test scores proportionate to the estimated dose exposure levels of these acrylates were found. Interestingly, individuals who had never smoked cigarettes but who had been exposed to acrylates were six times more likely than their nonexposed counterparts to evidence olfactory decrements. Prior experience with odors, particularly that obtained on taste and smell organoleptic panels, clearly influences measures of the ability to smell. For example, repeated testing within the perithreshold odorant concentration range results in decreased thresholds or enhancement of signal detection sensitivity measures (Doty et al., 1981; Engen, 1960; Rabin and Cain, 1986; Wysocki et al., 1989); practice with feedback influences the ability to name odors (Desor and Beauchamp, 1974; Engen and Ross, 1973). Interestingly, the hedonic quality of odorants can be influenced by repeated exposure, making unpleasant odors less unpleasant and pleasant odors less pleasant (Cain and Johnson, 1978). Assuming that adaptation is not the primary basis for this phenomenon, affective components of odors appear to habituate somewhat independently of odor intensity.

1995; Köster and De Wijk, 1991; Stuiver, 1958). First, the amount of adaptation induced is a function of the duration of exposure and the concentration of the adapting stimulus. Second, the subject’s attention level influences the degree of adaptation. Third, the rate and degree of recovery from adaptation are a function of the magnitude and duration of the adapting stimulus. Fourth, cross-adaptation is most commonly asymmetrical. For example, while exposure to odorant A decreases the perceived intensity of odorant B, exposure to odorant B may not decrease the exposure to odorant A to the same degree. Fifth, the sensitivity to a given odorant is typically reduced more by the exposure to that odorant than to any other odorant. Sixth, in rare instances an odorant may have a larger adapting effect on the sensitivity to another odorant than it does on itself. Seventh, the sensitivity to an odorant that self-adapts strongly is usually also reduced strongly by other odorants. Eighth, adaptation of one side of the nose produces adaptation, albeit less, in the other side of the nose. Ninth, adaptation to complex odorants (i.e., odorants made up of more than one chemical) is generally less than adaptation to single-component odorants. Finally, adaptation to odorants can be relatively rapid. For example, Aronsohn (1886) found that subjects continuously exposed to the vapors of lemon or orange oil reported complete loss of olfactory sensations, on average, in 3 minutes (range: 2.5–11 min). Recovery occurred in about the same time required to induce the loss.

D.

VI. THE PERCEPTION OF ODORANT MIXTURES

Adaptation

Exposure to an odorant, if recent and relatively continuous, can produce a temporary decrease in its ability to be perceived, empirically reflected, for example, by heightened detection threshold values or decreased intensity ratings (for a review, see Cometto-Muñiz and Cain, 1995). Some chemicals produce a decrement in the perception of other chemicals (termed cross-adaptation). Fortunately, most modern clinical olfactory tests are either little influenced by adaptation or operationally are standardized in such a way that any adaptation that occurs is unlikely to meaningfully influence the test results. For example, the UPSIT was designed to minimize adaptation by (1) employing largely multicomponent “natural” odorants, (2) requiring minimal sampling of each odorant, (3) having verbal, rather than odorous, response alternatives, (4) ordering the presentation of odorants such that dissimilar odorants follow one another (thereby minimizing cross-adaptation), and (5) allowing adequate time between the smelling of each odorant item (Doty et al., 1984a). Several general rules have emerged from studies of adaptation that are worthy of note (Cometto-Muñiz and Cain,

As noted above, a number of modern olfactory tests, including the UPSIT, employ stimuli that, for the most part, are complex mixtures of chemicals, mimicking stimuli encountered in everyday life. More often than not such stimuli are perceived as a unitary gestalt and given a name associated with the object or source from which they are known to emanate—cinnamon, pizza, cheese, gasoline, orange, lemon, walnut, etc. (see Livermore and Laing, 1998b). There is considerable clinical utility in using such tests, since many receptor types are activated. This is in contrast to threshold tests employing single odorants, as they presumably examine the responses of the olfactory system to a smaller subset of receptors. It has been shown that rodents who have sustained damage to 80–90% of their olfactory receptor cells still retain their ability to detect some single odorants. Similarly, odor sensitivity is retained unchanged when large lesions have been made in the bulb. Therefore, from at least a theoretical standpoint, major changes in the olfactory system can occur and not be detectable by the use of some single odorants. In contrast,

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the perception of mixtures invariably involves inhibitory interactions at the bulb (and possibly other olfactory centers) that occur through complex neural circuitry. Lesions that disrupt the circuitry are likely to alter the characteristic suppression effects observed between odors in mixtures. Rat data indicate that lesions involving much of the bulb can result in the failure to re-learn a mixture analysis task, compared to their successful retention of odor sensitivity and ability to discriminate between odor qualities (Slotnick et al., 1997). How is it that mixtures of chemicals end up providing a largely unitary perceptual gestalt? How much information, in terms of discriminating individual components, can humans obtain from complex mixtures? If one odorant suppresses the odor of another (as is seen in the case of deodorants or room fresheners), how does this relate to the relative concentrations of the odorants within the mixture? Are there psychophysical rules or laws explaining mixture relationships? These and other questions related to odorant mixtures are the basis of the remainder of the chapter. A. Effects of Mixing Odorants on Their Perceived Intensity Usually when two single compound odorants are mixed together, the perceived intensity of one or both is altered substantially, the net result being a lowering of the intensity of the components. However, on rare occasions enhancement may occur. In early mixture studies, Aronsohn (1886) reported that the odor of camphor was neutralized by such odors as gasoline, cologne water, and oil of juniper, and Nagel (1897) found that counteraction between two odorants could result in both being rendered almost odorless. Zwaardemaker (1900), the most famous of early olfactory scientists, confirmed these observations for a number of mixtures using an olfactometer and demonstrated that the extent of perceptual interactions between two odorants was more dependent on their concentrations than on their qualities. Similar results have been reported by others, including Moncrieff (1959) and Jones and Woskow (1964), the latter reporting that the perceived intensity of a binary mixture, although less than the sum of its component intensities, is more than a simple average of the two. Zwaardemaker (1930) conceptualized the mutual weakening of the perceived intensity of a mixture of two components as follows: “The two sensations can be imagined as two vectors representing two forces counteracting each other in our intellect.” The interaction between two odorants was later formalized by Berglund et al. (1973) in a mathematical model that incorporated the application of vector addition to odor mixtures for the prediction of the

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overall intensity of mixtures. Although the vector model has received widespread attention (e.g., Berglund, 1974; Berglund and Olsson 1993a; Berglund et al., 1976; Cain, 1975; Cain and Drexler, 1974; Moskowitz and Barbe, 1977; Laing et al. 1984; Olsson, 1994), after two decades of investigation its best predictions have been for simple binary mixtures. Other models for predicting the perceived intensity of simple mixtures have been proposed (e.g., the Strongest Component Model, the U Model, and the UPL Model; see Laffort and Dravnieks, 1982). Such models are modifications of the vector model, but have not been extended to multicomponent mixtures. The most recent model in this series was the UPL2 model (Laffort et al., 1989) which incorporated the power function that normally relates perceived odor intensity to concentration. The ERM model of Schiet and Frijters (1988) was also based on a power function relating these factors and, although applied with some success to simple gustatory mixtures, was not an improvement in the models just described for olfactory mixtures. As summarized by Cain et al. (1995), “the principle by which psychophysical information on single components reflects itself in a model of interaction seems to evade the psychophysical models presented here” (all the above). Clearly, none of the aforementioned models adequately describe the changes in perceived intensity for all pairs of odorants examined, and none have been demonstrated to reliably predict the intensity of mixtures containing more than two odorants. Booth (1995) provides an interesting critique on the modeling of odor interactions but provides no firm ground for future studies to proceed. Among a number of shortcomings, none of the above models have been based upon the receptive and neural processes that underlie the perception of mixtures, nor has attention been given to choosing odors that have physicochemical features that might provide some basis for antagonistic interactions. Furthermore, these models have provided no insight as to the nature of the sensory processes, and none adequately predicts the intensity of multicomponent mixtures. Present evidence suggests that addition or partial addition of the perceived intensities of the components of mixtures occurs with binary and ternary mixtures; beyond this number of components neural processes limit intensity addition (Berglund et al., 1976; Laing et al., 1994a; Moskowitz and Barbe, 1977). The interactions noted above concern suprathreshold concentrations of odorants and provide examples of where the sense of smell compresses rather than adds intensity information. In contrast, additivity of neural input appears to be inherent in mixtures containing only sub-threshold quantities of odorants (Laska and Hudson, 1991; Laska et al., 1990). Indeed, in mixtures with only three odorants,

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the magnitude of the addition was noted by Laska et al. to be substantial and to often exceed that obtained from simple summation. Patterson et al. (1993) reported instances of near-true additivity of subthreshold components and suggested that additivity may function to enhance sensitivity to the typically complex (and often subthreshold) odor stimuli encountered in everyday life. They noted that the number of chemicals activating the system could be as important as the strength of any one of the odorants, providing a type of “biological economy” of the input. B.

Discrimination of Components in Odorant Mixtures

Since, as mentioned earlier, odors are commonly encountered as mixtures in our environment, an important characteristic of the human sense of smell is to discriminate differences between mixtures. Discriminating the odors of fresh and “off ” milk, ripe and overripe fruit, cork taint in wine, and various perfumes are examples. In the area of pollution control, changes in the complex odor of sewage provide engineers with an insight as to the part of the treatment process that is not functioning properly; sulfides emanate if the anaerobic process is malfunctioning, and sour, rancid, and acid odors appear if the sludge treatment is inappropriate. In studies with binary mixtures, Rabin (1988) and Rabin and Cain (1989) showed that humans are particularly sensitive to the presence of small amounts of odorants that are not normally found in a stimulus. They reported that (1) high familiarity with a major component and the ability to label it consistently facilitates the detection of a minor component, (2) the minor component is not detected as readily if it is unfamiliar, and (3) unpleasant stimuli are more detectable than pleasant ones, although the effect was not as large as the effect of familiarity. Experience, therefore, and to a lesser extent pleasantness, improves discrimination between two single odorants or two mixtures, suggesting that similar cognitive processes operate with the two types of stimuli. Although the Rabin studies suggested that humans are very sensitive to small changes in an olfactory stimulus, Laska and Hudson (1992) reported that relatively large changes in the composition of mixtures are sometimes required for discrimination to occur. Thus, discrimination of 3-, 6-, or 12-component mixtures from the same mixtures minus 1 component produced error levels of 20–40%, with the level depending on the type of odorant that was removed. Accordingly, the dependence on the type of odor removed precluded defining a limit in the ability of humans to discriminate between two complex mixtures.

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C.

Identification of Components in Odorant Mixtures

Prior to studies of the abilities of humans to analyze mixtures, informal information from perfumers and flavorists suggested that between 5 and 30 components may be identified in mixtures (D. G. Laing, unpublished data). Over the past decade it has become clear that these numbers are an overestimate, as most individuals, including perfumers, are only able to identify up to 3 or, rarely, 4 components. An early hint that only a small number of odorants can be identified in mixtures was apparent in the report by Berglund (1974), who suggested from studies of the addition of the perceived intensities of components, that an analytic or additive process occurred up to 3 components, whereupon above this number an interactive process predominated. The latter was apparent as an asymptote in the total perceived intensity of a mixture, with little change occurring as the number of components increased. In accord with this notion, Moskowitz and Barbe (1977) found that in some instances the overall intensity of 5 component mixtures was less than that of mixtures with fewer components. In perhaps the first formal scientific studies on this topic, Laing and Francis (1989) and Livermore and Laing (1996) reported that training and experience did not increase the number of components identified with subjects that had been trained for a few minutes, 3 weeks, or who were perfumers and flavorists, the maximum still being 3–4. Varying the task or the odorants resulted in no improvement in the number identified; thus, in another study a selective attention procedure was not more efficient than a procedure that required subjects to identify as many components as possible during an ad lib sampling method (Laing and Glemarec, 1992). Futhermore, this maxima was not altered if the odorants used were those classified by perfumers as “poor blenders,” i.e., odorants that they used to “stand out” in mixtures (Livermore and Laing, 1998a). Schiet and Frijters (1988), using another approach to this problem, reported that subjects invariably underestimate the number of components in mixtures containing up to 4 components. A similar result was obtained by Jellinek and Köster (1979), whose subjects found the odor of single chemicals to be as complex as that of mixtures. Clearly, the data of the aforementioned studies indicate that there is a significant limitation in the olfactory system in the processing of information from more than about 3 odorants. Mixtures of complex odors tend to behave like mixtures of single odorants, with a maximum of about 3 being identified in stimuli containing up to 8 complex odors. The possibility that the entry of hundreds, perhaps thousands, of odorants into the nose would produce a

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nonidentifiable smell sensation has not yet eventuated (Livermore and Laing, 1998b). D.

Mechanisms Involved in Odor Mixture Perception

The limited ability of humans to discriminate and identify odorants in mixtures is likely due to a number of mechanisms. Changes in spatial processing arising from competition for receptor sites and cells at the periphery, and inhibition in the bulb and at other olfactory centers, would be expected to reduce the information within the activated receptor cell arrays, making it difficult to recognize the patterns of activation due to different odorants. Since temporal processing favors the first processed odorant, the initial odorant has the opportunity to act as an antagonist towards other odorants at the periphery and to inhibit neural activity arising from other odorants in the bulb. However, if the delivery of different odorants in ternary or more complex mixtures to the nose is less than the time for processing two odorants in working memory, the latter becomes the ultimate limiting factor as regards the number of odorants identified. These mechanisms are discussed in detail below. 1. Spatial Processing As noted at the beginning of this chapter and elsewhere in this volume, a given odorant activates unique arrays of receptor cells in the nose (Kauer, 1991; Mackay-Sim et al., 1982), which, in turn, are reflected by patterns of activation of glomeruli and mitral/tufted cells in the olfactory bulb. Different odors produce different arrays that represent the spatial codes of identification. However, when a mixture of two odorants is sensed and the perception of one or both is suppressed to some degree, the arrays representing the two stimuli in the bulb show a reduction in the number of glomeruli that are activated (Bell et al., 1987; Joerges et al., 1997). If the suppression of one of the odorants is such that it cannot be perceived, little of the normal array of activated glomeruli is seen (Bell et al., 1987). The suppression may be due to fewer receptor cells being activated (Ache et al., 1988; Kurahashi et al., 1994; Simon and Derby, 1995) because of competition by the odorants for the same receptor sites, resulting in less input to the bulb. Suppression can also be caused by lateral inhibition between glomeruli or mitral cells in the bulb (Pinching and Powell, 1971; Shepherd and Greer, 1990; White, 1979). The loss of identity of up to 5 odorants in 8-component mixtures (Livermore and Laing, 1998a) prompted Jinks and Laing (1999) to propose that the competition and inhibition between odorants could result in no odorant

being identified in mixtures containing double this number of components. Their psychophysical study showed that 1 and zero components were identified in 12- and 15-component stimuli, respectively. The fact that the 15-component stimulus had an odor, albeit not one that could be associated with any of the components or an object or source, indicates that neural input from some or all of the arrays characterizing the components was registered. In light of such observations, it is interesting to note that, in the rat, neural images of complex olfactory stimuli, including rat nest odors comprised of volatiles from urine, feces, and bodies (Stewart et al. 1979), have shown that the number of activated glomeruli is similar to that found with a simple single odorant such as limonene (Bell et al., 1987). Therefore, spatial processing of single and complex odorants involves both peripheral and bulbar interactions that reduce and simplify identification. Accordingly, the olfactory system uses spatial coding to analyze and identify single odorants when presented alone and in simple mixtures and simplifies identification of complex mixtures by combining the remaining parts of the arrays into a single characteristic array that is associated with the object or source of the stimulus. This interpretation is in agreement with the finding of Jellinek and Köster (1979) that single odors are perceived to be as complex as those of mixtures. But there is another aspect to spatial processing. An intriguing feature of single odorants and odor mixtures is that they can be characterized by several qualities or “notes” (Laing and Willcox, 1983; Moskowitz and Barbe, 1977). Hexenal, for example, is described as having “green” and “fatty” qualities, and ethyl butyrate as “sweet” and “fruity.” However, when single odorants are components of mixtures but cannot be identified, often one or more of their qualities can be discerned. Recently, Jinks and Laing (2001) investigated the qualities of binary, ternary, and quaternary mixtures of four dissimilar odorants to determine the information about odor quality that needs to be retained for identification of the odorant. The data indicated that failure to identify an odorant could occur with loss of some but not all of the qualities. However, failure could also occur when the major qualities were present but the ratios of their perceived intensities were substantially altered. This suggested that a different smell could be produced using the same qualities but in different ratios. Identification, therefore, was affected by the type and/or the perceived intensity of the qualities of an odorant. These results were interpreted in terms of a Configurational Hypothesis of Olfaction, in analogy with the Configurational Hypothesis of Facial Recognition (Enns and Shore, 1997; Rakover and Teucher, 1997). In brief, in the case of a face, identification of a person not only requires certain features to be present in a drawing or

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photograph, but these features must be in the correct proportion to each other. Similarly, identification of an odorant or a complex mixture requires some of the characteristic qualities in the correct proportions to be perceived. But what is the neural basis of a quality or “odor note,” and how is it represented in the spatial code? Thanks to advances in molecular biological studies of the chemoreceptive process, an insight into this problem is possible. For example, as noted at the beginning of this chapter, it is commonly accepted that each human receptor cell has only one type of receptor (Rawson et al., 1997) and that there are ~1000 receptor types, as indicated by the number of receptor genes (Buck and Axel, 1991). Stimulation of receptor cells by a single odorant will result in a variety of cells being activated in accordance with the degree or “ease of fit” of the odorant to each receptor site type. If the fit is predominantly to two or three receptor types, they will be the main inputs to the array of glomeruli and mitral/tufted cells activated in the bulb. However, the conformations adopted by an odorant to fit the two to three receptor types will be dictated by the structural features of the odorant and receptor molecule. In one conformation a molecule may be aligned within a receptor site according to its length and functional group, e.g., the 17 receptor for octanal (Araneda et al., 2000); in another it may sense a structural feature common to a number of odorants, e.g., an 8-carbon chain containing a terminal carbonyl group common to aliphatic aldehydes, acids, esters, and ketones (Imamura et al., 1992). Since the overall odors of these latter aliphatic carbonyl substances are easily discriminable (Laska et al., 2000), each odorant must require at least two receptor types to be occupied for this to occur. Accordingly, it is tempting to suggest that activation of the cells with the common receptor for these odorants results in an odor quality common to each odorant, while activation of the cells unique to each odorant produces a quality unique to each odorant. In addition, the spatial map of each odorant should show glomeruli or mitral/tufted cells that are activated by all four odorants and others that only one of the odorants will activate. From the limited data available, it is suggested that the conformations an odorant can adopt in different types of receptors defines the important structural features that provide the qualities perceived. This interpretation suggests that the spatial code for an odorant contains information about molecular structure and odor qualities. In contrast, the spatial map of complex mixtures such as chocolate aroma, where none of dozens of odorants can be identified, will be composed of input from receptor cells representing features of many odorants, and it may be the location and magnitude of the input to the bulb rather than molecular features that define its identity.

Nevertheless, several qualities can usually be discerned in complex aromas, and these are likely to be those that remain from individual odorants in the mixture which are insufficient to identify the latter but contribute to the overall aroma of the complex mixture. 2.

Temporal Processing

During the 1980s, Getchell et al. (1984) reported that odorants can differ by hundreds of milliseconds in the times they take to activate receptor cells, while Kuznicki and Turner (1986) showed that humans require different reaction times to recognize the four common tastants. These findings prompted Laing (1987) to propose that if the time differences between the activating times of odorants at the periphery were maintained as the neural message traveled through the bulb and other olfactory processing centers in the brain that dealt with memory and identification, then a “fast” odorant would have a number of advantages if presented in a mixture with a “slow” odorant. For example, the faster odorant may be more successful in competition for receptor sites and cells, and being the first to activate the bulb, it could trigger lateral inhibition between glomeruli or between mitral cells to further reduce neural input from the slower odorant. Accordingly, it was predicted that the faster odorant would be the first odorant identified in a mixture, the slower odorant would incur the greatest suppression of intensity, and the number of cells and glomeruli in spatial arrays activated by the latter odorant would be reduced. To investigate the first two of the above predictions, Laing et al. (1994b) used a specially designed computercontrolled olfactometer, which allowed odorants to be delivered together in a mixture or in series separated by intervals as small as 50 ms. By asking subjects which of two odorants was perceived first during a trial and varying the time between delivery of both odorants from 100 to 600 ms, the processing time difference between them was established as that which produced a chance response, i.e., 50% for the forced-choice yes/no task. The magnitude of the differences varied from zero to more than a second and was dependent on both the quality and perceived intensity of the odorants, with the latter being more important. Perceived intensity was also reduced more for the slower odorant. With both predictions upheld, the existence of temporal processing and its implications for mixture perception were demonstrated. A later study (Jinks and Laing, 1999b) confirmed that knowledge of processing time differences allowed predictions of which odor would be perceived first in other mixtures. Thus, they showed that when odor A was perceived before B and B was perceived before C, that A was

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Figure 7 Regression lines representing the proportion of trials an odor in a binary mixture was perceived “first” when presented with a time advantage, disadvantage, or as true mixture (0 ms interval). Arrows and times in boxes indicate when both odorants were perceived first on 50% of trials. (A) Stimulus of coniferan and triethylamine with coniferan being perceived 538 ms before triethylamine; (B) a stimulus of carvone/triethylamine with carvone perceived first 1739 ms before triethylamine; (C) a stimulus of carvone/coniferan with carvone perceived first 251 ms before coniferan.

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was needed before the usually faster odorant was perceived first (Fig. 8). With the other set, subjects recorded chance responses even with the 900 ms delay. The mean responses of subjects, when asked to identify the odorants in the mixture or delay conditions, showed that this was at chance level. The result is in agreement with the earlier studies of Laing and colleagues, who found that few subjects could identify all the components of ternary mixtures. Overall, the results with ternary mixtures indicated that a mechanism related to the speed of information retrieval about the identity or temporal order of the components was the cause. The most likely candidate appears to be the inability of olfactory working memory to process the information about the identity and order of the first two components before neural input from the third began to be processed. Although it is not fully understood, working memory is defined as the “system responsible for the temporary storage and manipulation of information, forming an important link between perception and controlled action” (Baddeley, 1998). The process of identifying an odor within working memory is likely to involve several steps: encoding of the odor by neurons, recalling of the

perceived before C, demonstrating that transitivity had occurred (Fig. 7). However, investigation of temporal processing in ternary mixtures revealed a substantial limitation in the ability of humans to indicate which odor is perceived first and the existence of a third mechanism that affects perception of components in odor mixtures (Jinks and Laing, 1999b). Temporal processing of ternary mixtures and the third mechanism, which is postulated to involve olfactory working memory, are discussed below. 3. The Role of Memory The perception of the order of processing odorants in ternary mixtures, however, has proved to be very difficult (Jinks and Laing, 1999b). Initial experiments indicated that subjects recorded chance level responses when asked to indicate which odorant was perceived first or last. To investigate whether the chance results were due to a limitation in the capacity of olfactory working memory to process both order and identity of the odorants, presentation of the third odorant was delayed by 300, 600, and 900 ms. With one of the two sets of three odorants studied, the results indicated that a delay of between 600 and 900 ms

Figure 8 Proportion of trials (numbers above bars) in which subjects selected an odor “coming first” in binary and ternary mixtures and mixtures where the presentation of triethylamine was delayed. Conditions: 1, binary mixture of carvone/coniferan; 2, ternary mixture of carvone/coniferan/triethylamine; 3,4, and 5, ternary mixture with the presentation of triethylamine delayed by 300, 600, and 900 ms, respectively. Open and shaded bars indicate that the means were significantly/not significantly different from 0.5 (chance), respectively.

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coded representation of the odor from long term memory, comparison of the two representations, and the judging and responding to the representations. This type of process has been proposed for visual information (Eskandar et al., 1992). Indeed, the interference of a third odorant with the perception of others is reminiscent of that reported for visual spatial memory, where it was proposed that an irrelevant visual stimulus may have obligatory access to a visual store and interfere with the storage and processing of other visual spatial information in working memory (Toms et al., 1994). Limitations in the capacity of olfactory working memory to process more than two odorants within 600 –900 ms appears to be a major factor limiting the discrimination and identification of odorants in multicomponent mixtures. Such a finding has implications for the perception of odorants released during an eating episode where many can be released within the processing time differences cited here, but only a few may be identified. Controlled release of odorants from different food media could, however, allow products to be developed with high flavor impact. VII.

CONCLUSIONS

The present chapter has provided an up-to-date review of the psychophysical means for testing the human sense of smell and has examined how the human olfactory system likely integrates information from complex arrays of odorant chemicals which, individually, would seem to produce conflicting odorous sensations. It is of interest that relatively high correlations exist among the scores derived from nominally distinct olfactory tests, regardless of whether they are based upon single- or multicomponent stimuli. Test reliability has been shown to be largely a result of test length, irrespective of the nature of the stimuli included in the tests. To what extent tests employing multicomponent odors are superior to ones employing single odorants is an empirical issue, although it would seem that by sampling more elements of the system, a test should be more sensitive. Continued efforts to refine the procedural elements of olfactory tests should help in the development of test batteries sensitive to wider ranges of olfactory deficits than those that are currently available. ACKNOWLEDGMENTS This paper was supported, in part, by Grants PO1 DC 00161, RO1 DC 04278, RO1 DC 02974, RO1 AG 27496, and RO1 AG 08148 from the National Institutes of Health, Bethesda, MD, to RLD, and an Australian Research Council Large Grant to DGL.

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11 Electrophysiological Measurement of Olfactory Function Gerd Kobal University of Erlangen, Erlangen, Germany

I.

INTRODUCTION

important to remember that methodological approaches employing nonhumans are usually simply surrogates or models for the human sense of smell. In some cases, data obtained from animals or cell cultures do not generalize well to humans. A case in point is a recent study that suggests that the distribution of the human olfactory epithelium is different from what would be predicted from animal investigations (Leopold et al., 2000). It is apparent that the collection of valid information about human olfactory processing requires the use of human subjects. The bulk of this chapter is devoted to the most common electrophysiological signal that has been measured to date, namely, the olfactory event–related potential (OERP). Other measures that are discussed in detail include the EOG, event-related changes in the background electroencephalogram (EEG), and signals derived using magnetic source imaging (MSI). A description of findings that have provided new insights into the olfactory system or have raised questions concerning functional properties and relationships of brain areas activated by odorants is also presented.

Unlike the situation in other sensory modalities, the field of human olfactory electrophysiology is rather poorly developed. For example, in vision, recordings of electroretinograms (ERG) or of visual event–related potentials (VERP) are routinely used diagnostically, in contrast to the situation in olfaction, where analogous potentials are rarely measured, even in university medical centers. However, as noted in this chapter, a number of laboratories are working in this field and have generated an impressive number of publications and a body of useful information for better understanding elements of the human olfactory process. Why should one want to obtain electrophysiological or other physiological measurements of the olfactory system in humans? First, there is a general need for more reliable data in all fields of science. Recording brain potentials, magnetic responses, changes in blood flow, etc., provides information that minimizes or eliminates potential biases related to conscious subject responses. For example, in some medical cases—particularly those associated with litigation—malingering may occur and electrophysiological assessment can greatly aid in the detection of such deception. Second, electrophysiological studies may help to determine the neural structures involved in pathological changes in sensory responsiveness, such as in hyposmia or dysosmia. For example, EOG measurement can be used, in some cases, to establish the involvement of the epithelial receptors in an olfactory deficit. Questions concerning such localization often arise, particularly in relation to pharmacological or surgical interventions. Finally, it is

II. A BRIEF HISTORY OF ELECTROPHYSIOLOGICAL RESEARCH ON THE HUMAN SENSE OF SMELL In 1883 Fleischl von Marxow observed that ammonia produced electrical brain potentials when presented to a rabbit’s nose (Fleischl von Marxow, 1890). Although Berger (1929) assumed that such potentials could also be found in the human EEG, he failed to demonstrate 229

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them. Indeed, it was not until the 1960s that OERPs were recorded by Finkenzeller (1965) and Allison and Goff (1967). Around this same time, the first electrophysiological recording from the human nasal mucosa (i.e., the EOG) was obtained by Osterhammel et al. (1969). This achievement was based upon the earlier animal studies of Hosoya and Yoshida (1937) and Ottoson (1956). Although suprisingly little work has subsequently been carried out on the EOG (see Hummel et al., 1996b; Leopold et al., 2000), research on human odor ERPs continued through the 1970s in various laboratories (e.g., Giesen and Mrowinski, 1970; Herberhold, 1976; Cianfrone and Subiaco, 1978). However, technical difficulties in producing defined olfactory stimuli with steep rise times and in analyzing the large amount of generated data hindered rapid progress. Moreover, data presented by Smith et al. (1971) suggested that an OERP could not be found in patients who had lost their trigeminal sensitivity, conceivably discouraging others to continue efforts along these lines. As described in detail below, subsequent studies by my group (e.g., Kobal and Plattig, 1978; Kobal, 1981) found that Smith et al.’s observation was likely due to experimental artifact. As a result of our early studies, a new era in the study of human OERPs, based upon sophisticated odorant presentation techniques, was born. III. EVENT-RELATED POTENTIALS VERSUS EVOKED POTENTIALS It should be noted that event-related potentials, which reflect high order processing, can be elicited by both external and internal stimuli. For example, when a series of stimuli is presented with a constant interstimulus interval, the omission of one stimulus may trigger an event-related potential, even though no physical, external stimulus is present. This is in contrast to “evoked potentials” (also termed “exogenous” or “obligatory” potentials), which reflect the very early components of the response and are largely independent of a subject’s mental state or arousal (Näätänen et al., 1993). The latter have not been recorded noninvasively in humans to odorants, although they have been obtained from the olfactory bulb and amygdala during surgery (Hughes et al., 1969, 1972; Hughes and Andy, 1979a,b; Kobal et al., 1998, unpublished). IV. OLFACTORY EVENT–RELATED POTENTIALS A.

Stimulation Requirements and Considerations

In general, precise stimulus control is crucial when recording event-related potentials. Why is this so? Event-related

potentials are EEG-derived polyphasic signals reflecting activation of cortical neurons which generate electromagnetic fields (Picton and Hillyard, 1988). The more neurons that are activated or synchronized, the larger the amplitude of the signal obtained at the surface of the scalp. Since the EEG is a noisy signal, which contains activity from many cortical neurons, ERPs need to be extracted from the background activity. The classical approach to this problem involves averaging of individual responses to olfactory stimuli such that random activity would cancel itself out, thereby leaving only nonrandom activity. Therefore, stimuli are typically presented repetitively with a steep onset (100 ms) in a well-controlled and homogeneous environment so that the stimulus onset synchronizes the activity of as many cortical neurons as possible. Three prerequisites must be met to obtain clear and accurate OERPs. First, as noted above, the stimulius must have a steep onset. Although a shallow stimulus onset may lead to a sensation, this sensation may not be reflected in an ERP as the cortical activity “drowns” in background noise. Second, the stimulus needs to be presented repetitively. This requires precise temporal control of stimulus onset in the range of milliseconds as fluctuations in the timing of stimulus onset will lead to differences in the peak latencies of individual ERPs (“jitter”). This jitter will lead to the modification/ cancellation of peaks in the averaged response. In addition, desensitization to repeated stimuli becomes an issue. Finally, to properly interpret the response it is necessary to know whether it is derived from intranasal chemical stimulation of the trigeminal (CN V) or olfactory (CN I) system.*

*The

recording of an ERP can be compared to the situation in a soccer stadium where, in analogy to an EEG electrode, a microphone is positioned over the middle of the field to record all sounds. The stadium is filled with thousands of people, similar to the millions of neurons sitting under a recording electrode on the scalp. During long sequences of the game, only noise is recorded coming from the soccer fans, talking to each other, laughing, commenting on the quality of the game etc. When the game becomes more exciting there may be more noise; when it is less exciting, people typically become quieter. But when a goal is scored communication between soccer fans becomes synchronized with many of them shouting—which compares to the synchronization of cortical neurons by the sudden onset of a stimulus. However, similar to the electrical fields of the EEG, it is a difficult to localize the site where the goal has been scored. If the noise comes from the left side this does not necessarily mean that the goal has been scored on the left. It may also be due to the fact that most of the fans of the scoring club sit on the left side, but the goal was actually scored on the right side (and this situation may change during the game, when teams switch sides). Finally, it may also be that the goal is not scored in this stadium but elsewhere. Specifically, the fans of one of our two teams may

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(a)

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(b)

Figure 1 (a) Schematic diagram of the switching device. When the odorant is switched on or off, the subject is unable to discern turbulences or changes in flow rate or pressure. The temperature and humidity or the carrier gas (air) are closely controlled. (b) The Burghart OM4/b olfactometer. Left. Subject being presented with odorants and performing a computerized visual attention task. Right. Data collection module. Center. olfactometer body. (Photo courtesy of the University of Pennsylvania Smell and Taste Center, Philadelphia, PA.)

How is it possible to produce chemical stimuli that have a rectangular shape with rapid onset, that are precisely controlled in terms of timing, duration, and intensity, and that do not simultaneously activate sensory systems other than olfaction? Based on the principles of air-dilution olfactometry (Prah et al., 1995), such a system was developed in the late 1970s and refined in the 1980s (Fig. 1) (Kobal and Plattig, 1978; Kobal, 1981; Kobal and Hummel, 1988). Odorants are applied intranasally by means of a canula with an inner diameter of 2–3 mm. This canula is inserted for approximately 1 cm into the naris such that its opening lies beyond the nasal valve. Presentation of odorants does not simultaneously activate mechano- or thermoreceptors in the nasal mucosa, as odor pulses are embedded in a constantly flowing, humidified air stream (typically 6–8 L/min). Hence, subjects do not perceive any change in flow rate when the stimulator is switched from a no-stimulus to a stimulus condition, and vice versa. In this system, two air streams are directed towards the outlet of the olfactometer. Both have the same flow rate, the same temperature, and the same humidity. One contains an odorant at a defined concentration (odorant
O have brought receivers to listen to the broadcast of a different, extremely important game; it may be that in this different stadium a goal is scored which turns the situation in the league in favor of one of our teams. This would then also create a synchronized outcry of the fans although nothing much has happened in the play we actually observe. In a similar way, pinpointing the source of an event-related potential can be a difficult task.

plus dilution
D), whereas the other contains odorless air (control
C). Different odorant concentrations are generated by means of air dilution; hence, a preestablished, fully odorant-saturated air stream (odorant
O) is mixed with an odorless air stream (dilution
D). While the sum of the two air streams is always constant (equal to the control air stream C), different O:D ratios produce different stimulus concentrations. A separate system of finely tuned pressure and vacuum is applied such that, similar to an air curtain, a small current of odorless air prevents molecules from O tubings to be drawn into other tubes. This cross current allows attaching several different odor lines to the same dilution line. During the interstimulus interval, a precisely tuned vacuum draws the odorant-containing air steam from the vacinity of the flowing air, ensuring that only odorless air enters the subject’s nose during this time. Employing this device, it is possible to switch between an odorized air steam and control air in less than 20 msec. Depending on the physicochemical properties of the odorants employed, a switch from one odorant to another can be made in less than 5 seconds without contamination from the previous stimulus. The constant airflow directed into a subject’s nose requires humidification (~80% relative humidity) and a stable temperature (36°C), since dry cool air produces nasal congestion, mucus discharge, and pain, which can interfere with the olfactory process (Mohammadian et al., 1997, 1999; Lötsch et al., 1998). The warmed and humidified air stream employed in our studies becomes unnoticable within a few seconds of its introduction; i.e., the subject adapts to the following air.

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In the commercially available olfactometers based upon our designs (Burghart Laboratories, Hamburg, Germany), airflow rates are determined by mass-flow controllers that, along with switching valves, are controlled by computer. The recording of stimulus-linked EEG segments (or any other physiological measure that can be transformed into electrical currents) is integrated into the same software that controls the olfactometer and stimulus presentation. This equipment also allows the setup of sequences of stimuli with different quality, intensity, duration, or interstimulus interval. Thus, the recording of the OERP becomes a routine procedure that can be carried out by any technician—a fact of particular importance in clinical applications. In contrast to the stimulus presentation procedures described earlier, some laboratories record OERPs in relation to stimuli that are puffed into the nasal cavity, a procedure that we do not recommend. What happens when odorants are puffed into the nose? Under these conditions, it is not only possible to obtain ERPs in anosmic subjects (Herberhold, 1976; Cianfrone and Subiaco, 1978; Swandulla, 1986; Sakuma et al., 1996; Bauer and Mott, 1996; Harada et al., 1997), but ERPs in normosmics that reflect the mixed activation of both the trigeminal and the olfactory systems. Such combined activity leads to numerous interactions at various levels of neuronal processing (for review, see Hummel and Livermore, 2001) which cannot be remedied by simple mathematical procedures. For example, the average of responses to individual stimulation with carbon dioxide and vanillin or hydrogen sulfide is significantly different from the response obtained after stimulation with the binary mixture of carbon dioxide and hydrogen sulfide (Kobal and Hummel, 1988; Livermore et al., 1992). Thus, it is difficult to interpret responses to olfactory stimuli contaminated by mechanical stimulation in patients with olfactory disorders. In addition, since the interactions between the trigeminal and olfactory systems are difficult to predict, it is misleading to interpret responses to mixed olfactory or trigeminal stimuli to reflect predominantly (and, implicitly, more or less exclusively) olfactory or trigeminal activation (Geisler and Murphy, 2000). Another approach to establishing OERPs involves the electrical activation of the olfactory epithelium (Sato et al., 1996; Ishimaru et al., 1997). While this would appear, at first glance, to be an extremely attractive technique— especially when considering the possibility that this would more definitively allow for the investigation of the integrity of axonal connections from the olfactory receptors to the olfactory bulb—it suffers from the fact that electrical stimulation also activates trigeminal nerve endings of the olfactory epithelium (Silver, 1991). Hence, it is not clear

Kobal

whether the observed cortical responses to peripheral electrical stimulation reflect olfactory or somatosensory activation. B.

Recording of Olfactory Event-Related Potentials

As noted earlier, ERPs are due to changes in electrical fields generated by large populations of cortical neurons. If they are recorded from the intact surface of the skull, their amplitudes are very small (50 V), and it becomes necessary to isolate them from the background activity by averaging and/or filtering. For averaging, a certain number of stimulus-synchronous EEG records of 1–2 sec duration are digitized by computer and transformed into a sequence of numerical values. Thus, averaging the stimulus-locked array of numbers results in visualized waveforms representing responses of synchronously reacting cortical neurons. However, if this technique is to yield reliable data, several prerequisites have to be met. For one thing, the background activity of the EEG has to be stationary and, at the same time, stochastic in regards to the concealed response. For another, the specific stimulus-induced response, i.e., the activity determined by the applied stimulus, has to be stable, especially if there are a number of consecutive positive and negative components. Even the slightest phase-shifting can easily cause the positive and negative components of the event-related responses to cancel each other, in the same way that can occur with background activity. As noted in the previous section, positive and negative waves are only separated by a fraction of a second. Therefore, the quality of the temporal presentation of the reproducible stimuli has to be excellent. The first positive peak of OERPs typically occurs at latencies of 250 msec. This peak is then followed by at least two other peaks—a major negativity and the late positive complex. As there has been confusion in the past on the nomenclature of OERPs, some have suggested naming each peak according to its polarity and mean latency at position Cz (e.g., Evans, 1993; Hummel, 2000). For example, a negativity at a mean latency of 340 msec would be called N340. In this review, peaks are named in the more conventional fashion to allow for comparisons across studies—i.e., P1, N1, and P2 (compare with Evans, 1993). Using data published in Kobal and Hummel (1988) for OERPs to vanillin, these peaks correspond to P383, N484, and P649 using the nomenclature based on peak latencies at position Cz. It should be noted that the P2 peak has also been described as P1 (Prah and Benignus, 1992) or P3 (Lorig, 1993; Barz et al., 1997). Why is it that OERPs appear later than auditory or visual OERPs? Unlike the case in audition, chemical stimulation needs approximately 100–200 msec utilization time at the

Electrophysiological Measurement of Olfactory Function

site of the receptors. For comparative purposes, this utilization time must be subtracted from the latencies (Getchell et al., 1984; Hummel et al., 1996b; Leopold et al., 2000). Thus, the N1 and P2 OERP peak latencies are, in fact, comparable to the N100 and P200/P300 latencies of audition and other sensory modalities. As briefly mentioned earlier in this chapter, unlike the case for audition or vision, no early ERPs have been recorded in response to olfactory stimuli, only late nearfield ERPs (i.e., responses from cortical neurons) (see Kobal and Hummel, 1991, for review). Peaks of the late near-field ERPs fall into two groups. Earlier peaks like N1 (N340) encode exogenous stimulus characteristics to a larger extent than later, so-called endogenous, components. That is, earlier components encode stimulus intensity or stimulus quality (e.g., “What is the nature of this stimulus?”), whereas later components are more related to the frequency, or the saliency of the stimulus (“What is the meaning of this stimulus?”) (Donchin, 1986; Picton and Hillyard, 1988; Pause et al., 1996b; Krauel et al., 1998). C.

Stimulation and Recording Parameters

Since the frequency spectrum of late near-field ERPs ranges between 1 and 8 Hz, both filtering and sampling frequency must be set accordingly. We prefer low-pass filtering at 30 or 70 Hz. As regards the number of averages per ERP, eight records are considered to be the absolute minimum.* However, some authors have used up to 200 records to average a single response, such as in the recording of late positive components in the so-called oddball paradigm, where two stimuli are presented with different probabilities of occurrence (Pause et al., 1996b). Given an ISI of 30–40 sec in single trial experiments, recording of such large numbers of responses requires at least 100 min. This is impractical and unnecessary in many situations, given evidence that improvement of the signal-to-noiseratio starts to reach a ceiling effect at 40 averages. In addition, the long testing periods can introduce other artifacts, e.g., changing levels of vigilance during recording. OERPs can be recorded from numerous scalp locations. Amplitudes exhibit characteristic patterns across scalp loci, with a centro-parietal maximum for both amplitudes N1 and P2 (compare Lorig et al., 1996; Pause et al., 1996b; Murphy et al., 1998). This specific topographical distribution can be used to differentiate between trigeminal and *The

use of only eight stimuli—while producing meaningful results—invites noise which, in turn, may only be mitigated by increasing the number of subjects. Hence, a larger number of trials is generally recommended.

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olfactory induced cortical activation (compare Spencer et al., 1999), as exemplified by the odor of nicotine (Hummel et al., 1992). Nicotine produces odor, burning, and stinging at increasing sensations. When presented at a concentration that mainly produces odor, maximum amplitudes N1 are found at centro-parietal sites. However, when used in concentrations that produce trigeminally mediated sensations, amplitudes are significantly larger at position Cz compared to all other recording sites. This relates, at least partly, to the different cortical areas that are activated by olfactory and trigeminal stimuli. D.

Control of Testing Conditions

During OERP recording, stable environmental conditions are important. This includes the visual and acoustical shielding of subjects. Visual shielding can be performed using drapes or blindfolds; for acoustical shielding, white noise (a mixture of all frequencies at the same loudness) is typically used. Further, a defined task has proven most helpful. Specifically, many labs use a tracking task, where, for example, subjects are requested to keep a small square controlled by a joystick inside a larger one, which randomly moves on a screen at a distance of ~1.5–2 m from the subject’s eyes. This task fulfills a number of purposes, including stabilization of eye movements (minimizing artifacts from eye movements), maintaining vigilance or attention, and providing quantitative assessments of how vigilant or attentive a subject was during various periods of the recording session (e.g., by assessing error rates). Typically, a subject’s performance increases during an experiment. Evaluating tracking performance also makes it possible to assess subtle differences in vigilance due to such experimental manipulations as the administration of sedatives or analgesics. During a test session, subjects are instructed to relax and sit as still as possible. This requires that they are comfortably seated in a chair equipped with arm, leg, and head rests. Often, subjects are trained, using simple biofeedback, in a specific breathing technique, the velopharyngeal closure, to prevent respiratory flow through the nose (Nagel, 1904; Kobal and Hummel, 1989). This technique, which is mastered in less than 5 min, helps to prevent respiratory flow through the nose by lifting the soft palate, which is under voluntary control. An alternative to velopharyngeal breathing is to present stimuli synchronously with inspiration (Tonoike et al., 1982; Lorig et al., 1996; Pause et al., 1999; Hummel et al., 2000). However, one must be aware that responses obtained under these circumstances are contaminated by the so-called “contingent negative variation” (Walter et al., 1964; Tecce, 1972; Torii et al., 1988; Lorig and Roberts, 1990;

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Auffermann et al., 1993). This negativity builds up as a consequence of the expectation of odorous stimulation, which is more likely to happen during inspiration. In addition, there is evidence that the processing of olfactory information differs between inspiratory and expiratory phases (Hummel et al., 2000). Considering these extensive prerequisites for an “ideal” session, it becomes clear why many experiments benefit from a specific adaptation or training session where subjects are thoroughly acquainted with the experimental procedures. While these efforts optimize recording conditions and, thus, the signal-to-noise ratio of the responses, they may be regarded as “overkill” in situations when subjects are only tested once, e.g., in clinical settings. Thus, normal mouth breathing, rather than velopharyngeal closure, may be less stressful to some patients. Stress itself may adversely effect the signal-to-noise ratio. E. Influences of Stimulus Characteristics and Stimulus Presentation Procedures 1.

Stimulus Intensity

There is controversy as to how OERP amplitudes relate to odorant concentration. In rats, it has been shown that OERP latencies shorten and amplitudes increase with rising odorant concentrations (Evans and Starr, 1992). In humans, some studies (e.g., Pause et al., 1996b) have used stimuli with mixed olfactory-trigeminal properties, such as linalool or citral (see Doty et al., 1978; Kobal and Hummel, 1988). Others have employed odorants with little or no trigeminal activity but have methodological shortcomings (Thiele and Kobal, 1984; Prah and Benignus, 1992; Pause et al., 1997). Still others have suffered from small sample sizes and the lack of statistical analyses (Köster, 1965) or too low of flow rates to observe all but the late OERP positivity (Prah and Benignus, 1992). In one study (Pause et al., 1997), low concentrations of linalool were presented in ascending sequence, conceivably superimposing adaptation or habituation on the obtained responses (Köster, 1965; Köster and de Wijk, 1991; Dalton and Wysocki, 1996). A recent study employing 15 subjects reported that OERP amplitudes (both early and late components) discriminate between different concentrations of vanillin (Tateyama et al., 1998). A similar finding was observed in a sample of 36 subjects using H2S (a stimulus that, at the concentrations used, activates only CN I afferents) (Hummel et al., 1998b). Thus, N1 and P2 amplitudes increased as H2S stimulus concentration increased. In addition, the OERP latencies were decreased in a concentration-related manner. In line with previous research (e.g.,

Pause et al., 1997; Covington et al., 1999), the latencies appeared to be more strongly related to changes in stimulus intensity than were the amplitudes. This observation, which is analogous to what is commonly observed in other sensory modalities, suggests that central olfactory processing may be more strongly tuned for discriminating among odor qualities than for differentiating odor intensities per se. 2.

Stimulus Duration

OERPs relate to stimulus onset (Fig. 2). Kobal (1981) presented the mixed olfactory/trigeminal stimuli isoamyl acetate and eucalyptol at a constant concentration, but at different stimulus durations (100, 300, 500, and 700 msec; ISI 40 sec), to a group of volunteers. While odor intensity ratings increased with increasing stimulus duration, there was no difference between OERPs. This indicated that the OERP—like ERPs in other sensory modalities—is predominantly determined by early segments of stimulus onset. 3.

Relation to Airflow Rate

OERPs are related to the flow rate used to transport odorants to the olfactory epithelium. This is expected, as the OERP depends on the number of odorous molecules

Figure 2 OERP in relation to stimulus duration; example from a single subject; OERP obtained at recording position Cz to eucalyptol stimuli of different duration, but identical concentration (100, 300, 500, and 700 msec; ISI 40 sec). Response shapes do not change significantly, although stimulus duration varies in the range of 1:7. The shape of the response depends on the onset of the stimulus, which is the same in all cases.

Electrophysiological Measurement of Olfactory Function

presented during early parts of the stimulus wave (see above); thus, both odor concentration and airflow should be determinants of the OERP. Kobal (1981) investigated this issue in healthy subjects using eucalyptol and linalool (at concentrations of 1287 and 6481 ppm, respectively; ISI 40 sec; stimulus duration 200 msec). Airflow varied between 5 and 277 mL/sec. Both amplitudes and latencies varied as a function of airflow (Fig. 3). 4.

Odor Quality

Attempts to relate differences in the shape of OERPs to differences in odor quality have not been successful (e.g., Kobal and Hummel, 1988). However, OERPs to different odorants exhibit differences in the topographical distribution over the scalp, even when normalized with respect to maximum OERP amplitudes (Hummel et al., 1992). When differences in the topographical distribution of OERP amplitudes and latencies are considered in relation to the stimulated naris (Kobal and Hummel, 1992), this complex pattern may be used to investigate stimulus quality. Specifically, differences between latencies and amplitudes of OERP to various odorous stimuli (e.g., acetaldehyde, phenyl ethyl alcohol, hydrogen sulfide, eugenol, and vanillin) were found in relation to lateralized stimulation

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(Kobal and Hummel, 1989, 1992; Hummel and Kobal 1994). As discussed above, this may be a reflection of the activation of different brain areas by different odorants (Ayabe-Kanamura et al., 1997; Kettenmann et al., 1997a,b) and/or their hedonic valence or emotional significance. 5.

Interstimulus Interval

Although the ISI is a significant determinant of OERPs, little research has been done in this area. In one study, the mixed olfactory/trigeminal stimulant eucalyptol (total flow 94 mL/sec; 12037 ppm) was used to investigate this issue (Kobal, 1981). ISIs of 12, 22, 32, 42, and 52 sec were investigated in 18 healthy subjects ranging in age from 20 to 41 years. An increase of the ISI from 12 to 42 sec was accompanied by a marked increase of amplitudes of the OERP. Further increase of the ISI had little, if any, effect. It was concluded that an ISI of 40–50 sec is ideal for such studies, as desensitization was minimal and the time required to collect an adequate number of potentials was reasonable. Using the odorant isoamyl acetate at concentrations that reportedly do not produce trigeminally mediated sensations, Morgan et al. (1997) confirmed the aforementioned findings, at least for young subjects (see also Murphy et al., 2000).

Figure 3 OERP to eucalyptol (200 msec stimulus duration, ISI 60 sec, 6481 ppm) presented at different flow rates (5, 85, 162, 235, 277 mL/sec; means, standard deviations, n
10). Results are shown for peaks N1 and P2. Correspondingly obtained intensity ratings (magnitude estimations; in estimation units, EU) are shown on the right. Amplitudes increase and latencies decrease when the flow rate is increased, i.e., the dose of the odorant is increased. Psychophysical results have a clearer dose dependency, because the response is related to the integrative evaluation of the total stimulus duration, whereas the OERP differences are only related to the differences in integrations during the onset of the stimulus.

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While the studies mentioned above investigated the optimal ISI for recording of OERPs, it clearly is possible to obtain meaningful data using ISIs of 20 sec or less (Durand-Lagarde and Kobal, 1991; Kettenmann et al., 1997b; Hummel et al., 1998a). Krauel (1999) varied the ISI to investigate the potential presence of a mismatch negativity (Naatanen et al., 1993) in the OERP. Using an ISI of 15 sec, a negative deflection was found that was not detectable at an ISI of 30 sec. The authors interpreted their findings in terms of the presence of a transient storage of olfactory information, as reflected by the mismatch negativity.

1998a; Covington et al., 1999). In the most extensive of these studies, Murphy et al. (2000) examined the OERP to amyl acetate in a group of 140 individuals whose ages spanned a wide range. A linear prolongation of peak latencies, accompanied by a decrease of amplitudes (amplitude N1P2: 1.5 V/year; amplitude P2: 2.0 V/year; latency P2 and P3: 2.0 msec/year), was noted across the age categories. These and other studies suggest that the changes in the processing of olfactory information may appear relatively early in life. For more details see Hummel and Kobal (2001). 4.

F.

Subject Variables that Influence OERPs

1. Arousal or Vigilance OERPs are related to arousal on different levels. On the one hand, they are dependent on the background signal, wheras on the other hand they are related to cognitive factors. Using linalool and eugenol in subjects, Krauel et al. (1998) found that allocation of attention led to a latency decrease of early components and a simultaneous increase of the amplitude of the late positivity of the OERP (compare to Spence et al., 2000). Similar findings for the OERP to amyl acetate were reported by Geisler and Murphy (2000). 2.

Ultradian Rhythms

Using OERPs evoked by citral, Pause et al. (1996a) found evidence that the processing of olfactory information varies during the menstrual cycle; they investigated subjects with cycle lengths ranging from 21 to 39 days. During the periovulatory period (monitored by changes in body temperature), stimuli were perceived as more complex/novel, as indicated by an increased amplitude P3-1 (see also below). A shortening of ERP latencies was noted near the time of expected ovulation, and a shortening of peak latencies was observed during the follicular phase. Consequently, the menstrual cycle should be considered when looking for subtle changes in the perception of odors using small sample sizes. 3. Age The well-established age-related decrease in chemosensory function is mirrored in OERP measures (Hummel et al., 2001). Specifically, the amplitude of the OERP’s late positivity (P2) and the composite amplitudes of the two major peaks (N1, P2) decrease with increasing age, whereas a prolongation of N1 peak latencies occurs (Murphy et al., 1994; Evans et al., 1995; Hummel et al.,

Gender

OERPs reflect differences in olfactory sensitivity in relation to gender. In study in 35 healthy subjects, ERP amplitudes to both H2S and vanillin were found to be larger in women than in men (Becker et al., 1993). Evans et al. (1995) found the peak-to-peak amplitude N1P2 to be larger in women than in men. G.

Reliability of OERP Measures

Despite the multitude of possible sources of variation, given a proper experimental environment the test-retest reliability of OERPs is regarded as high. For example, Kobal and Hummel (1988) reported very good reproducibility of OERPs in 13 subjects who were tested on 3 different days using different stimuli (compare Lago et al., 1998). Also, when measurements of OERPs to H2S (12 subjects) were repeated before and after application of a nonactive (placebo) nasal spray, there was no significant difference between olfactory OERP obtained during the two consecutive sessions (Hummel et al., 1998c). H. Relation of OERPs to Psychophysical and Neuropsychological Measures OERPs are generally correlated with psychophysical measures of olfactory function. Tateyama et al. (1998) found in a study of 16 subjects that correlations between olfactory threshold measures and OERP latencies to vanillin were larger when the highest concentrations had been used on the OERP determinations. This may relate to the poor detectability of OERP peaks at low odorant concentrations. In addition, correlations between latencies and thresholds were higher for the early ERP components (P1 and N1) than for the later positivities. Correlation coefficients were generally larger for OERP latencies than amplitudes. The ability of subjects to identify odors was found to correlate with P2 amplitudes (Hummel et al., 1998a).

Electrophysiological Measurement of Olfactory Function

Recently, Geisler et al. (1999a) reported that both amplitudes and latencies of the late positivity of the OERP correlated weakly, but significantly, with scores on a number of neuropsychological tests, including the Trail Making Test (a test of visual-motor attentional processing; Reitan, 1971) and the California Verbal Learning Test (a test of short- and long-term memory, free and cued recall, and delayed recognition). They also found that the agerelated decrease of OERP amplitudes and the corresponding increase of latencies were accompanied by reduced neuropsychological performance. I.

Applications of OERPs

An important application of OERPs is their use in diagnosing olfactory deficits and, in some cases, detecting malingering. A test procedure standardized by Kobal and

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Hummel (1991) includes the recording of responses to olfactory (e.g., hydrogen sulfide and vanillin) and trigeminal (e.g., CO2) stimuli (Fig. 4). All three stimulants are applied 15 times both to the left and the right nostril of the patient. The interstimulus interval is approximately 40 sec. The session, including preparation, lasts for 80 min. The EEG (filtering 0.1–30 Hz, electrode impedance 10 k) is recorded from positions of the international 10/20 system (Fz, Cz, Pz, and Fp2, referenced to A1A2). EEG records of 2048 ms duration are digitized (sampling frequency 250 Hz) and averaged in groups according to the three stimulants and the binasal stimulation. EEG records contaminated by eye-blinks (Fp2/A1A2) or motor artifacts are discarded from the average. This procedure (compare Evans, 1993) has recently been adopted by the working group “Olfaction and Gustation” of the German ORL Society (Hummel, 2000). So far, in all anosmic patients

Figure 4 Clinical olfactometry in a patient suffering from anosmia in the right (re) nasal cavity. Olfactory specific stimulants (vanillin and hydrogen sulfide) were used to stimulate both nostrils separately. Stimulation of the left (li) nostrils resulted in a rather large response to vanillin and in a less clear response to H2S, while there were no responses when the right (re) was stimulated. The use of carbon dioxide to stimulate specifically fibs of the trigeminal nerve resulted in responses on both sides, although somewhat different in shape and latency. Complete oflactory deficits on only one side are not that unusual. The patient noticed this deficit in a reduced ability to smell compared to times when both nostrils were equally sensitive.

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that have been investigated, intranasal trigeminal ERPs could be obtained after stimulation with CO2, although with significantly smaller amplitudes than those of healthy individuals (Kobal, 1982; Hummel et al., 1996a). In contrast, no OERPs could be detected after stimulation with the odorants hydrogen sulfide and vanillin (Kobal and Hummel, 1998). Using identical or slightly different protocols, this technique has been used in numerous labs (e.g., Leplow, 1994; Matern et al., 1995; Cui and Evans, 1997; Hawkes and Shephard, 1998; Mata et al., 1998; WelgeLüssen, 1999; Geisler et al., 1999b; Welge-Lüssen et al., 2000). J.

Assessment of Neurodegenerative Disorders

As noted in Chapters 23 and 24, a number of neurodegenerative disorders are associated with olfactory dysfunction. OERPs have been found to be altered in many such disorders, as described in detail below. 1. Parkinson’s Disease Using olfactory and trigeminal ERPs, Barz et al. (1997) investigated PD patients treated with anti-Parkinson drugs (n
13), PD patients who received no pharmacological treatment (n
18), and 38 age- and sex-matched controls. Odor identification was impaired in PD patients and was not influenced by treatment with anti-Parkinson drugs, in accord with earlier studies (e.g., Doty et al., 1988). OERP latencies were prolonged in both PD patients taking and not taking anti-Parkinson drugs (compare Hummel et al., 1993), although the effect was more pronounced in PD patients taking such drugs. In contrast to the OERP, the intranasal chemosensory trigeminal system was affected neither by the neuronal degeneration seen in PD nor by treatment with anti-Parkinson drugs (compare Hummel et al., 1993). Moreover, there is some suggestion that the OERP may be useful in assessing progression of PD, conceivably reflecting an association between a decreased number of neurons in the bulb and disease duration (Pearce et al., 1995; Hummel, 1999). The observation that some patients exhibit normal odor identification test scores but delayed OERPs suggests the possibility that OERPs may be a more sensitive measure of subclinical dysfunction within the olfactory pathways than some psychophysical tests (Hawkes et al., 1997). 2. Alzheimer’s Disease Despite considerable evidence that Alzheimer’s disease (AD) is associated with smell loss at its earliest stages, no formal studies of OERPs have been performed in AD patients to date. We have observed OERPs in a few AD

patients, although they were, in fact, anosmic upon psychophysical testing. Clearly, more research would be of value on this topic, especially since OERPs appear to be ideally suited to investigate patients who may have difficulties cooperating or paying attention to sensory stimuli. 3.

Multiple Sclerosis

Olfactory function has been reported to be impaired in patients with multiple sclerosis (MS). Hawkes (1996) found one quarter of 45 MS patients to have delayed OERPs, even though only 15% exhibited a decreased ability to identify odors. Similar to the findings reported in PD (see above), this suggests that OERPs may be more sensitive to olfactory than some psychophysical measures. 4.

Motor Neuron Disease

Hawkes et al. (1998) investigated olfactory function in 58 patients with motor neuron disease (MND) in comparison to 132 controls. According to psychophysical testing with an odor identification test, olfactory function was slightly decreased in MND patients (compare Wenning et al., 1995). OERP were found to be absent in 2 of 15 patients and delayed in 1 of 15 patients investigated. 5.

Other Disorders

Hummel et al. (1995) compared 12 temporal lobe epilepsy patients with a left-sided focus to 10 such patients with a right-sided focus. In both groups, longer ERP latencies were found following presentation of the trigeminal stimulant CO2 to the left naris than to the right naris. A different pattern emerged for olfactory stimuli. After right-side stimulation, latencies were prolonged in patients with right-sided epileptic foci. After left-side stimulation, latencies were prolonged in patients with left-sided epileptic foci (Fig. 5). Thus, the neocortical processing of olfactory, but not trigeminal, information seems to be affected by functional lesions of the temporal lobe. Moreover, analyses revealed nonoverlapping 95% confidence intervals for latency N1 when vanillin was applied to the right nostril. These results are in accord with the notion, derived from other studies, that the right temporal lobe may play a different role in the processing of olfactory information than the left temporal lobe. Finally, the data indicated that olfactory information is predominantly processed ipsilaterally to the stimulated nostril (see Doty et al., 1997, for review). OERPs have also been investigated in migraineurs (Grosser et al., 2000). Migraineurs have been found to exhibit greater ERP amplitudes N1 to trigeminal

Electrophysiological Measurement of Olfactory Function

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Figure 5 OERP in temporal lobe epilepsy (TLE). Twelve TLE patients with left-sided focus were compared with 10 TLE patients with a right-sided focus. After right-sided olfactory stimulation latencies were prolonged in patients with right-sided epileptical foci. When the left nostril was stimulated in patients with a left-sided focus, OERP latencies were prolonged. Latencies of responses induced by stimulation on the nonaffected sides were in the range of normal controls. Also, the topographical distribution in TLE patients was altered. While normals have their largest amplitudes of OERPs at Pz, patients had the largest amplitudes at the central site Cz. We interpret this as a consequence of plasticity effects in TLE resulting in a change of orientation of the underlying equivalent current dipole (see MSI).

stimulation, supporting the concept of trigeminal hyperexcitability. In contrast, OERP amplitudes P1N1 were significantly smaller in migraineurs. A leave-one-out classification procedure on the basis of these two parameters assigned 76% cases correctly. The OERP amplitude discriminated better between groups than trigeminal ERPs, emphasizing the significance of the olfactory system in migraine. Other uses of OERP have been in the assessment of patients with multiple chemical sensitivities (MCS)/idiopathic environmental intolerances (IEI) (Otto and Hudnell, 1993; Dalton and Hummel, 2000; Hummel and Livermore, 2001), and Down syndrome (Wetter and Murphy, 1999). Further, OERPs have been used to address chemosensory changes during the course of a common cold (Hummel et al., 1998b) or the investigation of drug effects on the olfactory system, e.g., local application of decongestive agents in the nasal cavity (Hummel et al., 1998c) or the systemic administration of diazepam (Hummel and Kobal, 1992).

K. Employment of OERPs to Investigate the Cognitive Processing of Odors As indicated above, one of the most important areas in the application of OERPs is the investigation of the cognitive processing of odors. Early investigations of the late positivity of the OERP were performed by DurandLagarde and Kobal (1991). Using the so-called oddball paradigm, stimuli were presented at an ISI of 6–8 sec such that a frequent stimulus (e.g., vanillin) alternated with a rare stimulus (e.g., H2S). Subjects were asked to count the occurrence of the rare stimulus (Fig. 6). As with other sensory modalities (Sutton et al., 1965), a late positivity occurred within the evoked potential in response to the rare stimulus (compare to Kobal and Hummel, 1991). Since this early work, a large number of studies has focused on the late positivity of the OERP (for review, see Pause and Krauel, 2000). Pause at al. (1996b) presented data suggesting that this component is modulated by

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Figure 6 Cognitive component. Examples from a single subject and a single patient with multiple chemical sensitivity. Late positive component (P3) in the normal subject to the target stimulus (H2S) compared to the standard stimulus (phenyl ethyl alcohol) probabilities were p (target)
0.2, p (standard)
0.8. In this case the late positive component of the patient was prolonged and different in shape. Patients suffering from MCS complain about an unusual hypersensitivity for odors. This is not a result of a lowered olfactory threshold. Hence, the earlier parts of the OERPs are not enhanced.

stimulus significance and stimulus probability. They found that “the P3 component elicited by meaningful stimuli is so large that the P2 component can be completely overlapped.” When odorants are presented at ISIs of 30–40 sec, comparable to a single-stimulus paradigm (Polich et al., 1994; Cass and Polich, 1997), it appears that the olfactory stimulus reaches a significant “meaningfulness,” which elicits a P3 component (Pritchard, 1981; Morgan et al., 1999) that determines the shape of the OERP and largely replaces the P2. In other words, what is frequently addressed as a “P2 peak” in the OERP (Kobal and Hummel, 1991) may resemble characteristics of a P300 component (Lorig, 1993). According to a hypothesis elucidated by Donchin and Coles (1988), the P3 signifies “context updating” related to the maintenance of the internal model of the external environment. From this perspective, the P3 amplitude would reflect the amount of change of the model, while the latency would reflect the duration of stimulus evaluation (compare Geisler et al., 1999a). Although alternative hypotheses have been put

forward (e.g., Verleger, 1988), they all are based on the view that P3 is largely a result of the cognitive processing of stimuli. Several major findings from this area of research are worth noting: (1) the P3 component is larger when subjects believe they have perceived the target odor, i.e., for both hits and false alarms (Pause et al., 1996b); (2) odors presented only rarely elicit larger amplitudes than frequent odors, independent of stimulus quality (Krauel et al., 1998; Pause, 1999), (3) the emotional significance of an odor may contribute to the generation of P3 (Pause et al., 1996b; Pause et al., 1997; Krug et al., 2000); and (4) in analogy to research in ERPs in other sensory modalities (Spencer et al., 1999), the P3 component can be divided into a P3-1 and a P3-2 peak, which differ in their topographical distribution (P3-1: fronto-central; P3-2: centro-parietal). P3-1 seems to be more related to the novelty and significance of an odor, whereas P3-2 more resembles features of the classical P3, e.g., its latency relates to the time required for stimulus evaluation (Pause et al., 1996b; Pause and Krauel, 2000).

Electrophysiological Measurement of Olfactory Function

V.

OLFACTORY MAGNETIC SOURCE IMAGING

A.

Description

The general goal of magnetic source imaging (MSI) is to localize generators of magnetic fields measured at the surface of the scalp (Cohen, 1972). These generators may be the number of cerebral neurons (electrically) active at the same time. Unlike positron emission tomography (PET) and functional magnetic resonance imaging (fMRI), MSI allows direct assessment of the activity of the neurons involved in the processing of sensory information. Assuming that the human head is a spherical volume conductor (Abraham-Fuchs et al., 1988) and electrical neuronal activity has the property of a current dipole possessing an electric field and a magnetic field, it becomes possible to calculate the location, the orientation, and the strength of the current sources creating the magnetic field measured at the surface of the sphere (Romani et al., 1982). In this model, not knowing the number of active dipoles, the magnetic field may be generated by one dipole or several different ones (the “inverse problem,” first discussed by von Helmholtz in 1853). Unfortunately, this is the situation in the active brain and therefore a unique solution cannot be obtained. Additionally, a radial dipole does not produce a magnetic field that can be measured with the usual order of the magnetometers. Such dipoles are “silent” and cannot be localized. On the basis of a measured magnetic field, a dipole position is estimated and followed by a nonlinear fit-strategy such as a Marquardt or Powell algorithm (Marquardt, 1963; Powell, 1964). Using these procedures, location, strength, and orientation of the theoretically determined dipole is iteratively changed such that the magnetic field produced by this calculated dipole corresponds to the dipole measured by the magnetometer. The statistical procedure leading to the so-called equivalent current dipole is a leastsquares search. As with most recording techniques, the accuracy strongly depends on the signal-to-noise ratio of the measured data, which decreases with increasing depth of the dipole within the brain. Also, localization accuracy is better for a dipole centered below the measurement grid than below the outer part of the measurement grid. With regard to the situation in the human brain, the size of this error can only be estimated by simulations (Barth et al., 1986; Janday and O’Connell, 1987; Meijs et al., 1988; Hari et al., 1988). It should be mentioned that brain-shaped head models, i.e., realistic head models, have also been used to calculate the source of the current dipole (Hämäläinen and Sarvas, 1987, 1989). By linking the magnetically defined equivalent current dipoles to the anatomical data provided by MRI, it is possible to visualize the location of activated areas in the

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subjects’ brain and to check them for both anatomical and physiological plausibility (e.g., Stefan et al., 1990). Aside from the aforementioned basic issues related to the physics of magnetic fields, the reliability of the estimations is influenced by external error sources, e.g., magnetic noise caused by electronic devices or artifacts caused by movements, heart activity, etc. (Abraham-Fuchs et al., 1988; for review see Hämäläinen et al., 1993). B.

Applications to Chemosensation

Employing a whole-head neuromagnetometer, Kettenmann et al. (1996) found bilateral activation in the superior temporal sulcus at approximately 700 msec using vanillin, phenyl ethyl alcohol, and hydrogen sulfide—three substances with minimal CN V stimulative properties. Another study confirmed the results obtained with the whole-head magnetometer by employing a planar 37-channel sensor array (Kettenmann et al., 1997b) and additionally identified the neuronal generators underlying the earlier components of the event-related potentials in the time interval between 200 and 700 msec following olfactory stimulation. Consistent magnetic fields were identified in both hemispheres following the stimulation of each nostril. In 60% of the measurements, reproducible dipolar field patterns were obtained 226–380 msec after stimulus onset, preceding or following the first major positive electric deflection P1 of the event-related potential (ERP). This equivalent current dipole was named ECD I. In 44% of the measurements a reproducible dipolar distribution was obtained 306–486 msec after stimulus onset, corresponding to the ascending or descending slope of the N1 component of the ERP, which was named ECD II. In the left hemisphere, this dipole was not identifiable in any of the subjects after stimulation with hydrogen sulfide. It was identifiable only in the right hemisphere in 36% of the measurements. The most stable dipolar field pattern appeared 518–730 msec after stimulus onset (in 66% of the measurements; ECD III) corresponding to the P2 component of the ERP (Fig. 7). Control measurements excluded the possible contamination of the measured signals by tactile or auditory artifacts. Humidified blanks delivered to the nostrils did not result in MEG or EEG activity. In 14% of all the measurements, all three ECDs could be identified during one session. ECD I was localized in the area between the superior temporal plane and the parainsular cortex. ECD II was localized in the anteriorcentral parts of the insula and ECD III was obtained in the superior temporal sulcus. Intraindividually, spatial differences of localization sites for one type of ECD were less than 20 mm. Individually, the angle of orientation varied

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Figure 7 Localization of olfactory activation in the temporal lobe and insula. Example from a single subject. Three main components of the activity could be discriminated in the temporal plane (ECD I), in the insula (ECD II), and in the superior temporal sulcus (ECD III). Other odorants, such as eugenol or hexenoic acid, activate more intensively the hippocampus and/or parainsular cortex.

between 10° and 30° in all three dimensions. The dipole strength varied between 0.009 and 0.03 mA*mm. In summary, localization results of the olfactory ERMFs demonstrated that odorants specifically activate neocortical areas, i.e., during the period of time when the ERP is obtained, areas that are activated include those between the superior temporal plane and the parainsular cortex, anterior-central parts of the insula, and the superior temporal sulcus. These electro(magneto)physiological data confirm the hypothesis that there is a direct connection between primary olfactory areas and the insular cortex

(Mesulam and Mufson, 1982), in accord with evidence from other functional imaging techniques. Only a few other groups have studied the olfactory system with MSI. Tonoike et al. (1998) employed a wholecortex 122-channel biomagnetometer and found generators of olfactory magnetic fields in two regions located fairly asymmetrically near the bilateral frontal deep areas. The results of Kettenmann and collagues were confirmed by Sakuma et al. (1997) in a study of 14 subjects that used pulses of odorant air containing amyl acetate or phenyl ethyl alcohol presented via a nasal tube Equivalent current

Electrophysiological Measurement of Olfactory Function

dipoles (ECDs) were estimated in the regions around the Sylvian fissure, symmetrically in both hemispheres.

VI.

THE ELECTRO-OLFACTOGRAM

Even today the recording of electro-olfactograms (EOGs) from the human nasal mucosa is very difficult. The placing of the electrodes is no easy task, since the intrusion of a foreign matter into the nose very often leads to sneezing and to excessive mucous discharge. Extensive local anesthesia has also to be avoided, since it might affect olfactory fibers and render the subject temporarily anosmic or hyposmic. This is probably the reason why so few publications on this topic exist. In an early experiment of two subjects in which coffeesaturated air was used as the stimulant, Osterhammel et al. (1969) discovered that the negativity recorded from the olfactory mucosa increased in relation to an incremental flow rate of the stimulus. Subsequently, Kobal (1981) employed the stimulation method described earlier in this chapter (which eliminates flow rate-related mechanical or thermal artifacts) and the odorants amyl acetate, H2S, and eugenol in a study of the EOGs of four subjects. The responses, all of which characteristically showed negative electrical potentials at the surface of the olfactory mucosa, were dependent on the concentration of the stimulus. When stimuli (e.g., H2S) of longer durations were applied, a temporal integration over a period of time of 10 sec was observed, a phenomenon that has been reproduced (Hummel et al., 1996b). In response to long-duration stimuli, a decrease in intensity estimates was found to be two to three times greater than the decrease of EOG amplitudes. Since the EOG reflects changes at the receptor level, it can be assumed that the observed decrement in perceived intensity reflects changes in central nervous processes rather than to peripheral adaptation, a well-established phenomenon (see Chapter 10). In the olfactory literature such a reduction in odor intensity is generally called adaptation. However, most of the time this concept is not distinguished from peripheral desensitization, which the author would prefer to call adaptation, and central desensitization, which the author would prefer to call habituation (Thompson and Spencer, 1966). Recently, Leopold et al. (2000) used EOG recordings to analyze the distribution of the olfactory epithelium. Up that the time of this study, there was general agreement that the olfactory epithelium is located high in the nasal cavity, predominantly on the dorsal aspects of the nasal vault, the septum, and the superior turbinate. However, when caring for patients with obstructed nasal cavities, there is some suggestion that some may be able to smell

243

with the affected nostril even when the olfactory cleft seems totally blocked with swollen mucosa, polyps, mucus, or tumor. One possible explanation for such spared sensory function would be the presence of functioning olfactory neuroepithelium located anterior to the usual boundaries of the olfactory cleft. Based on the topography of EOG recordings and histological and immunocytochemical evaluation of tissue from biopsy specimens, Leopold et al. (2000) reported that the olfactory neuroepithelium extends at least 1–2 cm anterior to the usually defined olfactory cleft. Other studies that have harvested olfactory receptor neurons report that the neuroepithelium may even extend to the anterior and middle parts of the middle turbinate (Restrepo et al., 1993; Thürauf et al., 1996). Clearly, more work needs to be done before EOG recordings can be meaningfully employed in clinical investigations or in a large numbers of subjects. Since the EOG represents the input signal into the olfactory channel, its recording is fundamental for the interpretation of more centrally generated olfactory responses (OERP, P300, CNV, and subjective ratings) in order to define the site of modulatory influences, when phenomena such as adaptation or habituation, anosmia or hyposmia, hypersensitivity, or parosmia, etc. are investigated.

VII.

MISCELLANEOUS TECHNIQUES

No major progress has been made in recent years in the recording of the contingent negative variation (CNV), the spontaneous electroencephalogram (EEG), the psychogalvanic skin response (PSR), and other types of reflexes. Therefore, only studies related to background EEG activity are discussed in this section. There is a large body of literature on the use of the EEG in the quantification of human olfactory sensations. In the 1950s and 1960s, the area of clinical applications was dominated by Italian scientists. Archilei and Moretti (1958) investigated electroencephalographic changes in 30 subjects after presentation of odorous stimuli. As a rule, they observed an arousal reaction in response to olfactory and trigeminal stimulation, i.e., slow EEG waves (thetaand alpha-band) were replaced by faster activity (betaband). Similar findings were reported by Moncrieff (1962) and by Motokizawa and Furuya (1973). These results were extended by Perbellini and Scolari (1966), who tested 50 patients using pyridine, vanillin, and “essence of rose.” They concluded that the method would appear to be particularly useful in the medico-legal field for the detection of deception. However, they also observed a number of cases where no arousal reaction could be recorded,

244

Kobal

although the subjects reported an olfactory sensation. Thus, when using this technique only positive responses can be viewed as an unambiguous result. In earlier work, Bartalena and Romeo (1962) had similarly noted, in 24 subjects, that weak odorous stimuli were unaccompanied by electroencephalographic changes. Brandl et al. (1980) performed frequency analyses of the EEG before and after administration of pleasant and unpleasant odorants. Significant correlations between a pattern of EEG spectra and the subject’s hedonic estimates were noted. Yoshidan et al. (1989) have also reported, in a preliminary study, that the alpha-band recorded at frontal sites appears to be an indicator of hedonic estimates of olfactory stimuli. However, Kobal et al. (1989) were unable to find changes of the stimulus-related EEG in relation to hedonic estimates of odorous stimuli, and Klemm et al. (1992) reported no direct effect of pleasant or unpleasant odorants on the theta-band. Lorig and Schwartz (1988) investigated intensityrelated effects of odorants on the EEG using topographical maps of theta activity. They reported changes of scalp patterns when the subjects were exposed to varying odor intensities. Differences were greatest over temporoparietal recording positions. Similarly, Van Toller (1987) reported intensity-related differences, although he compared the mixed olfactory-trigeminal stimulant ammonia to olfactory stimulants being perceived as less intense. Moreover, Van Toller and Kendall-Reed (1989) reported differential effects for olfactory versus trigeminal stimuli. Extending these findings in a follow-up study, Van Toller et al. (1983) reported that odors can be distinguished by means of changes in the alpha activity recorded at different sites. Interestingly, several authors have observed changes of the EEG in the presence of undetected odors (Lorig et al., 1991; Klemm et al., 1992). To summarize, analysis of the stimulus-related EEG is certainly of value in further understanding the relation of olfaction to the general state of cortical activity, e.g., how odors may modulate states such as arousal, mood, etc. (see Lorig, 1992). However, although the recording and analysis of stimulus-related EEG activity appears to be less difficult than, e.g., the recording of olfactory event-related potentials, there is still little evidence that these measures are specifically related to activities in the olfactory system. VIII.

CONCLUSIONS

At the present point in time, we are facing a new epoch of functional investigations of the brain (see also Chapter 12). Those who first started to use electrophysiological techniques have the feeling that their eyes have been uncovered and that—although not yet clear—one has gained a new

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247 Lötsch, J., Ahne, G., Kunder, J., Kobal, G., and Hummel, T. (1998). Factors affecting pain intensity in a pain model based upon tonic intranasal stimulation in humans. Inflamm. Res. 47(11):446–450. Marquardt, D. W. (1963). An algorithm for least-squares estimation of nonlinear parameters. J. Soc. Ind. Appl. Math. 11:431–441. Mata, J., Jimenez, J., and Postigo, A. (1998). Potenciales evocados corticales olfatorios. [Olfactory cortical evoked potentials]. Acta Otorrinolaringol Esp. 49(5):385–387. Matern, G., Matthias, C., and Mrowinski, D. (1995). Olfaktorisch evozierte Potentiale (OEP) und Contingent Negative Variation (CNV) bei der Begutachtung von Riechstörungen. [Olfactory evoked potentials and contingent negative variation in expert assessment of disordered sense of smell]. Laryngorhinootologie 74(2):118–121. Meijs, J. W., Peters, M. J., Boom, H. B., and Lopes da Silva, F. H. (1988). Relative influence of model assumptions and measurement procedures in the analysis of the MEG. Med. Biol. Eng. Comput. 26(2):136–142. Mesulam, M. M., and Mufson, E. J. (1982). Insula of the old world monkey. I. Architectonics in the insulo-orbito-temporal component of the paralimbic brain. J. Comp. Neurol. 212(1):1–22. Mohammadian, P., Hummel, T., Lötsch, J., and Kobal, G. (1997). Bilateral hyperalgesia to chemical stimulation of the nasal mucosa following unilateral inflammation. Pain 73(3):407–412. Mohammadian, P., Schaefer, D., Hummel, T., and Kobal, G. (1999). Experimentally induced nasal irritation. Rhinology 37(4):175–178. Moncrieff, R. W. (1962). Effect of odours on EEG records. Perfum. Essent. Oil. Rec. 53:757–760. Morgan, C. D., Covington, J. W., Geisler, M. W., Polich, J., and Murphy, C. (1997). Olfactory event-related potentials: older males demonstrate the greatest deficits. Electroenceph. Clin. Neurophysiol. 104(4):351–358. Morgan, C. D., Geisler, M. W., Covington, J. W., Polich, J., and Murphy, C. (1999). Olfactory P3 in young and older adults. Psychophysiology 36(3):281–287. Motokizawa, F., and Furuya, N. (1973). Neural pathway associated with the EEG arousal response by olfactory stimulation. Electroenceph. Clin. Neurophysiol. 35(1):83–91. Murphy, C., Nordin, S., de Wijk, R. A., Cain, W. S., and Polich, J. (1994). Olfactory-evoked potentials: assessment of young and elderly, and comparison to psychophysical threshold. Chem. Senses 19(1):47–56. Murphy, C., Wetter, S., Morgan, C. D., Ellison, D. W., and Geisler, M. W. (1998). Age effects on central nervous system activity reflected in the olfactory event-related potential. Evidence for decline in middle age. Ann. NY Acad. Sci. 855:598–607. Murphy, C., Morgan, C. D., Geisler, M. W., Wetter, S., Covington, J. W., Madowitz, M. D., Nordin, S., and Polich, J. M. (2000). Olfactory event-related potentials and aging: normative data. Int. J. Psychophysiol. 36(2):133–145.

248 Näätänen, R., Paavilainen, P., Tiitinen, H., Jiang, D., and Alho, K. (1993). Attention and mismatch negativity. Psychophysiology 30(5):436–450. Nagel, W. A. (1904). Einige Bemerkungen über nasales Schmecken. Ztschr. Psychol. 25:268. Osterhammel, P., Terkildsen, K., and Zilstorff, K. (1969). Electro-olfactograms in man. J. Laryng. 83:731–733. Otto, D. A., and Hudnell, H. K. (1993). The use of visual and chemosensory evoked potentials in environmental and occupational health. Environ. Res. 62(1):159–171. Ottoson, D. (1956). Analysis of the electrical activity of the olfactory epithelium. Acta Physiol. Scand. Suppl. 122:1–83. Pause, B. M. (1999). Body odor evoked potentials of self and non-self in humans. Genetica 104:285–294. Pause, B. M., and Krauel, K. (2000). Chemosensory event-related potentials (CSERP) as a key to the psychology of odors. Int. J. Psychophysiol. 36(2):105–122. Pause, B. M., Sojka, B., Krauel, K., Fehm Wolfsdorf, G., and Ferstl, R. (1996a). Olfactory information processing during the course of the menstrual cycle. Biol. Psychol. 44(1):31–54. Pause, B. M., Sojka, B., Krauel, K., and Ferstl, R. (1996b). The nature of the late positive complex within the olfactory eventrelated potential (OERP). Psychophysiology 33(4):376–384. Pause, B. M., Sojka, B., and Ferstl, R. (1997). Central processing of odor concentration is a temporal phenomenon as revealed by chemosensory event-related potentials (CSERP). Chem. Senses 22(1):9–26. Pause, B. M., Krauel, K., Sojka, B., and Ferstl, R. (1999). Is odor processing related to oral breathing? Int. J. Psychophysiol. 32(3):251–260. Pearce, R. K., Hawkes, C. H., and Daniel, S. E. (1995). The anterior olfactory nucleus in Parkinson’s disease. Mov. Disord. 10(3):283–287. Perbellini, D., and Scolari, R. (1966). L’elettroencefalo-olfattometria. Contributo clinico. [Electroencephalo-olfactometry. Clinical contribution]. Ann. Laringol. Otol. Rinol. Faringol. 65(4):421–429. Picton, T. W., and Hillyard, S. A. (1988). Endogenous event-related potentials. In EEG—Handbook, Vol. 3, T. W. Picton (Ed.). Elsevier, Amsterdam. Polich, J., Eischen, S. E., and Collins, G. E. (1994). P300 from a single auditory stimulus. Electroencephalogr. Clin. Neurophysiol. 92(3):253–261. Powell, M. J. D. (1964). An efficient method for finding the minimum of a function of several variables without calculating derivatives. Computer J. 7:155–162. Prah, J. D., and Benignus, V. A. (1992). Olfactory evoked responses to odorous stimuli of different intensities. Chem. Senses 17:417–425. Prah, J. D., Sears, S. B., Walker, J. C. (1995). Modern approaches to air dilution olfactometry. In R. L. Doty (Ed.). Handbook of Olfaction and Gustation, 1st Edition. Marcel Dekker, New York, pp. 227–255. Pritchard, W. S. (1981). Psychophysiology of P300. Psychol. Bull. 89(3):506–540. Reitan, R. M. (1971). Trail making test results for normal and brain-damaged children. Percept. Mot. Skills 33(2):575–581.

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12 Functional Neuroimaging of Human Olfaction Noam Sobel, Bradley N. Johnson, and Joel Mainland University of California, Berkeley, California, U.S.A.

David M. Yousem The Johns Hopkins University School of Medicine, Baltimore, Maryland, U.S.A.

I.

rently still lagging behind invasive methods in both spatial and temporal resolution, are mostly devoid of these drawbacks, and therefore promise to be the method of the future in the study of neural systems. The signal recorded with noninvasive functional neuroimaging can be either the direct electrical product of neural activity or an indirect product of this neural activity, such as metabolic products or blood flow. Different methods of neuroimaging have been developed to utilize these different sources of signal, each offering specific advantages and disadvantages. The most commonly used of these methods in humans are as follows:

INTRODUCTION

Scientific advance is largely dependent on methodological and technological advance. Neurobiology is currently experiencing a number of methodological advances of revolutionary proportions, not the least of which is the advent of methods for noninvasive functional neuroimaging. One may be able to indirectly infer a tremendous amount about how a neural system functions by both observing and quantifying its input (stimuli) and output (behavior). For the study of olfaction, the latter relates to olfactory psychophysics (reviewed in Chapter 10 of this volume). But to completely understand the function of a neural system, one must also have methods to directly measure and record its neural activity. For the case of olfaction, one would want to reliably measure where in the brain olfactory information is processed, and how this information is processed. A.

1.

Electroencephalography (EEG) and evoked potentials (EPs). These methods directly measure neural electrical activity. EEG and EPs can be noninvasively obtained, and offer high temporal resolution. These methods, however, provide very poor spatial resolution (the application of these methods to the study of human olfaction is covered in detail in Chapter 10 of this volume). 2. Magnetoencephalography (MEG). This method measures the electromagnetic fields produced by neural activity. MEG is noninvasive and offers high temporal resolution combined with moderate spatial resolution. Unlike the electrical currents recorded in EEG, the magnetic fields are not distorted by brain, skull, and scalp. MEG, however, can only detect sources producing fields oriented

Functional Neuroimaging

To date, most methods for measuring neural activity were invasive. The disadvantages of an invasive versus a noninvasive method are numerous. An invasive method, by definition, (1) alters (usually harms) the system that it sets out to measure, (2) often involves anesthesia that in itself alters patterns of neural activity, (3) inflicts suffering on animals, and (4) usually can not be used to study humans. Methods of noninvasive functional neuroimaging, although cur251

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parallel to the surface of the skull. MEG also usually requires a concomitant MRI scan upon which to register the MEG signals to anatomical space. 3. Single-photon emission computed tomography (SPECT), positron emission tomography (PET), and magnetic resonance spectroscopy (MRS). These three methods measure the biochemical components of neural transmission, for example, the distribution and density of a particular labeled neurotransmitter, metabolite, blood flow, or glucose utilization. These methods currently offer rather poor temporal and spatial resolution but give access to a type of information not available with other imaging methods, namely the highly sensitive measuring of biochemical processes—a PET camera can detect picomolar changes in labeled compounds. Both PET and SPECT are somewhat invasive in that they require administering to subjects a radioactive isotope and exposure to ionizing radiation. MRS is noninvasive, but the application of this method to humans is only in its early stages. PET and SPECT can also be used as an indirect measure of regional levels of neural activity through measuring either glucose metabolism or blood flow. One of the main advantages of PET is the absence of ferromagnetic or susceptibility artifacts at the skull base which plague fMRI studies of the olfactory system. Structural MRI scans often accompany PET and SPECT studies to provide a more detailed anatomical localization of activity. 4. Functional magnetic resonance imaging (fMRI). This method is a relatively recent modification to high-resolution structural magnetic resonance imaging (covered in Chapter 28 of this volume) that provides an indirect measure of neural activity through measuring blood flow. fMRI has high spatial resolution, fair temporal resolution, and is totally noninvasive. It does not require any other concomitant scanning, so intermodality registration issues are not a problem. It is these advantages— combined with relatively high and rapidly growing availability—that have made fMRI the most rapidly developing method of functional neuroimaging. Table 1 provides a comparison of temporal and spatial resolution of the above widely used methods of human neuroimaging. In this chapter we aim to acquaint the nonspecialist with the above modern methods of human functional neuroimaging, to briefly explain the source of the measured signal, and to stress some of the potential advantages

Sobel et al. Table 1 Range of Temporal and Spatial Resolution for Commonly Used Methods of Human Functional Neuroimaging Method

Temporal resolution

Spatial resolution

EEG MEG SPECT PET fMRI

~1 msec ~1 msec 60 sec ~45 sec ~3–5 sec

~10 mm ~5 mm ~6 mm ~4 mm ~1 mm

Source: Adapted from Volkow et al., 1997.

and disadvantages of each method. We then discuss the application of these methods to the study of human olfaction, reporting on the current state of the art. Considering that functional imaging of olfaction is in its infancy, we will relate in this chapter to work using all of the above methods. We will, however, concentrate on work using the blood flow–dependent methods of PET and fMRI, as those are the most commonly used methods, and the latter is also the method currently used by these authors. B.

A Brief History of Blood Flow Neuroimaging

The foundations of modern blood flow–dependent neuroimaging lay in the careful observations made in 1885 by the Italian physiologist Angelo Mosso (reviewed in Posner and Raichle, 1998; Raichle, 1998; Volkow et al., 1997). As anyone can observe by feeling the undeveloped skull of a newborn, the brain appears to pulsate in unison with heart rate and respiration. This pulsation is later obscured by the adult developed skull. Mosso had the opportunity to study this phenomenon in an adult patient named Bertino, who, due to head injury, had a skull defect that enabled simple recording and quantifying of this pulsation over the frontal lobes of the brain. Mosso noted that the rate of pulsation on the surface of Bertino’s brain increased during the performance of specific cognitive tasks, such as performing a mathematical multiplication task. Furthermore, this increase in rate of pulsation occurred independently of heart rate and blood pressure as measured on Bertino’s arm. These findings suggested that measuring brain blood flow might serve as a method to measure brain activity. Mosso’s measurements in a human were later corroborated by measurements Roy and Sherington made in animals (1890). Roy and Sherington describe a mechanism that automatically modulates the supply of blood to local areas of increased activity in the brain. This relationship between local increases in brain blood flow and local increases in neural activity was further solidified in a second famous early human case study published by John Fulton in 1928. Fulton reported on a patient known

Functional Neuroimaging of Human Olfaction

as Walter K., in whom a failed attempt at correcting an arteriovenous malformation in the occipital lobe resulted in a skull defect overlying the primary visual cortex. As the patient himself could later notice, the increased sound of blood flowing through the blood vessels could be heard from this location during performance of specific visual tasks. The physician could also hear, and record, this sound, simply by placing a stethoscope over the skull defect. Fulton reports that when the patient opened his eyes, there was only a moderate increase in the sound of blood flow. However, when the patient engaged in a demanding visual task, such as reading, there was a much larger increase in the sound of blood flow. The sound would reliably increase 20–30 seconds after the beginning of a task and would return to baseline within 2 minutes. The above pioneering studies in which the relationship between local increases in neural activity and local increases in blood flow was established laid the foundation for the later development of PET and fMRI functional neuroimaging techniques. C.

PET

Developments in animal autoradiography made by Kety, Sokoloff, Landau, and colleagues (Landau et al., 1955) enabled the quantification of the relationship between glucose metabolism, blood flow, and neural activity. These measurements enabled Lassen and colleagues to develop the application of an array of scintillation detectors to measure regional changes in blood flow in the human brain (Ingvar and Risberg, 1965; Lassen et al., 1978). These functional measurements of regional blood flow were later combined with structural x-ray computed tomography (Hounsfield, 1973) to result in the modern PET scan (Frackowiak et al., 1980; Raichle et al., 1983; TerPogossian et al., 1975). The PET scan is dependent on the radioactive decay of positrons emitted from unstable neutron-deficient atoms such as 15O. At the beginning of a PET experiment, this, or another, unstable tracer is injected into the subjects’ bloodstream. The tracer accumulates in the brain at concentrations directly proportional to regional blood flow, that is, regions with increased blood flow have higher concentrations of the tracer. The unstable tracer then decays (the half-life of 15O is 123 seconds), emitting positrons that are annihilated by negatively charged electrons present in tissue. The energy from this annihilation is emitted in the form of two photons that leave the brain in exactly opposite directions from the point of annihilation. By surrounding the brain with an array of radiation detectors coupled through coincidence circuits, it is possible to detect and map the exact location of the annihilation in the brain, or in other words, create a map of brain blood flow.

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The period that is best mapped is typically one minute following injection, and a typical PET experiment may consist of 12 such injections. The resulting blood flow map can then be overlaid on a structural image of the brain, such as that obtained with MRI. The above describes the use of PET to follow blood flow as a measure of neural activity. Other tracers are commonly used in PET to follow glucose metabolism as a measure of neural activity. D.

fMRI

Like PET, fMRI is also an indirect index of neural activity that measures blood flow, but rather than depend on an introduced tracer or contrast agent, fMRI depends on an intrinsic contrast agent—hemoglobin. The principals that underlie MRI were discovered independently by Block (1946) and Purcell and colleagues (1946) and were developed into an imaging method by Lauterbur (1973) (reviewed in Raichle, 1998). In an fMRI experiment, the subject is placed within a strong static homogeneous magnetic field. In such a field, the nuclei of elements in tissue that have an odd atomic weight, such as hydrogen, behave as little spinning magnets that tend to align their macroscopic spin axes with that of the external field. A brief pulse of radio frequency magnetic fields can then be used to tip the orientation of the spinning protons/magnets away from that of the external field. A given radio-frequency of pulse will tend to selectively tip a given atomic species. Because of the preponderance of protons and their high relative sensitivity, fMRI experiments selectively excite the protons in water. Following the pulse, these protons will then tend to realign in parallel with the external field (relax), while emitting energy at the same frequency as the excitation. This emitted energy is the source of the signal in MRI. The strength of the emitted energy increases with the strength of the static magnetic field. The relaxation of the protons orientation back to that of the static field is referred to as T1 relaxation, and the relaxation of proton’s spin from the orientation transverse to the static field is referred to as T2 relaxation. A third type of relaxation, which takes into account tissue and field inhomogenities, is referred to as T2*. These differences in relaxation time are the source of contrast in the structural MR image. But how do such differences in relaxation time give rise to a neurofunctional measure? As described by Fox et al. (1986, 1988), local changes in neural activity induce local changes in the amount of oxygen in tissue or, more specifically, in the ratio of oxyhemoglobin to deoxyhemoglobin. This change in ratio is counterintuitive in its direction. One might predict that a local increase in neural activity would induce a local increase in neurometabolic products, namely deoxyhemoglobin. But the dynamics of

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neural activity–induced blood flow are such that the local ratio of oxyhemoglobin to deoxyhemoglobin in fact increases by a few percent over baseline. This finding, combined with a previous finding by Pauling and Coryell (1936) that changing the amount of oxygen carried by hemoglobin changes the degree to which hemoglobin disturbs the magnetic field, suggested that MR relaxation times, specifically T2* relaxation, would differ in accordance with the amount of deoxyhemoglobin in blood. Such use of MRI to measure sensory task–induced local changes in brain blood flow in humans was first successfully reported by Ogawa and colleagues (1992) and has since become standard practice. This method is referred to as fMRI, and the signal is referred to as BOLD (blood oxygen level dependent). Other fMRI methodologies exist that utilize gadolinium-based contrast agents to measure blood flow or tag arterial blood with magnetic pulses to demonstrate increased flow to an activated state.

Sobel et al.

list of questions on localization of olfactory function that can be addressed using functional neuroimaging, but the following are examples of the type of questions that can be addressed: 1. Piriform cortex, one of the main components of the primary olfactory cortex (Price, 1990), is a cytoarchitechtonic definition. Therefore, piriform cortex cannot be delineated in vivo based on structural imaging information alone. Functional imaging, however, may enable in vivo delineation of human piriform cortex based on odorant-induced activation. A question such as this can best be addressed using fMRI, thanks to its high spatial resolution. 2. Secondary olfactory cortex is considered to reside primarily within the orbitofrontal gyri of the ventral frontal lobe. Which of the four gyri that primarily comprise the orbitofrontal complex is indeed part of human olfactory cortex, and the specific functional role played by this cortical region in olfactory processing, are currently not well understood. Questions such as these could best be addressed using either fMRI or PET. 3. What other brain regions are involved in olfaction, and what are their respective contributions to olfactory function? Whereas the previous localization question consists of refining a crude but existing picture of the human olfactory neural substrates, functional imaging can also be used to probe for novel regions of activity, namely, regions not previously known to subserve olfaction. Questions such as the above could best be addressed using PET, where whole brain data are regularly obtained (whole brain fMRI has also been studied using thicker and more numerous anatomical sections, but the fine anatomy of the primary olfactory cortex may be obscured when looking more globally).

II. APPLYING IMAGING TO THE STUDY OF OLFACTION: WHAT QUESTIONS CAN BE TESTED WITH THESE METHODS? Functional neuroimaging is primarily used to address questions within two major frameworks: questions on broad localization of function and questions on temporal and spatial properties of function. The distinction between a broad question on localization of function versus a question on spatial properties of function can best be delineated by a relevant example: asking where in the brain is primary olfactory cortex is an example of the former, whereas asking is there a spatial component in the encoding of odors in primary olfactory cortex is an example of the latter. This distinction represents more than just a difference in sensitivity and scale of measurement; it is asking “where” versus asking “how.” That said, this distinction does not represent mutually exclusive types of information, as indeed part of understanding “how” a neural subsystem functions is understanding “where” in the brain it functions. A. Questions on Broad Localization of Function: Functional Mapping of the Human Olfactory System Mapping consists of elucidating which brain regions are primarily responsible for carrying out specific tasks. This type of question represents a dominant paradigm in neuroscience prevalent since the work of Franz Joseph Gall and Paul Broca (reviewed in Harrington, 1995; Harris, 1995). This paradigm has dominated the field of human functional neuroimaging. We can not here offer an exhaustive

B.

Questions on Properties of Function

Functional neuroimaging can be used to address questions regarding both the spatial and temporal properties of neural activity. The following are examples of the types of questions that can be addressed: 1.

Is there a spatial component in the encoding of odor identity at the cortical level? A consistent strategy employed by the brain to encode sensory information is spatial mapping. The body surface is somatotopically represented in somatosensory cortex. Auditory frequencies are tonotopically mapped on the cochlea, and this spatial organization is

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maintained in auditory cortex (Gulick et al., 1989). Visual representations are retinotopically organized on the retina, and this spatial organization is maintained in primary visual cortex (Holmes, 1918; Horton and Hoyt, 1991). Although most cytoarchitectual evidence suggests no odortopically organized projection from the bulb to primary olfactory cortex (Price and Sprich, 1975), recording of activity in the rat piriform cortex made with optical imaging suggests that the piriform is divided into several functionally heterogeneous regions (Litaudon et al., 1997). Whereas odortopy is possible but unlikely in primary olfactory cortex, it has been in fact demonstrated to some extent in secondary olfactory cortex in monkeys (Tanabe et al., 1975). The nature and rules underlying this secondary cortical odortopy are currently unknown. Functional neuroimaging in humans promises to be an ideal tool to address this issue, because when studying the relationship between patterns of brain activity and odorants in humans rather than animals, there is the added dimension of the percept that humans can readily verbally report. Questions such as the above could best be addressed with fMRI thanks to its high spatial resolution. 2. What are the temporal properties of odor encoding at various levels of processing? Temporal parameters that can be probed range from standard measures, such as latency and duration of responses, to complex measures such as potential informationencoding (odorant encoding) in the phase and/or ordering of neural activity. Temporal measures can also be used to probe the nature of interaction between two or more regions processing olfactory information simultaneously. Questions such as the above could best be addressed using either MEG or single-trial fMRI thanks their high temporal resolution. 3. What are the differences in neural processing that correlate with differences in performance? This may include elucidating the neural substrates underlying differences in olfactory performance between groups such as men and women. In addition, functional neuroimaging may be used to probe patients with abnormalities of the olfactory system, be it anosmia, hyposmia, parosmia, olfactory hallucinations, or hyperosmia. In this realm fMRI is particularly useful because of the structural discrimination it provides, as well as its sensitivity to pathological alterations in anatomy. 4. Functional neuroimaging can be used to probe questions regarding higher olfactory function, such

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as what is the structural and functional neuroarchitecture that underlies the special interaction between odor and mood, and odor and memory. Questions such as this could best be addressed using either fMRI or PET.

III. TECHNICAL CONSIDERATIONS IN FUNCTIONAL NEUROIMAGING OF HUMAN OLFACTION The application of functional neuroimaging to the study of human olfaction consists of an unfortunate compounding of the technical difficulties inherent to both the field of olfaction and the field of neuroimaging. A.

Stimulus Generation

Generating olfactory stimuli is a complex task as it is, but this complexity is increased in the MRI environment, where one cannot introduce ferrous materials due to the strong magnetic field. Thus, valves, canisters, tubing and piping, etc., that are part of the olfactory stimulus–generating apparatus must all be either nonferrous or at a distance from the subject. Aside from this added complication, the rules that apply to stimulus generation in the imaging environment are similar to those in other experimental designs (see Chapters 10 and 11) (see also Prah et al., 1995). Because imaging studies usually consist of comparing two olfactory conditions (e.g., odorant versus no odorant, high concentration versus low concentration, etc.), it is imperative that the difference between these conditions is restricted to the process of interest alone. Any additional sources of variance, such as valve noise, airflow rate change, airflow temperature change, humidity change, that are associated with one condition and not the other may contaminate the imaging result. (A detailed description of simple, yet sufficient, olfactometers for fMRI studies can be found in Sobel et al., 1997, and Lorig et al., 1999.) The necessary quality and accuracy of the stimulusgenerating equipment to be used in any given study is related to the type of questions asked. For example, considering the questions previously described, one may study the emotional response to an odor using relatively simple methods of odorant generation; however, to study the temporal aspects of the neural response to odors, one must use a higher quality stimulus-generating device. In this regard, one of the major current controversies regarding the methodology of imaging human olfaction is whether subjects should sniff or not sniff during the experiment. The advantages of not sniffing are in reducing the risk of

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motion-related artifacts as well as reducing the non–odorant-related sources of activation, such as the somatosensory or motor components of the sniff. In turn, natural olfaction consists of sniffing. The sniff is in itself part of the olfactory percept, modulating patterns of activity in olfactory cortex, as well as the quantity (Laing, 1983; Sobel et al., 2000a) and type (Sobel et al., 2000b) of odorant sampled. Thus, to fully characterize olfactory processing, ideally both sniffing and nonsniffing paradigms should be employed, with close monitoring of respiration. In the nonsniffing experiments, such monitoring is necessary to assure that odorants did not induce an unwanted automatic respiratory response (Jackson, 1976; Warren et al., 1992, 1994), and in sniffing experiments such monitoring is necessary so that the sniff itself can later be factored out in the statistical analysis of the functional imaging data. A procedure that achieves this is as follows: subjects are instructed to sniff by either a visual or auditory cue that appears at a predetermined rate. Subjects are further instructed to maintain their sniff for the duration of the projected message or tone, typically set to 800 msec. Thus, sniffing can be equalized for all conditions, e.g., sniffs of odorant versus sniffs of no odorant. Finally, the olfactometer can be coupled to a pneumatotachograph offering a precise measurement of airflow rate, duration, and volume of each sniff during the entire experiment. This enables both post hoc and on-line assurances that the sniffs were indeed equal across all conditions and can thus be subtracted as a factor in later statistical analysis. In sniffing designs, the use of a bite bar to prevent sniff-related head motion is imperative. An alternative odor presentation paradigm, suggested by Cerf-Ducastel and Murphy (2001), avoids many of the logistical challenges presented by using an olfactometer in the MRI environment. They presented odorants in aqueous solution to the oral cavity and showed that cortical activation via retronasal olfaction is qualitatively similar to activation due to airborne stimuli. This method may be especially suitable for studying flavor, or for direct comparisons of olfaction and gustation. B.

Choice of Odorants

Considering that functional neuroimaging studies consist of prolonged exposure to odorants, odorants with minimal toxicity should be chosen. To this end, employing odorants that have been safely used for extensive periods of time in human psychophysical studies would seem preferable. The latter strategy will also readily enable comparison of results across studies and methods. Studies of areas sensitive to rapid habituation must also take into account the fact that long temporal windows may

obscure transient activity at the beginning of odorant exposure. These problems are reduced if multiple odorants are used during a single scan period, but this method can prove problematic due to inconsistencies in psychoperceptual characteristics of different odorants. Adaptation may still occur even with multiple odorants in the form of crossadaptation, whereby exposure to one odorant decreases the response to a second odorant (Cometto-Muñiz and Cain, 1995; Köster and de Wik, 1991) (see Chapter 10). The advantage of a long temporal window is that precise timing of odorant presentation is less important. While ERP, MEG, and some fMRI paradigms require sophisticated olfactometer presentation, less precise stimulus presentation techniques can be used with PET. The sensation of smell is often the result of stimulation in more than just the olfactory nerve. For example, trigeminal nerve stimulation also contributes to the sensation of some smells (reviewed in Chapter 47 of this volume). For this reason, and in order to elucidate the separate neural substrates that may underlie trigeminal versus pure olfactory perception, it is imperative that experimenters be aware of the extent of trigeminal stimulation potentially present in the stimuli used. Doty and colleagues (1978) studied congenitally anosmic patients to identify stimuli that can be detected by these patients and thus presumably have trigeminal components. Yousem and colleagues confirmed the results of this behavioral approach using functional neuroimaging. When they tested congenital anosmics with fMRI and olfactory nerve stimulants, no activation was seen, and when anosmic patients with Alzheimer’s disease were given olfactory nerve odorants, again no stimulation was observed (Yousem et al., 1997a, 1998). C.

Imaging Parameters

It is an unfortunate coincidence that the olfactory regions of the brain, namely the ventral portions of the temporal and frontal lobes, are the regions most susceptible to artifact in fMRI. This is because the MRI image is highly susceptible to distortion at sharp borders of signal intensity. A common source for such sharp borders is the interface of tissue to air-filled cavities, and such cavities abound around the olfactory regions of the brain (i.e., the paranasal sinuses, the petrous apices, and the temporal bones). Various strategies can be employed to address this common source of artifact (Yang et al., 1997), and here we will describe only a few of the very basic imaging parameters that are conducive to minimizing such artifacts. First, it is advised to use as thin a slice as possible during acquisition of the data. This reduces the probability of averaging areas of very different signal intensity within the same voxel.

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minimizing artifacts in the ventral temporal region are only the very first basic approach and should be augmented by various methods of image postprocessing (e.g., Yang et al., 1997). Another technique for performing fMRI for olfaction has been advocated by Levy and colleagues. They use a spoiled T1-weighted gradient echo (FLASH) scan utilizing gadolinium injections to demonstrate the increased blood flow to the activated area. This is analogous to a perfusion scan; however, the T1-weighting, ultra-short echo time, and spoiling reduces the susceptibility artifact that interferes with BOLD fMRI at the skull base (Levy et al., 1997, 1998 a,b, 1999). The limitation of this technique is that very few slices are allowed per acquisition due to longer scan times when not using echoplanar or spiral imaging. Regarding comparison of imaging methods, it is important to note that PET is not susceptible to these artifacts in the ventral portions of the brain. D.

Figure 1 The olfactory slice: the oblique orientation at which we recommend obtaining functional MR data from the olfactory system. Obtaining data at this orientation reduces ventral-temporal–related artifacts. The data can then be displayed at this, or any other, orientation.

This is advised despite the signal-loss attributed to using thinner slices. Second, it has proven helpful to acquire the data at an oblique orientation such as seen in Figure 1. For a standard coronal or horizontal acquisition to contain the olfactory regions, it would necessarily also contain the paranasal sinuses and temporal bone. By contrast, this oblique orientation enables covering the entire olfactory regions, with only minimal coverage of these bony and airfilled structures. An added advantage of this slice orientation is that both primary and secondary olfactory cortex appears within the same slice, which is very convenient for both analysis and presentation. Finally, what has proved as helpful in some efforts at imaging the olfactory regions is using a specific method, or pulse sequence, for sampling the three-dimensional imaging space (referred to as k-space). Specifically, the commonly used fMRI method referred to as echo-planer imaging (EPI) samples the imaged space in a zigzag trajectory. An alternative method, referred to as the spiral trajectory, or spiral sequence (Glover and Lai, 1998), samples the imaged space in a spiral fashion. For reasons beyond the scope of this chapter, the spiral sequence appears to be less susceptible to the artifacts typical to the ventral temporal region. The above strategies for

Experimental Design and Statistical Analysis

There are currently two standard designs for functional neuroimaging experiments: the block design and the single-trial design (also referred to as event-related design) (Fig. 2). The block design consists of extended alternating epochs of a control and experimental condition, for example, an odorant condition versus a no-odorant condition. This type of design is best suited for methods with relatively low temporal resolution, such as PET. A typical PET study may consist of two blocks, each lasting a minute. A typical fMRI block design study may consist of blocks ranging between 20 and 40 seconds in duration that are repeated four to six times within an experiment. In contrast, a single-trial design consists of random presentations of brief experimental and control epochs. This type of design is best suited for methods with higher temporal resolution such as MEG or fMRI. A typical fMRI singletrial study may consist of 40 experimental and 40 control stimuli, with stimuli presented at 16-second intervals in a randomized fashion. The single-trial design has several advantages over the block design in the study of human olfaction. In the single-trial design, one negates the potential expectation of an upcoming experimental (odorant) condition, and more importantly, one can minimize the habituation that is an inevitable occurrence during a prolonged odorant epoch in a block design experiment. Once functional imaging data are obtained using either of the above experiment designs, they are subjected to statistical analysis. This analysis typically consists of parsing the data into a matrix of voxels representing the anatomical area that was imaged. The time course of activity, or signal, in each such voxel is then correlated with the time course of the task

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Figure 2 Typical time-course of odorant generation. Whereas a block design experiment consists of alternating extended epochs of odorant presence and odorant absence, the single trial design consists of random presentations of brief odorant or no-odorant epochs.

(e.g., odorant presence vs. odorant absence). Voxels in which the frequency of the signal is significantly correlated with the frequency of the task are considered to be involved in the neural processing underlying that task. For purposes of display, such voxels are commonly color-coded and overlaid on a gray-scale anatomical image in order to depict the region of activity. In cases where only one experimental and one control block are defined, as in most PET studies, the above process amounts to subtracting the activation pattern in the control block (e.g., no-odorant) from activation in the experimental block (e.g., odorant), in order to reveal activation related to the process of interest alone. The analysis of these data may be performed for individual subjects, individual stimuli, or groups of subjects and stimuli, and several different software packages are available as share-ware for the performing of such analysis. It is beyond the scope of this chapter to exceed this simple overview on how functional neuroimaging data are analyzed. For a more complete review of this topic, the interested reader is referred to Bandettini et al. (1993), Friston et al. (1994, 1996), Buckner et al. (1996), Zarahn et al. (1997), Aguirre et al. (1997), Josephs and Henson (1999), and the references therein.

IV.

STATE-OF-THE-ART

In a comprehensive chapter on the neuroanatomy of the human olfactory system, Joseph Price (1990) reviews the olfactory structures in an order that follows the neuroanatomical flow chart of olfactory processing, starting from peripheral and extending to central structures. Here we will follow the same neuroanatomical flow chart, reviewing functional, rather

than structural, neuroimaging. We will concentrate on studies addressing properties of basic sensory processing. For functional neuroimaging studies of higher olfactory processing, such as odor memory or semantic processing of odors, we refer the interested reader to the work of Royet et al. (1999), Dade et al. (1998, 2001), and Savic et al. (2000). For a review on functional imaging of taste, we refer the interested reader to the work of Small et al. (1997 a,b, 1999). A.

Olfactory Epithelium and Bulbs

When an odorant enters the human nasal passages, it travels a distance of about 7 cm, crosses a mucous barrier, and is then transduced at olfactory receptors that line the upper nasal cavity on a sheet termed the olfactory epithelium (see Chapter 2). The odorant-induced neural signal then progresses along the processes of bipolar sensory neurons to the first synapse within the olfactory bulb. The location of the human olfactory epithelium and bulbs amid air-filled cavities, combined with the small size of the olfactory bulbs, renders them inaccessible to current human functional neuroimaging methods. We have, in fact, tried several times to acquire functional data from the olfactory bulbs with fMRI, at both 1.5T and 3T magnetic field strengths, but have not succeeded in obtaining an artifact-free image. For this reason, under this heading we will deviate from the main scope of this review, namely human studies, and report on some imaging studies of the olfactory bulb in animals. Yang and colleagues (1998) used a 7T fMRI magnet to study odorant-induced activation in the olfactory bulbs of rats. The 7T field was utilized to maximize spatial resolution that was set at 220  220  1000 m. Following

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stimulation with iso-amyl acetate, these authors reported highly significant activations at the level of individual, or small groups of, glomeruli. These activations were reproducible both across and within animals. These findings corroborate previous findings with other methods suggesting that there is a spatial component to the encoding of odors at the level of the olfactory bulb (reviewed in Buck, 1996). This notion has recently received further support in an imaging study by Rubin and Katz (1999). These authors used optical imaging in rats to find specific patterns of glomerulus activity that relate to specific odorant-features and odorant-concentrations. Optical imaging, however, is an imaging method not currently applicable to noninvasive study of adult humans, and is therefore not reviewed in this chapter. (For more on optical imaging, see Grinvald, 1992; Malonek and Grinvald, 1997. For more on the application of optical imaging to study the cortical processing of olfaction in rodents, see Litaudon et al., 1997.) B.

Primary Olfactory Regions

The primary olfactory cortex is currently defined as the regions that receive direct projections from the olfactory bulb. 1.

Piriform Cortex

Price (1990) refers to the piriform cortex as “the largest and most distinctive olfactory cortical area in most mammals.” The piriform cortex is at the end of the lateral olfactory tract, inhabiting a small portion of both frontal and temporal lobes at the ventral junction of the two. Paradoxically, several attempts to visualize odorantinduced fMRI activation in human piriform cortex have yielded, at best, only weak activations (e.g., Fulbright et al., 1998; Koizuka et al., 1994; O’Doherty et al., 2000; Sobel et al., 1998a, 1999; Yousem et al., 1997, 1999 a,b). MEG studies have also not reported piriform cortex activation (Kettenmann et al., 1996; 1997; Kobal and Kettemann, 1999; Sakuma et al., 1997). PET studies have had varying levels of success at imaging odorant-induced activation in piriform cortex, ranging from significant activation (Fig. 3, see color plate) (Dade et al., 1998; Savic et al., 2000; Zatorre et al., 1992) to minimal activation (Zald and Pardo, 1997) to no activation at all (Dade et al., 2001; Rouby et al., 2000). These differences in the results with PET are likely not related to differences in methodology, as most of these attempts at imaging piriform cortex activation with PET are in fact out of the careful work of the same laboratory, headed by Robert Zatorre and Marilyn Jones-Gotman in Montreal. Recently, Sobel and colleagues have shed some light on this paradox regarding the inability to record odorant-

Figure 3 Odorant-induced activation revealed by PET. Odorantinduced activation is seen bilaterally in the region of the piriform cortex and unilaterally in the right orbitofrontal cortex. (Image courtesy of R. Zatore, M. Jones-Gotman, and colleagues.) (See color insert.)

induced activation in piriform cortex using fMRI (Sobel et al., 2000c). Initially, they found that the somatosensory stimulation induced by sniffing nonodorized air was sufficient in itself to induce significant activation in the ventral temporal regions (Sobel et al., 1998a). They then hypothesized that this sniff-delineated region is primary olfactory cortex and that the effect of odorants may be measurable within this sniff-delineated region (Sobel et al., 2000c). To test this possibility, subjects were first scanned while sniffing, and a region of interest (ROI) was drawn out of the sniff-activated region. Activity in this region was then measured in separate scans with odorants. It was found that odorants induce a rapid onset and short-lived response in about 8% of this region—an area that appeared to correspond well with the location of the human piriform cortex, as suggested in recent atlases (Mai et al., 1997). This response decreased rapidly throughout each individual 40-second odorant epoch of a block-design study and also decreased in initial amplitude from block to block over a 4-block experiment (Fig. 4). By using statistical measures that took this habituation into account, they were able to consistently and reliably measure fMRI odorant-induced activation in piriform cortex (Fig. 5; see color plate) (Sobel et al., 2000c). Considering that event-

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Figure 4 Time course of odorant-induced activity in human piriform cortex: the averaged time course from piriform cortex (8% of the region responsive to sniffing alone) in 8 subjects exposed to the odorant vanillin. The response shows a rapid habituation within the first 40 seconds of odorant presence as well as a continued habituation throughout the 320 seconds of the experiment.

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related experimental designs could minimize habituation, the above findings suggest that an event-related design would best fit for measuring odorant-induced activation in piriform cortex. Recently, Poellinger et al. (2001) probed temporal differences that may underlie habituation. Using an event-related fMRI design, they showed that piriform cortex, entorhinal cortex, and the amygdala exhibit short phasic increases in signal followed by a prolonged decrease in signal below baseline. In contrast, the orbitofrontal cortex exhibited a sustained increase in activity over nearly 60 seconds of odor presentation. The rapid habituation of odorant-induced activity shown in piriform cortex explains why it was not previously evident in many fMRI and PET studies, but why was such activity evident in yet other PET studies? One possibility, discussed in detail by Sobel et al. (2000c), is related to the differences in statistical analysis commonly used in PET and fMRI studies. The PET analysis may lend significant weight to the early transient odorantinduced activity, whereas commonly used fMRI analysis packages may have obscured this phenomenon. A second potential source for the difference between different PET studies may be related to subject behavior. In the PET studies, subjects knew which block would be an odorant block and which block would contain a diluent only. It is quite conceivable that when knowingly presented with a

Figure 5 Odorant-induced activation revealed by fMRI. Odorant-induced activation in piriform cortex and additiona olfactory regions. The activation is a composite of 8 subjects stimulated with the odorant vanillin. Activation was analyzed using statistical methods that took into account the rapid habituation in piriform cortex. (From Sobel et al., 2000c.) (See color insert.)

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foil, subjects made less of an effort to scan the olfactory environment or, in other words, sniff. As previously shown (Sobel et al., 1998a, 2000c), sniffing alone is sufficient to induce activity in the ventral temporal regions, and therefore it could be that the difference between the odorant and no-odorant conditions was in fact partially a difference in sniffing between conditions in these studies. Although the above suggestions may indeed underlie the between-study variability in piriform cortex activation, it is still nevertheless evident that the rules underlying piriform cortex activity are complex. This activity appears to significantly change throughout continued stimulation, as well as change as a result of previous experience, as suggested in other animals as well (Stopfer and Laurent, 1999). Now that we can consistently image such activity using either the method that accounts for habituation (Sobel et al., 2000c) or the event-related design in fMRI, we should be able to delineate human piriform cortex in vivo as well as better characterize the rules underlying piriform cortex activity. 2. Anterior Olfactory Nucleus and Olfactory Tubercle These two small structures that receive direct projections from the olfactory bulbs are not well defined in the human (Price, 1990). However, significant odorantinduced activation in the area of these structures is consistently measured (Sobel et al., 2000c). Figure 6 (Fig. 6; see color plate) depicts activation induced by the odorants vanillin, decanoic acid, propionic acid, and valeric acid in composite images of eight subjects. All four odorants induced significant activation in this region. Although it would appear that this activation is primarily in the region of the anterior olfactory nucleus, one cannot structurally delineate these bodies on the MRI image and therefore cannot rule out the possibility that the source of this activation was in fact the olfactory tubercle or other neighboring structures. These activation images suggest that these regions may play a considerable role in processing olfactory information in humans, in spite of the fact that they are not very well defined in the human from a structural and/or cytoarchitectural point of view. What role these structures play in human olfactory processing, and how this role is carried out, remains unknown. 3. Amygdala The anterior cortical nucleus of the amygdala and the periamygdaloid cortex receive direct projections from the olfactory bulbs (Price, 1987). Odorant-induced amygdala activation has indeed been reported in both PET and fMRI

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studies (e.g., Birbaumer et al., 1998; Royet et al., 2000; Zald and Pardo, 1997). The amygdala plays a major role in processing of emotionally significant stimuli. For this reason, Zald and Pardo used an aversive sulfide cocktail to study the odorant-induced response in the amygdala. The sulfide odorants produced significant bilateral amygdala activation (Fig. 6; See color insert). By contrast, pleasant odorants did not induce as significant activation in the amygdala (Zald and Pardo, 2000). This dissociation of amygdaloid activation induced by unpleasant but not pleasant odorants may be related to the findings that seizures originating in the amygdala often induce olfactory hallucinations of unpleasant but not pleasant odors (Andy, 1967; Chitanodh, 1966). The latter relationship, however, must be addressed with caution, as it remains to be determined if the increased amygdala response was related to odorant hedonic value per se, unconfounded by odorant intensity or odorant species. The amygdala activation induced by unpleasant odorants shows a tendency towards asymmetry, whereby increased unpleasantness is associated with increased left amygdala activation as measured in right-handed subjects. This asymmetry was evident in a PET study (Zald and Pardo, 2000) (Fig. 7; see color plate) and in a pilot fMRI study (Prabhakaran et al., 1999), but not in an fMRI study by Birbaumer et al. (1998) that compared odorant-induced amygdala activation in social phobics and nonphobic controls. 4.

Entorhinal Cortex

The rostral portion of the entorhinal cortex receives a direct projection from the olfactory bulbs (Price, 1990). Reporting on odorant-induced activation in the entorhinal cortex is somewhat neglected, we think for the following reason: often, activation in the amygdala will also cover a portion of neighboring entorhinal cortex. However, because the amygdala is usually a specific target of interest, researchers tend to report on “amygdala and neighboring cortex.” The same is true for activation that occurs in the hippocampus, which also often covers parts of the entorhinal cortex. Thus, although entorhinal cortex odorant-induced activation is commonly seen (as reported in Sobel et al., 1998a; Zald and Pardo, 2000), it is not carefully analyzed. In this, we as an olfactory imaging community are falling into an unfortunate, yet common, trap. By concentrating our efforts on some regions where we expect something important is happening vis-á-vis olfactory processing (e.g., amygdala), we may be overlooking areas that are perhaps less trendy but nevertheless are of equal or greater importance in olfactory function.

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Figure 6 Odorant-induced activation in the region of the anterior olfactory nucleus and olfactory tubercle. The top row shows composite images of activation from 8 subjects in slice 5 of the olfactory acquisition (see Fig. 1). All odorants induced significant activation in the region of the anterior olfactory nucleus and olfactory tubercle (green arrows). To assist in localization of this, the average centroid of the activation was transferred to the corresponding point on a standard coronal plane acquisition, using a threedimensional cross-referencing program (red circle). The centroid of activation was within a coronal plane 2 mm anterior to the anterior commisure, in the region of the anterior olfactory nucleus (AON), bordering with the olfactory tubercle (Tu), the medial tip of the frontal portion of the piriform cortex (PirT), and the lateral tip of the diagonal band nucleus (Db). The activated region occasionally spanned down to the uncus (Un) and occasionally down to the area of the basomedial amygdaloid nucleus. Additional activations seen in this slice are in the claustrum (Cl), peri-insular region (Ins), cingulate gyrus (Cg), and inferior (IFG), and middle (MFG) fontal gyri. (See color insert.)

C.

Secondary Olfactory Regions

1. Hippocampus The hippocampus receives an olfactory input from the entorhinal cortex (Price, 1990). The hippocampus has been the focus of several human neuroimaging studies of memory

(Brewer et al., 1998; Gabrieli et al., 1997), but no neuroimaging studies of olfaction have focused on hippocampal activity. Odorant-induced hippocampal activity, however, has been reported in several human neuroimaging studies using either PET, SPECT, or fMRI (e.g., Malaspina et al. 1998; Savic et al., 2000; Small, 1997; Sobel et al., 2000c)

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induced by the compounds remains unclear. The existence of human pheromones is still very controversial (Sobel and Brown, 2001) (see Chapter 17), but the compounds used by Savic et al. clearly differ from more traditional odorants in the pattern of hypothalamic activation they induced. 3.

Figure 7 Odorant-induced activation in the amygdala. PET activation induced by unpleasant odorants in the amygdala and neighboring regions. Activation can be seen in the amydala (Amg) bilaterally and in the left claustrum (Cl) and insula (I). (Image courtesy of D. Zald and colleagues.) (See color insert.)

2.

Hypothalamus

The hypothalamus receives extensive olfactory input, most prominently from the piriform cortex and anterior olfactory nucleus, and also from the amygdala (Price, 1990). Electrophysiological studies in monkeys suggest narrowly tuned odorant-responsive cells in the hypothalamus (Takagi, 1986; Tazawa et al., 1987). Hypothalamic odorant-induced activation has been reported in some human neuroimaging studies (e.g., Rouby et al., 2000; Savic et al., 2001; Sobel et al., 1999). Rouby et al. (2000) were the first to use imaging to address the specific role of the hypothalamic relay in human olfactory processing. Using PET, these authors measured greater hypothalamic activity during pleasantness judgments than during intensity judgments of the same odorants. The authors speculate that the hypothalamus may be involved in the affective processing of olfactory information that requires access to information about internal state. Using PET, Savic et al. (2001) recently measured robust sex-specific activation of the hypothalamus. Smelling an androgen-like compound produced activation in the hypothalamus of women, but not men; smelling an estrogenlike compound activated the hypothalamus of men, but not women. This surprising pattern of activation is likely due to the nature of the odorants used. Both compounds are considered by some as putative human pheromones, and the hypothalamus mediates a wide variety of so-called pheromonal effects in other animals, but the specific effect

Thalamus

The thalamus receives olfactory projections from the piriform cortex, periamygdaloid cortex, entorhinal cortex, and olfactory tubercle (Price, 1985). Most of these projections are thought to synapse onto the mediodorsal thalamic nuclei, and some onto the submedial nucleus. In monkeys, odorantinduced electrophysiological activity has been recorded from both of these thalamic sites (Benjamin and Jackson, 1974; Russchen et al., 1987). In contrast to recordings from the hypothalamus, odorant-induced responses in the thalamus appear to be broadly tuned (Yarita et al., 1980). Such thalamic activation is seen in human functional neuroimaging studies (e.g., Savic et al., 2000; Sobel et al., 2000c), but this activation has yet to be carefully characterized. It is, however, our impression, that the thalamic odorant-induced activation in humans is medial, yet more anterior than that recorded in monkeys (Sobel et al., 1999). This impression awaits the scrutiny of a more careful specific characterization of thalamic odorant-induced patterns of activation in humans. 4.

Orbitofrontal Cortex

The orbitofrontal cortex is considered the main site of secondary olfactory processing in humans. This cortical region, located on the ventral portion of the frontal lobe, in fact evolved as part of a series of concentric rings around primary olfactory cortex (Carmichael et al., 1994; Zald and Kim, 1996a,b). The orbitofrontal cortex receives both direct and indirect input from primary olfactory cortex. Direct projections from piriform cortex form a transsynaptic input to the posterior orbitofrontal region (Price, 1990), and indirect projections from primary olfactory cortex reach the orbitofrontal cortex via the thalamus (Yarita et al., 1980). In monkeys, cells with selective odorant-specific responses have been characterized in orbitofrontal cortex (Critchley and Rolls, 1996; Rolls et al., 1996; Tanabe et al., 1975), and in humans, orbitofrontal lesions lead to impairments in odorant discrimination and identification (Jones-Gotman and Zatorre, 1988; Zatorre and Jones-Gotman, 1991). In contrast to the previously described difficulties in imaging odorant-induced activation in primary olfactory cortex, odorant-induced activation in orbitofrontal cortex has been routinely reported in both PET and fMRI studies. The localization of activation within the orbitofrontal gyri

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is quite consistent across these studies and methods (reviewed in Zald and Pardo, 2000; Zatorre and JonesGotman, 2000). As stressed by Zald and Pardo (2000), the orbitofrontal cortex should not be treated as an homogeneous region vis-á-vis olfactory processing. Electrophysiological studies in monkeys suggest a distinction between lateral and central olfactory zones in posterior orbitofrontal cortex (Takagi, 1986; Tanabe et al., 1975; Yarta et al., 1980). Human neuroimaging studies have reported odorant-induced activation in various parts of the orbitofrontal cortex, most commonly in the intermediate and posterior orbital region, and in the lateral orbitofrontal gyrus (Sobel et al., 1998a, 2000c; Zald and Pardo, 1997), and less commonly in the medial orbitofrontal region (Royet et al., 1999; Small et al., 1997). Review of the literature suggests a trend whereby tasks related to olfactory memory induce activation more medially, and tasks related to olfactory hedonics induce activation more laterally, but this distinction is far from concrete, and more work is necessary in order to elucidate which orbitofrontal area is primarily involved in which olfactory task. Lesions to the right orbitofrontal cortex lead to greater olfactory impairment than lesions to the left orbitofrontal cortex (Jones-Gotman and Zatorre, 1993). This functional asymmetry implied by lesion findings is indeed reflected in patterns of odorant-induced activation as measured with neuroimaging. Specifically, activation is usually more significant in and occurs in a larger portion of the right than left orbitofrontal cortex. This asymmetry was first shown in the pioneering PET study reported by Zatorre and colleagues in 1992, and later replicated with fMRI (Royet et al., 1999; Sobel et al., 1998a). Similar asymmetry has been reported in additional frontal regions (Savic and Gulyas, 2000; Yousem et al., 1999a,b). Lesion studies have also suggested greater right than left orbitofrontal cortex involvement in olfactory memory tasks (Jones-Gotman and Zatorre, 1993), and this is indeed also reflected in greater right than left activation in olfactory memory neuroimaging studies (Dade et al., 1998; Savic et al., 2000). Some evidence suggests that the asymmetry in orbitofrontal cortex activation seen in most imaging studies may be odorant-specific. Using PET, Zald and colleagues found that odorants with strong negative hedonic characteristics appear to break down the functional coupling between the right and left orbitofrontal cortex (Zald et al., 1998) and induce greater activation in the left than right orbitofrontal cortex (Royet et al., 2000; Zald and Pardo, 1997). A similar suggestion has recently been made for additional frontal regions as well (Fulbright et al., 1998). Although the possibility of hedonic-based laterality of olfactory activation is appealing, Zald and Pardo (2000) later noted that the

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same group of subjects exhibited greater left than right orbitofrontal activation when exposed to pleasant odorants as well, suggesting that the leftwards shift in orbitofrontal cortex activation that they witnessed earlier is not solely the result of the hedonic value of the odorants used. Furthermore, Rouby et al. (2000) used PET to compare activation induced by odorant pleasantness ratings versus odorant intensity ratings and also found predominantly right orbitofrontal activation for both types of olfactory assessments. The only significant difference in brain activation induced by these two tasks was in the hypothalamus, where greater activation was measured following pleasantness judgments. Royet et al. (2001) asked subjects to make judgments about the presence (odor detection), intensity, pleasantness, familiarity, and edibility of various odorants. They found that left orbitofrontal cortex activity increased significantly during the pleasantness and familiarity tasks. Right orbitofrontal cortex activity increased in all five tasks but was highest during familiarity judgments and lowest during the detection task. Considering the high degree of variability regarding orbitofrontal cortex laterality, the rules underlying asymmetry remain unclear. The orbitofrontal cortex is also the site of extensive cross-modal integration (Rolls, 1996). An example of the orbitofrontal integration of smell and taste is the electrophysiological findings in monkeys suggesting that orbitofrontal neurons decrease their response to the odors Women

Men

Subject group

Left frontal voxels activated

Right frontal Left temporal voxels voxels activated activated

Female Male Ratio female to male

157 19 8.3

730 152 4.8

303 55 5.5

Right temporal voxels activated 465 31 15.0

Figure 8 Odorant-induced activation is greater in women than in men. The composite activation of maps of eight right-handed women are compared with those of eight right-handed men, given the same olfactory stimuli in an fMRI experiment at 1.5 Tesla. The women’s group-averaged activation maps showed up to eight times more activated voxels than did those of men for specific regions of the brain. The difference was most striking in the right temporal (peri-insular) regions. (From Yousem et al., 1999b.) (See color insert.)

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of food that had been eaten to satiety (Critchley and Rolls, 1996). O’Doherty and colleagues (2000) conducted a replication of this finding in humans using fMRI. They found that the activation induced by the odor of banana, but not by the odor of vanillin, was significantly decreased after eating banana to satiety. The site of decreased activation appeared to be variable within the orbitofrontal region. Odorant-induced activation has been consistently witnessed in additional regions within the frontal lobe.

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These regions include the frontal polar area (Dade et al., 2001), superior frontal gyrus (Dade et al., 2001; Fulbright et al., 1998; Royet et al., 1999; Sobel et al., 2000c; Yousem et al., 1999a), middle frontal gyrus (Dade et al., 2001; Fulbright et al., 1998; Malaspina et al. 1998; Sobel et al., 2000c), and inferior frontal gyrus (Dade et al., 2001; Fulbright et al., 1998; Malaspina et al. 1998; Royet et al., 1999; Sobel et al., 2000c), which was also activated in response to a subthreshold stimulus (Sobel et al., 1999).

Figure 9 Odorant-induced activation is greater in young than in older subjects. A full-brain acquisition of a young (top) versus older (bottom) subject, given the same olfactory stimuli in an fMRI experiment at 1.5 Tesla. Significantly greater activation is seen in the younger versus the older subject. (From Yousem, 1999a.) (See color insert.)

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The specific role that each of these different frontal regions plays in olfactory processing remains unclear. Interestingly, Yousem and colleagues (1999b) reported that the extant of frontal odorant-induced activation was eight fold greater in women than in men (Fig. 8; see color plate) and also age-dependant—greater in young than in old adults (Fig. 9; see color plate) (Yousem et al., 1999a). Although this can be taken to suggest that the role frontal regions play in olfaction may be related to the advantage in olfactory performance often attributed to women over men (e.g., Doty et al., 1985), it remains unclear if this lesser frontal activation represents less frontal processing of the same input or is simply a reflection of lesser input from the peripheral olfactory structures. 5.

Insula

In the rat, a direct projection from piriform cortex to agranular insular cortical areas has been described (Price, 1985). It is also assumed that olfactory information reaches the insular area via a thalamic relay (Price, 1990). Electrical responses to olfactory stimuli have been recorded from insular areas in monkeys (Takagi, 1986), and odorantinduced insular activation is indeed commonly measured in functional neuroimaging studies of human olfaction using both PET and fMRI (Fulbright et al., 1998; O’Doherty et al., 2000; Savic et al., 2000; Sobel et al., 2000c). Activation has been reported in both the anterior and posterior portions of the insula, and in some cases activation has been reported as borderline between the insula and claustrum (Savic et al., 2000; Zatorre et al., 1992). The role of the insular region in olfactory processing remains unknown, but it may be related to the integration of olfactory and other sensory information. Taste, for example, has been shown by functional neuroimaging to also be represented in the insular region (Kinomura et al., 1994; Small et al., 1997, 1999). Fulbright et al. (1998) found that the extent of fMRI activation in the left insula was correlated with the hedonic ratings of the odorant isovaleric acid whereby activation was greater when subjective ratings of unpleasantness were more intense, thus suggesting an insular role in the hedonic assessment of odors. The perisylvian region has also been noted by Yousem et al. (1999a,b) to be a large area of odorant-induced activation, which again shows gender and age variability (Fig. 8). D. Regions Not Previously Associated with the Olfactory System Odorant-induced activation has been consistently recorded with both fMRI and PET from several brain regions not previously associated with olfactory processing (in addition to the various frontal lobe regions previously

described). These include various parietal lobe activations (Dade et al., 2001; Malaspina et al. 1998; Royet et al., 1999; Sakuma et al., 1997; Savic et al., 2000; Yousem et al., 1999a), the precentral gyrus (Dade et al., 2001; Fulbright et al., 1998; O’Doherty et al., 2000), superior temporal gyrus (Kettenmann et al., 1996; Kobal and Kettenmann, 2000; Malaspina et al. 1998; Sakuma et al., 1997), inferior temporal gyrus (Malaspina et al., 1998), cingulate gyrus (Dade et al., 2001; Fulbright et al., 1998; Levy et al., 1997; O’Doherty et al., 2000; Royet et al., 1999; Savic et al., 2000; Sobel et al., 2000c; Yousem et al., 1999), occipital lobe (Royet et al., 1999; Savic et al., 2000; Yousem et al., 1999b), and cerebellum (Qureshy et al., 2000; Savic et al., 2000; Small et al., 1997; Sobel et al., 1998b; Yousem et al., 1997). The odorant-induced activation in most of these regions has not been carefully characterized; thus, it remains unclear to what extent these activations are genuinely odorant-induced or perhaps related to additional resources supporting performance in olfactory tasks. A case in point may be the activation seen in cingulate gyrus, which more likely reflects the attentional demands generated by performing any task, olfactory or other, rather than odorants per se. One such region not previously associated with olfactory processing that has received some direct experimental attention is the cerebellum, as discussed below. 1.

Cerebellum

The cerebellum is a large brain structure located at the back of the brain that in the human contains more neurons than the rest of the brain combined (Williams and Herrup, 1988). The cerebellum has classically been considered as primarily a motor control organ (Ito, 1984) (for alternative views, see Bower, 1997; Ivry, 1997). Cerebellar functions in visual- and auditory-related tasks have been extensively described (Huang and Liu, 1991; Stein and Glickstein, 1992). Recently, Qureshy et al. (2000) provided evidence that the cerebellum is also active during cognitive processes in olfaction. In one task subjects were asked to name odors (naming task), and in another subjects were asked to compare the presented odorant with a previously memorized odorant to determine if the odors were the same (matching task). Both tasks activated the right posteromedial and left anterolateral portions of the cerebellum. As olfaction is a sensory process largely dependent upon the fine motor process of sniffing (Laing, 1983; Le Magnen, 1945; Mozell et al., 1983; Rehn, 1978), the cerebellum may also play a role in motor control of olfaction. Sniffing plays a major role not only in transport of the olfactory stimulus (Hahn et al., 1994), but also in patterns of neural activity in primary olfactory cortex in the human (Sobel et al., 1998a, 2000c). A fine reciprocal interaction persists whereby sniff-

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ing strategy and timing modulate odorant intake and, in turn, odorant intake content modulates further sniffing. For example, in response to increasing odorant concentration, there is a decrease in sniff volume (Laing, 1983; Sobel et al., 2000a; Youngentob et al., 1987). Cerebellar involvement in respiration (Colebatch et al., 1991; Mansfeld and Tyukody, 1936) suggests that sniff-motor/sensory circuits may be in part controlled by the cerebellum. Thus, considering that odor content affects sniffing, odor content information may also be relayed to the cerebellum. Preliminary reports using fMRI, suggesting that odorants may indeed activate the cerebellum (Small et al., 1997; Sobel et al., 1997b; Yousem et al., 1997), merited a more careful examination of this possibility. To this end, we studied cerebellar patterns of activation in response to various concentrations of the pure olfactant vanillin and the strongly trigeminal odorant propionic acid (Sobel et al., 1998b) (Fig. 10, see color plate). The odorants vanillin and propionic acid both induced significant activation, primarily in the posterior lateral hemispheres. Activation was concentration dependent, greater following stimulation with higher concentration odorants (Fig. 10). By contrast, the action of sniffing nonodorized air induced significant activation in the anterior cerebellum, primarily in the central lobule. Cerebellar activation in response to odorants has now been reported in several imaging studies using both PET and fMRI (Qureshy et al., 2000; Savic et al., 2000; Small et al., 1997; Sobel et al., 1998b; Yousem et al., 1997; Zatorre and Jones-Gotman, 2000). Before these imaging studies, it was unknown whether the cerebellum contributes in any way to olfactory processing. What may be the role of the cerebellum in olfaction? The following is a working hypothesis: sniff volume is inversely proportional to odor concentration

Figure 10 Odorant-induced activation is concentration-dependent in the cerebellum. Activation induced by low, intermediate, and high concentrations of propionic acid in the lateral posterior portion of the cerebellum. Greater odorant concentration induced greater activation in this, but not other, cerebellar regions. (From Sobel et al., 1998b.) (See color insert.)

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(Laing, 1983; Sobel et al., 2000a). Maintaining this inverse proportionality calls for an accurate rapid feedback mechanism that monitors the sensory input (odor concentration) and modulates the motor output (sniff volume). Cerebellar maintenance of such feedback mechanisms has been extensively described for tactile information, as well as for vision and audition, and here we suggest the same cerebellar function in olfaction. In this capacity, the cerebellum could be subserving maintenance of the Teghtsoonian model of olfactory size constancy (Teghtsoonian et al., 1978).

V.

CONCLUDING REMARKS

A.

The Naive Observer

Current efforts at functional neuroimaging of human olfaction have measured odorant-induced activation in most of the classically defined olfactory regions of the brain and thereby have further validated this new research tool. However, visual inspection of these imaging efforts leaves one with the following striking impression: if we were to display the results of all these studies before a person who knows a lot about imaging and nothing at all about olfaction and ask this person to pick out the brain regions responsible for olfactory processing, a surprising result may occur. A naïve observer would likely pick one of several potential regions based on the extensive odorantinduced activation in those regions (e.g., the superior temporal gyrus), but probably none of those regions would be the piriform cortex, since this region exhibits only a very small activation. This state of affairs has significant implications for the imaging community regarding how it treats functional imaging results and significant implications for the olfaction community regarding how it views cortical processing of olfaction. On one hand, it is important to keep in mind that the size and significance of an activation are not a linear reflection of its importance or centrality in sensory processing. For example, there is no doubt that piriform cortex plays a major role in olfactory processing, even though it took quite an effort to even measure this activity with functional neuroimaging, and even when now consistently measured it is not very great in extent. Thus, one does not want to be an overly naïve observer. On the other hand, ridding oneself of preconceptions when approaching the results of olfactory neuroimaging studies may provide new insights into the cortical processing of olfaction. Thus, approaching the data like an uninformed, naïve observer is at times quite beneficial, a point in case being the recently revealed cerebellar role in olfactory processing.

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B.

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What Is Primary About Primary Olfactory Cortex? Time to Add a Functional Definition

Human primary olfactory cortex is generally considered to be composed of several structures and areas that inhabit the ventral junction of frontal and temporal lobes (Allison, 1954; Eslinger et al., 1982; Haberly and Price, 1978; Jones-Gotman et al., 1997; Price, 1973, 1990). A cortical region processing sensory information is generally classified as “primary” when it is the first cortical region to receive input from the peripheral receptors. In olfaction, the peripheral receptors synapse only onto the olfactory bulb. The olfactory bulb then synapses onto extensive cortical regions. For example, anterograde tracers placed into the olfactory bulb of macaque monkeys labeled axons in the following cortical areas: the anterior olfactory nucleus, piriform cortex, ventral tenia tecta, olfactory tubercle, anterior cortical nucleus of the amygdala, periamygdaloid cortex, and olfactory division of the entorhinal cortex (Carmichael et al., 1994). Additional studies, although controversial, suggest some projections from the olfactory bulb directly to frontal lobe (Cinelli et al., 1987; Shipley and Adamek, 1984). Are all these regions that receive direct bulbar projections, including the possible frontal portions, to be considered primary olfactory cortex? If so, what value for us as a field is there in this definition that encompasses almost all the cortical olfactory areas and, in fact, a rather large portion of the brain! Human primary olfactory cortex as it is currently defined relates to a rather large group of dispersed structures, some of which may have very distinct roles in olfactory processing (e.g., amygdala vs. piriform cortex). An alternative approach to classifying a cortical region as “primary” would be to add functional to structural criteria. One such potential criterion would reflect levels of processing. By this approach, primary olfactory cortex would be the cortical region responsible for the extraction of the earliest features in the olfactory stimulus space, for example, olfactory detection without any further odor-content processing. One may note that such a functional classification was possible even before the advent of functional neuroimaging, through the interpretation of human lesion outcome. The effect of ventral temporal lesions on olfactory processing, however, has proven to be quite variable (Doty et al., 1997; Eskenazi et al., 1983, 1986; Eslinger et al., 1982; Henkin et al., 1977; Jones-Gotman and Zatorre, 1988; Rausch and Serafetinides, 1975). Furthermore, human lesion case studies usually cannot offer precise localization, nor do they enable studying normal olfactory processing. In contrast, functional neuroimaging is devoid of such concerns. Thus, we could now use functional neuroimaging to redefine and localize

primary olfactory cortex in the human based on functional criteria. Such functional criteria may be a hierarchy of functional capacity, such as the levels of processing criteria described above, or in turn, such functional criteria may be a temporal hierarchy, i.e., primary olfactory cortex can be defined as those areas that first process information from the olfactory bulb, from a temporal rather than purely anatomical connectivity standpoint. Either of these potential methods for functional classification of cortical regions can now be applied to the human olfactory system by using functional neuroimaging. A first step in this direction has recently been made by Savic et al. (2000), who used PET to study activation induced by olfactory tasks hierarchically organized in terms of demands. Savic and colleagues reported a task-specific recruitment of cortical regions subserving olfactory tasks, starting from olfactory detection (amygdala-piriform, orbitofrontal, cingulate, thalamus), discrimination of odor intensity (additional recruitment of insula and cerebellum), discrimination of odor quality (additional recruitment of prefrontal cortex, frontal operculum, caudate, and subiculum), and, finally, odor recognition memory (additional recruitment of temporal and parietal regions). This PET study, however, was designed to address global levels of processing and not to specifically extract regions involved in odorant detection alone. To do the latter, one must use extremely low odorant concentrations that are above chance detection threshold, but under chance identification threshold. When using high-concentration odorants, subjects automatically also perform odorant identification, and the activation associated with detection alone cannot be discerned. We would like to here put forth a suggestion for a functional criterion defining primary olfactory cortex that is in fact a combination of the traditional neuroanatomical definition, combined with the above hierarchical functional criteria of processes and time. In our view, one might consider the cortical regions that both receive direct projections from the olfactory bulb and are activated by sniffing alone (i.e., with no odorant content) as primary olfactory cortex. The sniff is the attentional spotlight of olfaction, and it appears to prepare a specific subset of olfactory cortex for the upcoming olfactory input. It is the directing of this olfactory searchlight that we consider the most primary olfactory task and therefore suggest that it reflect primary olfactory cortex. The above is our suggestion for a functional definition of primary olfactory cortex. We can, however, imagine several alternative functional criteria for defining primary olfactory cortex and hope that our fellow neuroimagers of olfaction will tackle this task.

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C.

Final Word

This review chapter has been written at an awkward point in the time of functional neuroimaging of human olfaction. Functional MRI of human sensory processing was only first achieved 9 years ago (Ogawa et al., 1992). The first effort at fMRI of human olfaction was only 7 years ago (Koizuka et al., 1994). Thus, the number of human olfactory neuroimaging studies is quite limited. That said, this very small number of studies has in fact already given rise to some new insights into olfactory processing, and first attempts have now been made by Levy and others to introduce functional neuroimaging to the olfactory clinical arena (Crespo-Facorro et al., 2001; Levy et al., 1998 a,b, 1999; Moberg et al., 1999; Turetsky et al., 2000; Yousem et al., abstract). Furthermore, considering the current explosion in proliferation of fMRI machines in research institutions around the world, if we were to wait for the next edition of the Handbook of Olfaction and Gustation in order to introduce the topic of functional neuroimaging of olfaction, it would be considerably too late. Thus, we opted to be a bit early rather than quite late and hopefully have given the reader some insight into how functional neuroimaging works, how it has been applied to the study of human olfaction, and in what way it may be applied in the future. The methodology of functional neuroimaging is improving at an exceptionally fast rate. One of the most exciting recent developments is the use of simultaneous MEG and fMRI, offering the temporal resolution of the former combined with the spatial resolution of the latter (e.g., Dale et al., 2000). This and other technical developments lead us to predict that functional neuroimaging methods will revolutionize our understanding of cortical processing of olfaction over the next few years. REFERENCES Aguirre, G. K., Zarahn, E., and D’Esposito, M. (1997). Empirical analyses of BOLD fMRI statistics. II. Spatially smoothed data collected under null-hypothesis and experimental conditions. Neuroimage 5(3):199–212. Allison, A. C. (1954). The secondary olfactory areas in the human brain. J. Anat. 88:481–488. Andy, O. J. (1967). The amygdala and hippocampus in olfactory aura. Electroencephalogr. Clin. Neurophysiol. 23(3):292. Bandettini, P. A., Jesmanowicz, A., Wong, E. C., and Hyde, J. S. (1993). Processing strategies for time-course data sets in functional MRI of the human brain. Magn. Reson. Med. 30: 161–173. Benjamin, R. M., and Jackson, J. C. (1974). Unit discharges in the mediodorsal nucleus of the squirrel monkey evoked by electrical stimulation of the olfactory bulb. Brain Res. 75(2):181–191.

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273 Youngentob, S. L., Mozell, M. M., Sheehe, P. R., and Hornung, D. E. (1987). A quantitative analysis of sniffing strategies in rats performing odor detection tasks. Physiol. Behav. 41(1): 59–69. Yousem, D. M., Williams, S. C., Howard, R. O., Andrew, C., Simmons, A., Allin, M., Geckle, R. J., Suskind, D., Bullmore, E. T., Brammer, M. J., and Doty, R. L. (1997a). Functional MR imaging during odor stimulation: preliminary data. Radiology 204:833–838. Yousem, D. M., Williams, S. C. R., Simmons, A., Doty, R. L., and Kroger, H. (1997b). Functional magnetic resonance imaging using olfactory stimulants. International Symposium on Smell and Taste, San Diego, CA, July 7–13. Yousem, D. M., Geckle. R. J., and Doty, R. L. (1998). Differences between olfactory and trigeminally mediated simulants on FMRI studies. ASNR, Philadelphia, May 20. Yousem, D. M., Maldjian, J. A., Hummel, T., Alsop, D. C., Geckle, R. J., Kraut, M. A., and Doty, R. L. (1999a). The effect of age on odor-stimulated functional MR imaging. Am. J. Neuroradiol. 20:600–608. Yousem, D. M., Maldjian, J. A., Siddiqi, F., Hummel, T., Alsop, D. C., Geckle, R. J., Bilker, W. B., and Doty, R. L. (1999b). Gender effects on odor-stimulated functional magnetic resonance imaging. Brain Res. 818:480–487. Zald, D. H., and Kim, S. W. (1996a). Anatomy and function of the orbital frontal cortex, I: Anatomy, neurocircuitry; and obsessive-compulsive disorder. Neuropsychiatry Clin. Neurosci. 8(2):25–38. Zald, D. H., and Kim, S. W. (1996b). Anatomy and function of the orbital frontal cortex, II: Function and relevance to obsessive-compulsive disorder. J. Neuropsychiatry Clin. Neurosci. 8(3):249–261. Zald, D. H., and Pardo, J. V. (1997). Emotion, olfaction, and the human amygdala: amygdala activation during aversive olfactory stimulation. Proc. Natl. Acad. Sci. USA 94: 4119–4124. Zald, D. H., and Pardo, J. V. (2000). Functional neuroimaging of the olfactory system in humans. Int. J. Psychophysiol. 36(2): 165–181. Zald, D. H., Donndelinger, M. J., Pardo, J. V. (1998). Elucidating dynamic brain interactions with across-subjects correlational analyses of positron emission tomographic data: the functional connectivity of the amygdala and orbitofrontal cortex during olfactory tasks. J. Cerebr. Blood Flow Metab. 18: 896–905. Zarahn, E., Aguirre, G. K., and D’Esposito, M. (1997). Empirical analyses of BOLD fMRI statistics. I. Spatially unsmoothed data collected under null-hypothesis conditions. Neuroimage 5(3):179–197. Zatorre, R. J., and Jones-Gotman, M. (1991). Human olfactory discrimination after unilateral frontal or temporal lobectomy. Brain 114(Pt 1A):71–84. Zatorre, R. J., Jones-Gotman, M., Evans, A. C., and Meyer, E. (1992). Functional localization and lateralization of human olfactory cortex. Nature 360:339–340. Zatorre R. J., Jones-Gotman, M., and Rouby C. (2000). Neural mechanisms involved in odor pleasantness and intensity judgments. Neuroreport 11(12):2711–2716.

13 Structure–Odor Relationships: A Modern Perspective Luca Turin University College, London, United Kingdom

Fumiko Yoshii Niigata University, Niigata, Japan

I.

INTRODUCTION

necessarily shared by others. It is striking how few experiments in which odorants are applied to biological preparations take into account the perceived odor of the molecules. We hope that biologists will realize that, once a vocabulary is agreed upon, odor is as reliable a sensation as pitch or color.

This review is intended as an introduction for nonspecialists to structure–odor relationships (SOR), and as a critique of the field rather than a compendium. The perspective will be that of biology rather than fragrance chemistry. In other words, we are more interested in what SORs tell us about the mechanisms of human olfaction than about the synthetic chemistry of odorants. We believe that the recent advances (see Mombaerts, 1999a, for review) that followed Buck and Axel’s 1991 discovery of odorant receptors will someday make odorant design a rational process. In the meantime, we want to highlight a few salient findings which we feel a successful SOR theory must account for, in the hope that this will help researchers design experiments to elucidate the mystery of primary olfactory reception. A perennial difficulty of structure–odor relationships has been that both structure and odor have proved hard to pin down. Considered as a structure-activity problem, olfaction is several orders of magnitude more complicated than its conventional pharmacological counterparts because there are many more structures and a vast number of odors. There is also an additional problem: as a sensation, olfaction does not seem to enjoy the same status as, say, vision. Most biologists—in fact, most people not directly involved with fragrances or flavors—seem to think that odor sensation is “subjective” and not

II.

THE CURRENT STATE OF SORS

Chemists have, by design and by accident, been producing odorants since the dawn of organic chemistry 200 years ago, and a vast database of odorants and their corresponding odor profiles has built up. This seems a good place to state what is perhaps the most surprising fact of SORs: no two odorants have ever been found to have exactly the same odor. Despite figures often mentioned in the literature of “a few thousand,” as far as we know, the resolution of the human olfactory system is infinite. The field of fragrance synthesis, though still small in comparison to, say, pharmaceuticals, is an $8 billion industry dominated by a few large firms: in alphabetical order, Dragoco (Germany), Firmenich (Switzerland), Givaudan-Roure (Switzerland), Haarmann & Reimer (Germany), International Flavors and Fragrances (United States), Quest (United Kingdom), and Takasago (Japan). Each of these firms has a library of tens of thousands of odorants. Understandably, most of these data 275

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Figure 1 (Left) Ethyl citronellyl oxalate, a molecule possessing a macrocyclic musk odor but linear in shape. (Right) A macrocyclic musk, cyclopentadecanolide. Shape-based theories assume that the linear musk assumes a conformation close to that of the macrocyclic when binding to the receptor, hence the similarity in odor.

are proprietary and not available to the scientific community. Nevertheless, many hundreds of odorants have been described in the literature, and their SORs have been extensively reviewed, most recently by Rossiter (1996). Most reviews of SORs are collections of disparate facts with no unifying theme save a basic postulate: odor must be related to molecular structure. The search for a predictive theory based on this assumption has been frustrating: Bedoukian (1966) stated that “it is not possible to predict the odor of a substance with any degree of accuracy.” McCartney (1968) felt that “the difficulties in the way of uncovering the connection [between structure and odor] have been very great.” Hornstein and Teranishi (1967) considered the results of such searches “disappointing.” More recently, Frater et al. (1998) described the state of SORs as “sorry.” Indeed Sell (1999) recently suggested that there may be no connection at all between structure and odor, and that the wiring from receptor to brain may be arbitrary. The reader interested in getting a feel for the fascinating regularities and irregularities of the structure–odor map is referred to the excellent review by Boelens (1974) and the monograph by Ohloff (1991). Attempts have been made to accommodate discrepant structure–odor relationships by a process known as conformational analysis (Yoshii et al. 1994). This involves exploring the space of conformations adopted by the odorant molecule when deformed away from its energy minimum. The fraction of configuration space allowed depends on the energy arbitrarily assigned to molecular motions. The value of conformational analysis is unclear since it is usually a directed process in which the molecule is bent purposely to resemble another odorant. An example of this is given by the study of linear musk citronellyl oxalate (Yoshii et al. 1994), whose lowest-energy conformer resembles a macrocyclic musk. At room temperature, however, the linear musk must also also explore a vast range of conformations, which resemble dozens of other odorants (Fig. 1). The complexity of structure–odor relationships, and the fact that the three-dimensional structure of the receptor site

is unknown, make it very difficult to apply conventional quantitative structure-activity relationships. QSARs have proved very useful in many areas of pharmacology (Balbes et al., 1994; Dearden and James, 1998). They work best when the structure of the site to which the molecule binds is known exactly from crystallographic measurements. Then the full force of computational chemistry can be brought to bear on designing molecules. Some studies have attempted to calculate both the three-dimensional structure of the receptor and its interaction with odorants (Floriano et al., 2000; Singer, 2000). These studies will undoubtedly become increasingly useful as our knowledge of receptor structure increases and modeling techniques become more realistic. In the meantime, most of the work proceeds by examining the structures of the odorants alone. It is not clear how many odorants have been designed using QSAR alone, or even as a principal tool to guide synthesis. Fragrance companies are reluctant to discuss the subject. Perhaps the best indication of this is that new odorant synthesis in the firms still proceeds by trial and error. It is our impression that QSAR has strong competition, particularly from combinatorial chemistry techniques that now make it easier to synthesize large numbers of molecules.

III.

WHAT MAKES AN ODORANT?

The general requirements for an odorant are that it should be volatile, hydrophobic, and have a molecular weight less than approximately 300 daltons. Ohloff (1994) has stated that the largest known odorant is a labdane, with a molecular weight of 296. The first two requirements make physical sense, for the molecule has to reach the nose* and may need to cross membranes. The size requirement

*Note

that some hydrophobic compounds of low volatility can reach the nose from the bloodstream. The garlicky smell of IV thiopental is perceived by anesthesia subjects seconds before they lose consciousness.

Structure–Odor Relationships

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Figure 2 Comparison of molecular size between a benzenoid musk (left) derived from acetophenone and its sila counterpart (right) in which the central carbon atom in the t-butyl groups has been replaced with Si. The carbon musk is a strong odorant, the sila musk odorless.

appears to be a biological constraint. To be sure, vapor pressure (volatility) falls rapidly with molecular size, but that cannot be the reason why larger molecules have no smell, since some of the strongest odorants (e.g., some steroids) are large molecules. In addition, the cut-off is very sharp indeed: for example, substitution of the slightly larger silicon atom for a carbon in a benzenoid musk causes it to become odorless (Wrobel and Wannagat, 1982d) (Fig. 2). A further indication that the size limit has something to do with the chemoreception mechanism comes from the fact that specific anosmias become more frequent as molecular size increases. At the “ragged edge” of the size limit, subjects become anosmic to large numbers of molecules. An informal poll among perfumers, for example, has elicited the fact that most of them are completely anosmic to one or more musks (e.g., Galaxolide®; MW 244.38) or, less commonly, ambergris odorants such as Ambrox® or the larger esters of salicylic acid (Fig. 3). One can probably infer from this that the receptors cannot accommodate molecules larger than a certain size and that this size is genetically determined (Whissel-Buechy and Amoore, 1973) and varies from individual to individual.

Figure 3 Two molecules that are occasionally odorless to humans-galaxolide (MW 244.38) and Ambrox (MW 236.40).

A.

Odor Descriptors and Odor Profiles

Odor descriptors are the words that come to mind when smelling a substance. The more generally understood the words are, the more useful they are as descriptors. An untrained observer may use, for example, “Grandma’s linen cupboard” as an accurate descriptor, whereas the professional would be more analytical and say woody (the cupboard), musky (the linen), or camphoraceous (the mothballs). Note that these descriptors may be applied to a single, pure odorant. Nevertheless, odor description always works by analogy since there is no objective alternative. Odor description seems to have acquired the reputation of being arcane, even fanciful, perhaps in part as a result of the hoopla surrounding fine wines and fragrances. In practice, it is easy for any observer, after a little training, to use the standard descriptors of fragrance chemistry. Accordingly, almost all the examples in this review are chosen from among those commercially available, and we urge the interested reader to obtain some of them and check the odor. Anosmias aside, outright disagreements between observers are, in our experience, rare. One exception is Karanal® an ambergris odorant that is perceived as animalic by some observers (C. Sell, personal communication) (Fig. 4). Another is trans-2-hexenal, perceived as green by some (Arctander, 1991) and bitter almond by others (Ohloff, 1994). The much more common and oft-quoted cases of perceptual disagreements, e.g., phenylacetic acid, are probably due to ambiguity, not difference. Phenylacetic acid smells both of honey and of fresh urine. When asked to use either descriptor, subjects will opt for one or the

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Figure 4 Two molecules whose odor appears to differ between observers. Karanal (left) is a woody-amber to most observers but smells unpleasantly urinous to some. Trans-2-hexenal (right) is described in the literature either as a green (Arctander, 1994) or bitter almonds (Ohloff, 1994) odorant. To the authors, it smells of bitter almonds.

other without hesitation. When asked whether the other descriptor might also apply, however, they will always agree that there is a honey or urine “side” to the smell. This is not so strange when one considers a color analogy. Ask a group whether an appropriate shade of turquoise is blue or green, and you may get half giving each answer. This does not mean that they perceive it differently. The reader wishing to become familiar with odorants and their descriptors can peruse Aldrich’s Flavors and Fragrances catalog in which odorants are listed by chemical type and by principal descriptor. Kits of esters and heterocycles are also available from the same firm, which provide an excellent introduction to the raw data of SORs, i.e., structure and odor. It is unfortunate that the vast majority of commercial odorants are not represented in catalogs of chemical suppliers familiar to the biologist. Nevertheless, fragrance firms will on request

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provide researchers with samples. For those wishing to delve more deeply into the subject, Arctander’s handbook (Arctander, 1994) lists thousands of molecules and their odor profiles and represents a mine of reliable and largely untapped information on SORs. Unfortunately, the chemical structure drawings in Arctander are antiquated and often unclear, and the book contains no descriptor index. In addition, two companies (Leffingwell and Boelens) offer independent information on fragrances and flavors at www.leffingwell.com and www.xs4all.nl/~bacis.

B.

Some Odor Categories and Their Representative Molecules, Chosen to Illustrate Structural Diversity

1.

Musk

Musk is perhaps the most famous of all odor categories because of its universal inclusion in fragrance and its exotic origin in the secretions of the musk deer. In fact, because of expense and legislation, musks have been synthetic for a long time. Musk odor descriptors might be “smooth, clean, sweet, and powdery.” The molecules that possess this odor character are exceptionally diverse in structure. Macrocyclic musks contain a 15- to 18-carbon cycle closed either by a carbonyl or by a lactone and smell similar but fresher and more natural, often with fruity overtones (cyclopentadecanolide, ambrettolide). Nitro musks, discovered originally as a byproduct of explosives chemistry, smell sweeter and are reminiscent of old-fashioned barbershop smells (Fig. 5).

Figure 5 Representatives from five chemical classes that yield musk odors: (1) androst-16-en-3-ol, a steroid musk; (2) ambrettolide, a macrocyclic musk; (3) Musk Bauer, a nitro musk; (4) Tonalid, a tetralin musk; (5) Traseolide, an indane musk.

Structure–Odor Relationships

2.

Ambergris

Originally derived from concretions spat out by whales and aged in the sun, ambergris odorants smell nothing like natural ambergris tincture, which has a weak animalic marine smell. The smell of ambergris odorants was once aptly described to us by a chemist-perfumer as “glorified isopropanol.” Ambergris odorants are of interest to the student of SORs because they provide an interesting combination of very closely related smells with widely different structures: amberketal, timberol, karanal, and cedramber are close enough that a perfumer will occasionally mistake them for each other (Fig. 6). 3.

Figure 8 Some examples of green odorants. Clockwise from top left: cis-3-hexenol, ligustral, nonadienal, and ethylmethoxypyrazine.

Camphoraceous

Camphoraceous (mothball) notes are seldom used in perfumery, but they are of interest of SORs because they formed the basis for one of the early attempts at smell classification by Amoore (1971). Camphor, cyclooctane, cineole are good examples of camphoraceous smells and smell rather similar to each other (Fig. 7). 4.

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Figure 9 Two bitter-almond odorants: benzaldehyde and hydrogen cyanide.

Green

This category includes cut grass, fresh green bean notes with a sharp, almost aggressive feel. Diverse compounds possess this descriptor, ranging from classic grassy notes of cis-3-hexenol and ligustral to the cucumber peel of nonadienal and the bell-pepper green note of some pyrazines (Fig. 8).

5.

This easily recognized category is interesting to students of SORs because it includes a small molecule (HCN) that smells metallic, not almond-like, to a large fraction of observers (Fig. 9). Benzaldehyde, nitrobenzene, and trans2-hexenal (but see above) are good examples. 6.

Figure 6 Two ambergris odorants: timberol (left) and cedramber (right).

Other Categories

Many other categories such as musty, spicy, aldehydic, lactonic, indolic, and marine exist, each with subdivisions. It must be emphasized that the odor categories above are merely convenient descriptors and only cover a very small fraction of odor “space.” In fact, especially when one steps out of perfumery materials proper into smells noticed by chemists in the course of organic and inorganic syntheses, the most frequent descriptor appears to be sui generis, i.e., a smell associated with nothing in particular. IV.

Figure 7 Three camphoraceous odorants: (left) 1,8-cineole, (center) camphor, and (right) cyclooctane.

Bitter Almonds

PLAUSIBLE THEORIES OF ODOR

Many theories of SORs have been proposed in the past (reviewed in Moncrieff, 1951), but advances in biological understanding, not least the discovery of odorant receptors, have gradually ruled them out. Leaving aside the pessimistic view outlined above, according to which there may be no relationship between structure and odor, there

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appear to be two possible types of SOR theory left standing. One is based on fragments of molecular shape or odotopes (Mori and Shepherd, 1994), the other on molecular vibrations (Turin, 1996, 2002). A.

Shape-Based Theories: Odotopes

Most enzyme-substrate and receptor-ligand binding relies on molecular recognition between protein and ligand. Recognition depends on interactions that can be either attractive or repulsive (Davies and Timms, 1998). All attractive chemical interactions are ultimately electrostatic in nature, whether they occur between fixed charges, dipoles, induced dipoles, or atoms able to form weak electron bonds (e.g., hydrogen bonds). Repulsive interactions can be electrostatic or quantum-mechanical (electron shell exchange repulsion). Almost every change in molecular structure (with some exceptions, described below) alters the set of surface features capable of forming such attractive or repulsive interactions and thus affects what we loosely call molecular shape. The range of known molecular recognition mechanisms in biology is vast. At one extreme might be a vast set of immune- type receptors, each able to bind to a single odorant molecule. At the other end of the spectrum, some binding sites such as those of odorant-binding proteins (Bianchet et al., 1996), albumins (Curry et al., 1999) and cytochromes P450 (Lawton and Philpot, 1993) are rather nonspecific. When odorant receptors were first identified, their large number was taken by some as evidence for immune-like recognition. However, in vivo and, more recently, in vitro studies have shown (Firestein et al., 1993; Duchamp-Viret et al., 1999; Malnic et al., 1999) that, with one notable exception (Wetzel et al., 1999), receptors respond to more than one odorant, suggesting that they detect the presence not of the whole molecule but of a partial structural feature thereof, hence odotopes. According to odotope theory, the smell of a molecule is then due to the pattern, i.e., the relative excitation of a number N of receptors to which it binds. Even if one assumes that receptors are only on or off, this scheme gives considerable combinatorial room. Consider, for instance, a molecule having 20 exposed atoms and assume that each odotope involves three of these. A binary (on-off) oneodotope recognition system would then be able to detect 1140 molecules. If odotopes involved four atoms, the number would rise to 4850, etc. Combining odotopes, and adding to this basic scheme a measure of intensity of excitation for each receptor, clearly enables it to detect a vast number of odorants. If the large number of odorant receptors is taken to represent odotope categories, the combinatorial possibilities become astronomical. A more sophisticated argument has been made by Lancet et al. (1993). They make plausible assumptions

about the number of “subsites” (odotopes) and their variability, combined with calculations of the energetics of binding. Assuming a very low affinity of 105 M 1 for odorant binding, they arrive at the conclusion that in order to achieve recognition, 300–1000 receptors are needed, in line with current estimates of receptor number (see Sec. VI for further discussion of this point). Higher affinities, more consistent with olfactory thresholds, lead to greater still receptor numbers.

B.

Vibration Theories

The idea that the nose operates as a vibrational spectroscope was first proposed by Dyson (1938) and later taken up and refined by Wright (1982). What makes it attractive in principle is that vibrational spectra share three properties with human olfaction: (1) no two molecular spectra are exactly alike, particularly in the aptly named “fingerprint region”; (2) many functional groups are easily identified by their specific vibrational frequencies (and by smell, see below); and (3) a system utilizing a physical property as basic as vibration will be ready for never-before-smelled molecules, i.e., does not depend on a repertory of existing or expected structures. In that sense, it does not rely on molecular recognition. Several difficulties beset vibration theories and ultimately caused their demise 20 years ago: 1. Enantiomers, which have identical vibrational spectra in solution, sometimes have different odors (see Boelens and van Gemert, 1993). Wright countered this by emphasizing that while laboratory spectroscopes were achiral, and thus unable to distinguish between enantiomers, a protein receptor would be intrinsically chiral and would thus respond differently to enantiomers. A modified version of this argument is described in Section V.C. 2. No mechanism was ever found for a plausible protein-based spectroscope, infrared optics being obviously out of the question. 3. Wright assumed that receptors were mechanical vibration sensors and that the receptors in the nose would only be able to feel vibrations excited by thermal motions at body temperature. He then restricted his search for correlations between structure and odor to the region below 600 cm 1. These were somewhat unconvincing and appeared to have little predictive value. The situation changed a few years ago with the proposal that electron tunneling might be a plausible mechanism enabling proteins to act as vibrational spectroscopes.

Structure–Odor Relationships

C.

A Biological “Spectroscope”

Inelastic electron tunneling spectroscopy (IETS) is a nonoptical form of vibrational spectroscopy (Jaklevic and Lambe, 1966; Hansma, 1982; Adkins and Phillips, 1985). It relies on the interaction between electrons tunneling across a narrow gap between metallic electrodes. When the gap is empty, tunneling electrons cross the gap at constant energy, and the tunneling current is proportional to the overlap between filled and empty electronic states in the metals. If a molecule is present in the gap, tunneling electrons will be scattered by the partial charges on the molecule’s constituent atoms and lose energy to the molecule by exciting one of its vibrational modes. When this happens, electrons can follow an indirect path, first exciting the molecular vibration and then tunneling to the second metal at a lower energy. The new tunneling path causes an increase in the conductance of the junction. Metallic conductors are absent in biology, but electron transfer is ubiquitous. Doing IETS with proteins (Fig. 10) would involve addition and removal of electrons at well-defined energy levels on either side of an odorant-sized (300 daltons) binding site, which serves as the tunneling gap. On one side of the gap, a donor site with occupied donor levels is present, while an acceptor site with empty acceptor levels is on the other side of the tunneling gap. If there is nothing between the electron source (donor) and sink (acceptor), then for direct tunneling to occur there must be an (occupied) energy level in the source that matches the energy of an (empty) state in the sink. If there is a molecule between the electron source and electron sink, and if that molecule vibrates, then indirect

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tunneling can occur only if there is an energy level in the source with energy E above that in the sink. In other words, tunneling occurs only when a molecular vibrational energy E matches the energy difference between the energy level of the donor and the energy level of the acceptor. The receptor then operates as a spectrometer, which allows it to detect a single well-defined energy, E. If there are several vibrational modes, which one(s) get excited will depend on the relative strengths of the coupling. That may be expected to depend on, among other things, the partial charges on the atoms and the relative orientation of the charge movements with respect to the electron tunneling path (Fig. 10). Unlike conventional IETS, “biological IETS” does not involve scanning of the energy range, which would probably be unfeasible in a biological system. Instead, the range of vibrational energies is covered piecewise by a series of receptors tuned to different energies. The energy range is limited only by the emf (reducing power) of the electron source. An estimate of biological reducing power is 500 mV (1 e V
8086 cm 1) (Frausto da Silva and Williams, 1993), which means that the entire vibrational range to 4000 cm 1 could be sampled. To cover the vibrational spectrum, several receptor classes would be required, each tuned to a different segment of the vibrational spectrum. A small number might be sufficient, much as three pigments with broad, partially overlapping absorption spectra suffice for color vision. One essential feature of the biological spectrometer is its relatively poor resolution. A biological system must work at ambient or body temperature, i.e., around 300 K. Donor and acceptor levels across the tunneling gap will therefore have a minimum width of 2kT (艐400 cm 1). The range 0–4000 cm 1

Figure 10 Schematic of the proposed transduction mechanism: the receptor protein accepts electrons from a soluble electron donor (NADPH). When the receptor binding site is empty (top), electrons are unable to tunnel across the binding site because no empty levels are available at the appropriate energy. The disulfide bridge between the receptor and its associated G-protein remains in the oxidized state. When an odorant (here represented as an elastic dipole) occupies the binding site (bottom), electrons can lose energy during tunneling by exciting its vibrational mode. This only happens if the energy of the vibrational mode equals the energy gap between the filled and empty levels. Electrons then flow through the protein and reduce the disulfide bridge via a zinc ion, thus releasing the G-protein for further transduction steps.

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could thus be covered by 10 or so receptor types. A similar arrangement exists in the other spectral senses, vision and hearing, in which broadly tuned receptors classes cover segments of the complete spectrum. V.

ODOTOPES VS. VIBRATIONS: HOW THEY FIT THE FACTS

In a field as vast and amorphous as that of SORs, observations can be found to lend support to almost any theory. In what follows, we shall therefore try to stick to observations that are potentially able to disprove one or the other of the two contenders. A.

Smelling Chemical Groups

A fact that has, in our opinion, received too little attention from olfaction researchers is the ability of humans to detect the presence of functional groups with great reliability (see Klopping, 1971, for review). The case of thiols (–SH) is familiar, but other chemical groups such as nitriles (–CN), isonitriles (–NC), oximes (–NOH), nitro groups (NO2), and aldehydes (C – O(H)) can be reliably identified once the odor character the functional group character confers is known. When nitriles are used as chemically stable replacement for aldehydes, they impart a metallic character to any smell: cumin nitrile smells like metallic cumin (cuminaldehyde), citronellyl nitrile smells like metallic lemongrass (citronellal), and nonadienylnitrile smells like metallic cucumber (nonadienal). Oximes give a green-camphoraceous character, isonitriles a flat metallic character of great power and unpleasantness, nitro groups a sweet-ethereal character, etc. Remarkably, even bonds between atoms can be detected: the acetylenic C–C triple bond of -ynes imparts an isothiocyanate-like mustard-like smell to molecules that is clearly recognizable— for example, in acetylene and in methyloctynoate. 1.

What could make the SH infallibly distinctive as an odotope, as compared to the OH group? Partial charge, bond length, bond angle, and atom size are somewhat different between R-SH and R-OH, but it is hard to see how these can be detected with absolute reliability by, say, an amino acid side chain in the presence of thermal motion. A more distinctive property of sulfur lies in the energy of its lone pair orbitals, as witnessed by the specificity with which it forms complexes with certain metals. If a metal is involved, then it becomes hard to explain that (1) dimethyl sulfide has no thiol character and (2) a thioether (–S–) link can often replace a –C
C– with very little change in smell and no sulfuraceous character (Boelens and van Gemert, 1993b) (Fig. 11). The same problem applies to other smellable functional groups and can be stated more generally: if functional groups are odotopes, then they are so small as to only be able to form one or two interactions, e.g., hydrogen bonds, with odotope receptors. Their small size will similarly restrict the number of repulsive interactions. Therefore small molecules should bind with various degrees of affinity to many odotope receptors, and small molecules should have similar odors, particularly at high concentrations (Klopping, 1971). That is not the case: small molecules like methylnitrile and methylnitrate smell distinctively different at all concentrations. Indeed, still smaller ones— e.g., ozone, sulfur hexafluoride, and carbon disulfide— also have this property. 2.

Functional Groups and Vibrational Theory

By contrast, the distinctive smell of functional groups is a natural feature of a vibrational theory. Above 1800 wavenumbers, IR absorption lines are diagnostic of the stretch frequencies of diatomic functional groups. The aldehyde-nitrile replacement rule can be understood from the closeness of their stretch vibration. Similarly, the similarity in smell between acetylenic bonds and isothiocyanates can be explained by their respective stretch frequencies.

Functional Groups as Odotopes

An odotope theory can explain these regularities only by assuming that the functional group is an odotope. In the older structure–odor literature, this used to be described as electronic factors (as opposed to steric). The idea was that, given that many functional groups were similar in size, the recognition mechanism must somehow be sensitive to the fine structure of the electron distribution (orbital energies, charge density, etc.) of the functional group. This seemingly reasonable notion runs into problems on closer examination. Consider, for instance, the SH group in methanethiol. Alcohols never smell of sulfur, whereas thiols always do.

Figure 11 Replacing a C
C bond with a sulfur atom does not change odor character, suggesting that “electronic” properties of sulfur are not sufficient for molecular recognition.

Structure–Odor Relationships

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Figure 12 The dependence of the sulfuraceous character on molecular vibrations and atomic partial charges, as predicted by a vibrational theory. Decaborane (left) smells sulfuraceous, and its terminal B-H bonds have a stretch frequency of ~2500 wavenumbers. In triethylamine-borane (middle), the B-H stretch is shifted to 2300 wavenumbers and the sulfuraceous smell is no longer present. In p-carborane (right) the near-neutral partial charges make the SH bond odorless. (See color insert.)

The clearest example so far is that of boranes. The terminal B–H bond in boranes has a stretch frequency whose range overlaps with that of thiols. Turin (1996) therefore predicted that boranes should smell sulfuraceous, despite the complete absence of similarity, both structurally and chemically, between boron and sulfur. A comparison between borane and thiol smells is best made using decaborane.* Decaborane smells strongly of boiled onion, a typical SH smell. Curiously, its smell was described as “chocolate-like” (chocolate contains some thiols) in papers reporting its synthesis, which may account for the fact that the similarity was not noticed earlier. Other, less stable boranes share this sulfuraceous smell character (Fig. 12). There are three possible nonvibrational interpretations of this finding: (1) boranes do not in fact smell of sulfur, (2) the similarity in smell between B–H and S–H, while real, is pure coincidence, and (3) BH and SH activate the same odotope receptor by some unknown mechanism, despite the difference in shape. In answer to objection 1, we advise the interested reader not to take the authors’ word and to smell decaborane observing due precautions. Objection 2 is harder to answer, because the odds against such a thing happening, while large, are impossible to calculate exactly. Predictions are rare in SOR theories, and this is a conspicuously successful one.

*This

experiment requires caution: although stable at room temperature in air, decaborane is reported to be highly toxic and has a high vapor pressure. It is therefore best to open the container in a fume cupboard, close it again after a few minutes, and smell the very small amount of decaborane condensed on the outside of the cap. One of us (LT) has been doing this periodically for some time with no apparent ill effects.

Objection 3, by contrast, can be answered rather simply. If terminal BH groups activate the same odotope as SH, then all BH-containing compounds should have a sulfuraceous character. This is not the case: as was pointed out to one of us (LT) by R. H. Biddulph (personal communication), triethylamine-borane does not smell sulfuraceous. Remarkably, the vibrational frequency of the BH bond in triethylamine-borane is shifted downward by 200 wavenumbers, i.e., out of the thiol range. Another instance is that of the three isomers of carborane, which smell camphoraceous, though o-carborane has a faint onion-like (sulfuraceous) smell. The reason for this is not yet clear, but their extraordinary chemical stability is consistent with a low polarity of the B-H bond, and this would tend to reduce the intensity of the BH stretch vibration to the point where it may be no longer detectable. 3.

Hindered Functional Groups

Molecules could in principle be designed to settle the issue of whether functional groups are perceived as odotopes or by their vibrations. Suppose, for example, that a functional group possessing a distinctive odor was present in a molecule, but was buried in such a way as to be inaccessible to molecular recognition. Because tunneling electrons penetrate the molecule, the vibrational theory would predict that it should still smell, whereas odotope theory would not. The ideal molecule in this respect would include, say, an SH group within its innards, completely shielded from touch. Such a molecule does not yet exist and may be impossible to construct given the maximum size requirement for odorants. Sterically hindered phenols provide a first approximation to this goal. The presence of an OH group on a substituted benzene ring gives the molecule a distinctive

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Figure 13 Space-filling models of 2,4-(left) and 2,6-di-t-butyl phenols. These two molecules smell equally phenolic despite the OH group’s being accessible in one and sterically hindered in the other.

“phenolic” odor, which the corresponding benzene does not have. Once again, if one assumes that the OH group is an odotope, then making it less accessible to molecular recognition should silence its smell. This idea is easily tested by comparing the smell of di-tert-butyl derivatives of phenol, which are readily available commercially. The results go against the odotope theory. 2,6-Di-tert-butyl phenol, in which the OH group is strongly hindered, smells as phenolic as, say, the 2,4 derivative in which it is more accessible (Fig. 13). It may be argued that the OH group is insufficiently buried in this molecule and remains accessible to some molecular interaction. Designing molecules with buried functional groups—for example, the trimethylsilyl analogs of phenols and thiophenols—could, in principle, settle this question. B.

Figure 14 Electron-density maps of ferrocene (left) and nickelocene (right) with electrostatic potential mapped onto the surface. Structure, electron density and potential surfaces calculated by semiempirical methods using Spartan with PM3 parameters. Red is more negative. There are small differences in ring spacing and charge distribution, whereas the odor of these two molecules is radically different: ferrocene smells spicy-camphoraceous; nickelocene smells oily-chemical. (See color insert.)

Isosteric Molecules

A strong test of vibrational vs. odotope theories would be the odor comparison of molecules identical in atom composition, shape, weight, electron distribution, and all other physical properties, differing only in vibrations. That is, of course, an unattainable ideal, but one can come quite close, either by element substitution (Ni for Fe inside a metallocene, Si for C) or by isotope substitution (D for H) in normal odorants. 1. Metallocenes Ferrocene and nickelocene have very similar structures and very different smells. Vibrationally, the main difference is in the internal movements of the metal ion between the rings (Fig. 14). 2. Sila Compounds Silicon (and in some cases Ge and Sn) can replace carbon in odorants (Mundstedt and Wannagat, 1985; Wannagat

et al., 1985, 1993; Wrobel and Wannagat, 1982a–d, 1983). Because of the high polarity and consequent instability of the Si-H group, only C atoms linked to four carbons can be substituted in this fashion. The geometry of Si–C bonds is tetrahedral. Though very similar in overall geometry, sila compounds will differ somewhat from the parent carbon compound. The Si-C bond is 1.8 Å long, as compared to 1.5 Å for a typical C–C bond, and the Si–C bond is more polar. By contrast, the vibrations of the molecule will be markedly altered by the Si and Ge substitution. For example, the Si-C stretch vibration is around 650 wavenumbers instead of 1000 (Fig. 15). In all cases, there was some change in odor, though it was sometimes subtle and sometimes striking. For example, sila substitution in linalool “light and refreshing, floral-woody odor with a citrusy note” (Wrobel and Wannagat, 1982a) to give sila-linalool makes it “more hyacinth-like, sweeter.” Similarly, sila-terpineol smells more muguet-like and less lilac-like than the parent

Structure–Odor Relationships

Figure 15 Three representative examples of molecules in which Si replacement for C causes a marked change in odor. Left: Sila linalool; center: sila terpineol; right: sila cyclocitral.

compound, but sila-carvomenthene smells “similar” to the parent carbon compound. Interestingly, though the largest jump in size and other properties occurs between C and Si, Ge derivatives are again different in odor from both. Silacyclocitral smells “camphoraceous, sweet, earthy with a green tea note,” whereas the parent compound smells “minty, turpentine-like” (Mundstedt and Wannagat, 1985). In her comprehensive review of SORs, Rossiter (1996) summarized these results by saying: “Those examples where the odor of the sila analogue is similar to that of the carbon counterpart are interesting anomalies for . . . vibrational theories.” These data could also be interpreted as interesting anomalies for odotope theories. The reader interested in exploring the differences in odor between sila and parent compounds can readily obtain 1,1-dimethyl-1silacyclohexane (艐cyclopentamethylene dimethylsilane) and its parent compound 1,1-dimethyl cyclohexane from either Aldrich or Lancaster (United Kingdom). The difference in smell between the two compounds is striking. The odor profiles, assessed by a professional perfumer, are as follows: 1,1-dimethylcyclohexane, camphoraceous, with a faint sweet fruity, powdery background; 1,1-dimethyl-1silacyclohexane, intense, chemical-green note reminiscent of cis-3-hexenol, with a faint camphoraceous background (Fig. 16).

Figure 16 The calculated structures of two commercially available compounds with similar shape and very different odors. Left: 1,1-Dimethylcyclohexane; right: 1,1-dimethylsila–cyclohexane.

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In summary, the results on sila and germa compounds are consistent with both theories. Odotope theory does not adequately account for the very large difference in smell between C and Si, and especially between Si and Ge compounds. Neither theory adequately explains why odor differences should be so marked in some cases and weak in others. 3.

Isotope Substitution

Isotope substitution is in principle the best way to make perfectly isosteric compounds differing “only” in molecular vibrations. The “only” in the sentence above illustrates the fact that, as Wade has pointed out in his comprehensive review of isotope effects in biology, there are in fact subtle differences in the physical and chemical properties of isotopes as compared to the parent compound (Wade, 1999). Their hydrophobicity will be slightly different because of the small difference in size and polarizability of the electron cloud surrounding the heavier nuclei. In addition, the range of conformations that the compound will explore during thermal motion will be different, because the altered masses respond differently to thermal excitations. Nevertheless, these effects are small: isotope separations on chromatography columns require long elution times, and the lowest energy conformation (i.e., molecular shape) will in all cases be unaffected by isotope substitutions. By contrast, effects on molecular vibrations can be large: substitution of D for H reduces the X–H stretch frequencies by a factor of approximately 兹苶2—for CH, for example, from 3000 to 2200 wavenumbers. Effects of isotope substitution (deuterium for hydrogen) on animal olfaction have been known for a long time. Hara (1977) showed that fish could reliably distinguish deuterated glycine from the parent compound. Meloan and collaborators have performed a remarkable series of studies (Meloan et al., 1988; Kuo, 1982; Scriven, 1984; Havens, 1993; DeCou, 1993) in which they showed that insects could distinguish between isotopes. For example, cyclohexanone is a powerful cockroach repellent, whereas deuterated cyclohexanone is inactive. No human counterpart of these effects was appreciated until it was reported that deuterated acetophenone could be distinguished from the parent compound by smell (Turin, 1996). These experiments were performed on a gas chromatograph using a smelling port to eliminate the possibility that impurities might be responsible for the smell difference. The difference in smell to trained observers was subtle, but definite. We have recently found a more striking isotope odor difference in dimethyl sulfide. Arctander describes the odor of dimethyl sulfide as “repulsive, sharp, green,

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cabbage-like” at high concentrations. Dimethylsulfide-d6 clearly smells cleaner, more truffle-like without the gassy cabbage-like note of the parent compound. This is a particularly easy experiment to replicate because (1) both dimethyl sulfide and dimethylsulfide-d6 are safe to smell (despite its unpromising descriptors, DMS is a perfumery raw material!) and available at very high purity from Aldrich, and (2) dimethylsulfide is a very strong odorant, so impurities will be unlikely to influence the overall odor. We urge interested readers to do the experiment. The antisymmetric and symmetric C–S stretch vibrations are shifted from 710 and 654 wavenumbers, respectively,* to 670 and 608 wavenumbers. Finally, one of us (LT) has obtained a sample of deuterated decaborane. Unlike those of thiols, the terminal hydrogens of boranes are not readily exchangeable. This allows one to test whether the stretch frequency of boranes is genuinely necessary to their sulfurous smell. Fully deuterated decaborane ( 90% D) smells distinctively different, more mustard-like, pungent, and less sulfuraceous than its H counterpart. In summary, available evidence from isotope experiments appears to be inconsistent with odotope theory and in broad agreement with vibrational theory. In order for the odotope theory to apply, one would have to postulate additional factors to be involved. For example, a very high differential sensitivity of the odotope receptors to small changes in odorant hydrophobicity might account for the results. Alternatively, one might suppose that the fact that different low-lying vibrational states with energies around kT (艐240 wavenumbers) are excited in the deuterated odorant will cause its average conformation to be slightly different and thereby cause a difference in odor. There is at present no evidence for either mechanism. C.

Enantiomers

Most enantiomeric pairs of odorants smell identical, but there are many examples of enantiomer pairs that smell completely different (Boelens and van Gemert, 1993b). The best known of these outside fragrance chemistry are R and S carvone: R carvone smells of mint, S carvone of caraway (Fig. 17). Differences in smell between enantiomers have in the past been considered strong evidence against vibrational theories of olfaction, because solution IR spectra of enantiomers probed with unpolarized light are of course

Figure 17 The enantiomers of carvone. (S)-() Carvone (left) smells of caraway; (R)-() carvone (right) smells of spearmint.

identical. By contrast, if the IR absorption of a regular solid (crystal) is probed with polarized light, then the spectrum depends on the relative orientation of the molecular dipoles in the crystal to the plane of light polarization. Turin (1996) has argued that a “biological spectroscope” resembles the latter case, and that the smell of carvone can be explained by a polarization effect. In a tunneling mechanism for detection of molecular vibrations, the odorant is bound in a fixed orientation in the receptor and is probed by tunneling electrons which are polarized, i.e., deflected in specific directions by the odorant. Strong dipoles (the C
O group in the case of carvone) will be most likely to show polarization effects. It could be that in mint carvone, the C
O is not detected because it is wrongly oriented. One would then expect that “adding back” the carbonyl vibration by smelling simultaneously a small carbonyl-bearing odorant (e.g., acetone, butanone) with mint carvone would change the smell from mint to caraway, which it does.* It remains to be seen whether similar experiments can be devised for other enantiomer pairs. Interestingly, the smell of enantiomers poses a very serious problem for odotope theory, although this fact seems to have received no attention. The reason is analogous to the problem with odorless molecules discussed above. Suppose that an odorant is probed by several different odotope receptors. Each of those receptors is likely to be chiral to some extent, because it is near-impossible to engineer a specific protein-binding site without chirality. A good indication of this comes from drug-receptor interactions, where drug enantiomers almost always have different actions (Hutt, 1998). Consider now the case of two enantiomers with identical smells. To account for this, odotope theory needs to make one of two assumptions. Either the enantiomer binds equally well and with the same affinities to the n (chiral) odotope receptors or a *The

*Computed

ab initio using a 3-21G* basis set and corrected to 0.9 of the calculated value.

demonstration is easy to perform: mix three parts of butanone with two parts of mint carvone and smell immediately, because the butanone evaporates rapidly. The mint smell is gone, replaced by a good approximation to caraway.

Structure–Odor Relationships

completely different set of odotope receptors with opposite chirality are wired in the same fashion to give the same pattern of nerve excitation. Both are unlikely. D.

The Evidence from Receptor Expression Studies: Patterns of Receptor Activation

In the past few years, following the early lead of Raming et al. (1993), several receptor expression studies have been published. The advantage of receptor expression is that the response of a single receptor type to different odorants can be assessed directly. The results so far are still somewhat contradictory, and the field is evolving very rapidly. Zhao et al. (1998) have expressed receptors in olfactory neurons and found a broad response spectrum. Their particular receptor subtype was optimally stimulated by heptanal, less so by aldehydes of a shorter or longer chain length, consistent with an odotope-based model. Breer et al. (1998), Kaluza and Breer (2000), and Touhara et al. (1999) also found that different receptors had relatively broad ligand specificity. These results are in agreement with the in vivo responses of olfactory receptor neurons (Firestein et al., 1993; Duchamp-Viret et al., 1999). By contrast, Krautwurst et al. (1998) reported greater specificity in odorant receptor responses, and Wetzel et al. (1999) reported that a receptor only responded to one odorant (helional) at very low concentrations but not to the closely related molecule piperonal (Fig. 18). These remarkable results differ from those reported by other groups, including in vivo studies (see Firestein et al., 1993). If confirmed and extended, they would suggest that odorant-receptor interactions are far more specific than has been hitherto supposed. To date, however, the most comprehensive set of data comes from the elegant work of Malnic et al. (1999), in which 14 receptor types were expressed and their responses to a set of 19 odorants compared. Figure 19 illustrates the spectrum of response of the 14 different receptor types to 19 different odorants. The odorants are arranged in series:

Figure 18 Helional (left) and the related molecule piperonal. A recent study (Wetzel et al. 1999) has suggested that helional alone, out of 100 odorants, can activate an olfactory receptor, with even closely related molecules being 1000 times less potent.

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carboxylic acids, alcohols, bromo-carboxylic acids, and dicarboxylic acids with carbon chains of varying lengths. A remarkable pattern emerges: the matrix of receptor responses to odorants is sufficiently complex that even with this small number of receptors, an odorant in the list can be identified from the pattern of receptors it is capable of activating. They conclude that “different odorants are recognized by distinct combinations of receptors” and interpret this data according to an “odotope” model, though they do not use the term and do not specify which odotopes may be involved. There is, however, another pattern in their data, namely that in each series the number of receptor types that respond increases as one lengthens the carbon chain. This immediately suggests that the spectrum of responses may be related in part to the hydrophobicity of the odorant. The latter can easily and accurately be calculated as logP, where P is the (calculated) partition coefficient between octanol and water. When the number of receptor types activated (a crude measure of the potency of the odorant) is plotted against logP (Fig. 20), a new pattern is evident. First, the number of receptors activated is roughly proportional to logP for each series of odorants. This suggests that partition into a hydrophobic site, possibly the receptor itself, governs “affinity” within a series. The different series appear to have different efficacies, however: dicar-

Figure 19 Pattern of responses (black circles) of different olfactory receptors (columns) to different odorants (rows). (From Malnic et al., 1999.)

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Figure 20 Response of expressed olfactory receptors to a variety of odorants. When the number of different receptor classes activated (ordinate) is plotted against the water-octanol partition coefficient (log P, abscissa), it becomes clear that a determining factor in molecular selectivity is hydrophobicity. When the data are corrected for scattering intensity in a vibrational mechanism (right), the correlation improves. Weak responses obtained at 100 M (small circles in original figure) were treated as 0.5. (From data in Malnic et al., 1999.)

boxylics, while considerably less soluble in octanol than alkanols, are clearly more potent. Such a pattern would be expected from a vibrational mechanism. In this model the affinity of the odorant for the receptor is governed by logP because the receptor site is hydrophobic. The efficacy, by contrast, is governed by the electron-tunneling cross section of the molecule, i.e., its ability to scatter electrons. That in turn is proportional to S
q2·x2 where q and x are, respectively, the calculated electrostatic partial charges and atom displacements for each vibrational mode, and the summation is carried out over all vibrational modes. In other words, the larger the charges on the component atoms and the bigger their displacements, the stronger the odorant will be. This makes sense when interpreting Figure 20 (left): the least potent odorants are the alkanols (partial charge largely on OH), then come the carboxylics (partial charges on the acid group), then the bromocarboxylics (additional charge from the C-Br bond), and finally the dicarboxylics (two sets of acid group charges). The calculated values of S for octanol, octanoic acid, bromooctanoic acid, and octanedioic acid are 1.38, 3.50, 3.97, and 7.48, respectively.* When the logs of these values are used to correct the left-hand graph in Figure 20, the

right-hand graph is obtained. The curves now follow roughly the same linear relationship. This suggests that the data of Malnic et al. (1999) are equally consistent with a mechanism invoking odotopes and by a vibrational mechanism involving the physical quantities logP and S. VI.

WHY ARE THERE SO MANY RECEPTORS?

The large number of receptor sequences found in humans (347 at the last count, see Zozulya et al., 2001) is often taken as evidence in favor of molecular recognition mechanism based on shape. This is not necessarily so. First, if— as seems likely—odorants bind to a specific binding site in the receptor, then the variability needed to accommodate different odorants of MW 300 daltons should be restricted to a dozen or so neighboring amino acids which are in direct contact with the odorant (Floriano et al. 2000). Second, a large number of receptors would be more consistent with highly specific responses (one receptor, one odorant). Most of the evidence points to broadly tuned receptors, which removes the need for a large number. By way of comparison, several thousand colors can be distin*Partial

charges (electrostatic fit) and atom displacements were calculated for the lowest homolog in the series using semiempirical methods with AM1 parameters (Mac Spartan, Wavefunction, Inc.).

Structure–Odor Relationships

guished using the relative intensity of signals coming from only three types of broadly tuned retinal cones. Third, some of the variability seems to be related to the developmental role receptors play in guiding olfactory receptor neurons to the correct place in the bulb (Wang et al., 1998). Olfactory receptor-like proteins have been found in nonolfactory tissues and may serve a general developmental purpose (Dreyer, 1998). It is worth bearing in mind that in higher vertebrates, including humans, some of the olfactory receptor neurons renew themselves throughout life, which may require developmental clues. A large number of receptors is also expected from a vibrational mechanism. An idealized receptor would have an odorant-binding site that is as unspecific as possible, analogous to the cuvette of an ordinary spectrometer. At a molecular level of course this cannot be achieved, because the cuvette needs to be molecule-sized and will thus always incorporate some element of selectivity and chirality. Conversely, if it were made large enough, say the size of a lipid droplet, in order to accommodate all odorants, it would be too large to allow electron tunneling to occur. A biological spectroscope that wishes to accommodate a broad range of odorants therefore needs a large variety of odorant-binding pockets. Thus, both a shape-based and a vibrational theory may require a large number of receptors, and the main difference is in the way the receptors are wired. The arguments set out in Lancet et al. (1993) (see Sec. VI.A), which enable them to calculate the number of receptors required to achieve a target affinity for a large set of ligands, apply equally well to odotope and vibrational theories. In a shape-based theory the receptors are wired by odotope, whereas in a vibrational theory the receptors are wired by spectral class. All receptors binding molecules of different shapes but probing the same part of the vibrational spectrum would be expected, say, to project to the same part of the olfactory bulb. There is some evidence that different parts of the rat olfactory epithelium respond to the presence of different functional groups (Scott et al., 1996, 1997), but it is not clear whether the differences follow odotopes or vibrations. Not enough is known about the relationship between bulbar responses and either shape or vibration to decide the issue at the moment. VII. A.

THE PUZZLE OF ODORANT INTENSITY Odorless Molecules

The question of odorant intensity (strong vs. weak) as distinct from odor character (the sum of descriptors) raises issues of unexpected subtlety. Odotope theories implicitly assume that odorant intensity is part of the odor character,

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i.e., that a molecule can be described legitimately as, say, green–weak or green–powerful. When traced back to its intellectual roots, this idea originates from pharmacology, where a ligand, irrespective of its affinity for the receptor, may have a low or high efficacy. This seems reasonable enough, but is actually quite hard to reconcile with odotope theory. For a molecule to be odorless, it would have to be simultaneously odorless to all the odotope receptors that it binds to. While this is possible in principle, it would be more likely to occur with small molecules bearing few odotopes, and therefore binding to few receptors, and gradually less likely as molecular size increases. What is observed is precisely the opposite: with some exceptions (see below), odorless molecules appear only as molecular size nears its upper limit, i.e., when the number of possible odotopes available for binding is at its maximum. An odotope theory modified to account for this might include a size selectivity filter in each receptor, such that the molecule has to fulfill two criteria to be odorant: fit in the filter and bind to the receptor. The difficulty with this ad hoc hypothesis is that it then requires perfect uniformity in the size of the selectivity filters. Were this not the case, one would expect the most frequent outcome to be not an odorless musk but a different smell altogether if only a subset of odotope receptors are still able to smell it. This is not the case—musks are either odorant or odorless to different subjects without change in smell character. A vibrational theory has the opposite problem, namely, that no molecule that has nonzero partial charges on its component atoms should be odorless, since all molecules have a vibrational spectrum. This agrees (Turin, 1996) with the fact that all small molecules are odorous, except for the ones with either very weak or zero charge (e.g., hydrogen or oxygen gas) and those for which all the vibrations are below kT, since they will be indistinguishable from thermal noise. In a vibrational theory, the odorless character of a molecule can arise only from the fact that it does not bind to a receptor. The problem of odor behavior near the size cutoff also applies to a vibrational theory, but in a less extreme fashion than with odotopes. First of all, there need to be fewer receptor types, ideally only enough to cover the vibrational spectrum piecewise. Turin (1994) has estimated their minimum number to be 10. Second, the odorant-binding site could in principle be completely nonspecific, since all that is required is that the vibrations of the odorant be probed adequately by the spectroscopic receptor. Indeed, all receptor sites could be identical in structure and differ only in the segment of the spectrum that they probe, which makes it easier to understand why they would all have the same size cutoff. How this might be reconciled with the large number of odorant receptors found has been discussed above.

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B.

Turin and Yoshii

Weak and Strong Odorants

There are few reliable data sets on threshold detection values in the literature (van Gemert, 2000). The largest data set appears to have been laboriously collected over many years by the fragrance firm Givaudan-Roure. Their odor-value chart, which would be of considerable interest to researchers, is (understandably) proprietary. Even when differences in volatility are taken into account, odorants seem to differ in intensity by at least eight orders of magnitude. As was discussed above, odotope and vibrational theories differ crucially in how they account for this fact. Odotope theories regard odorless, weak, and strong odorants, respectively, as inactive, weak, and strong agonists. In other words, in an odotope theory, odorant intensity is in part related to the efficacy with which the molecule activates the olfactory receptors, not necessarily just to its affinity for the receptors. The difference between affinity and efficacy has been elegantly summed up by Colquhoun (1998): the affinity of a drug for its receptor is “simply the microscopic equilibrium constant for binding to the inactive state.” Efficacy is “the set of all the other equilibrium constants which describe the transduction events that follow the initial binding reaction.” For example, a molecule that binds best to the active receptor conformation will favor the conformation change from inactive to active (agonist), whereas one that binds tightly to the inactive state will be an antagonist.

Figure 21 undecanal.

Two extremes of odorant polarity: maltol and

In a vibrational theory, efficacy has a different interpretation. All molecules vibrate, and spectral intensities are likely to differ by only a factor of 20 or so between the smallest molecule with the weakest charges (e.g., methane) and a large molecule with large partial charges (e.g., a nitro musk). To account for the several orders of magnitude in odorant intensity, a vibrational theory must therefore assume that the strongest odorants simply bind most tightly to the receptors, i.e., that efficacy is proportional to affinity. There is, however, a physicochemical difficulty in accounting for the vast range in intensity of odorants simply by assuming that the strong ones bind more tightly. To be sure, odorants differ greatly in polarity, as reflected in their water-octanol partition (logP). LogP varies by six orders of magnitude: for example, maltol and undecanal—respectively, polar and hydrophobic strong odorants—have calculated logP values of 0.07 and 3.20 (Kantola et al., 1991) (implemented in Spartan, Wavefunction, Inc.) (Fig. 21). However, many very strong odorants, such as diacetyl and vanillin, are relatively polar. Clearly, some interaction

Figure 22 The strong (left) and “odorless” (right) isomers of lyral bound to a zinc ion via the carbonyl oxygen lone pair and the pi orbital on the double bond. In the weak isomer, the geometry is unfavorable to zinc binding, as reflected by the angle formed by the two bonds to zinc: 102° for the strong isomer, 75° for the weak one. Structures determined ab initio using Spartan software with 3-21G(*) parameters. (See color insert.)

Structure–Odor Relationships

other than hydrophobic partition is required to account for their intensity. C.

Structural Correlates of Odor Intensity

It has long been known empirically that certain structural features of molecules tend to make them stronger odorants. Moncrieff (1967) has listed many of these. The clearest correlates seem to be: (1) polar functional groups (OH, C
O, CN, SH, –O–, etc.) increase intensity; (2) unsaturation generally increases intensity; (3) steric shielding of a functional group decreases intensity; and (4) when two hydrogen bond acceptors are present, the odorant is stronger when they are close to each other (Ohloff bifunctional rule) (Ohloff, 1994). Taken together, these rules suggest that odorants may be binding to some ligand that has a high affinity for double bonds and lone pairs. Ohloff’s bifunctional rule (see, e.g., Fig. 22) is particularly interesting because it applies to a large number of structurally unrelated odorants and it is consistent with both functional groups binding to the same ligand. We propose that a zinc ion coordinated to the receptor protein may function as a ligand for odorants, as was suggested independently by Turin (1996) and more recently by Rakow and Suslick (2000).

291

Kollmann and his collaborators (Kuntz et al., 1999) have pointed out that there are three strategies to achieve high-affinity binding: perfect steric fit, e.g., biotin-avidin, unlikely with a set of molecules as diverse as odorants; lots of hydrogen bonds, difficult to achieve simultaneously with high-volatility hydrophobic molecules; and metal binding, which we believe is the strategy evolution has followed to achieve high-strength odorants. D.

Zinc Binding Is a Good Predictor of Odorant Intensity

A large number of olfactory receptor sequences have now been published, and new ones appear every month. While many of these sequences may be pseudogenes (Mombaerts, 1999b; Rouquier et al., 2000; Zozulya et al., 2001), it is now possible to form an accurate impression of their relationship to other known 7-TM receptors. Recently, a thorough study by Skoufos (1999) has shown that one of the most conserved regions (Region 3 at the cytoplasmic end of TM helix 6) corresponds to the zincbinding site proposed by Turin (1996). Interestingly, the histidine that binds the zinc is completely conserved (Zozulya et al., 2001). This is what would be expected if the zinc-binding site were essential to the operation of the

Figure 23 A sample of strong-weak isomer pairs: 1, methylanthranilate; 2, eudesmol, 3, neron, 4, p-menthane derivatives, 5, caparrapi oxide, 6, iridanes. In every case, the strong isomer (left) is a bidentate ligand for zinc, whereas the weak isomer has unfavorable geometry for zinc- binding. (From Ohloff, 1994.)

292

receptor, and in particular if it were the odorant-binding site itself. Remarkably, Sheikh et al. (1999) have shown that if two histidine zinc-binding sites are engineered at the cytoplasmic end of helices 3 and 6, then the presence of zinc prevents receptor activaion in two different types of 7-TNM receptors, suggesting that relative movement of helices 3 and 6 is essential. Furthermore, there is a good deal of circumstantial evidence linking zinc with olfaction and gustation. Turin (1996) pointed out that many strong odorants possessed structural features capable of bidentate binding to a metal ligand. This was recently confirmed (Suslick, 2000) by colorimetric measurements of odorant binding to metalloporphyrins. This binding accounts in a straightforward fashion for the fact that a hydrophobic zinc salt, zinc ricinoleate, is a very effective deodorant. There is also a good deal of circumstantial evidence linking zinc with olfaction and gustation. Zinc deficiency, either dietary (Alpers, 1994) or caused by treatment with histidine (Henkin et al., 1975), thiocarbamides (Erikssen et al., 1975), or captopril (Zumkley et al., 1985), is unique in causing a complete and rapidly reversible anosmia. We propose that this notion can be usefully extended by including pi-bonding from double bonds, triple bonds, and cyclopropane rings (Bader, 1990) as possible metal ligands. To test this, an unbiased data set is required. Ohloff’s (1990) review of strong-weak stereoisomer pairs provides such a set, since it was selected without this theory in mind and the odor threshold data are reliable. A good example is provided by double-bond isomers of lyral (Fig. 22). When the three-dimensional structure of these molecules is calculated and bidentate binding to zinc between the carbonyl oxygen and the double bond is included (Fig. 22), it becomes clear that only the strong isomer (mol) can bind to zinc in this fashion. The same idea explains intensity differences between many other isomer pairs described by Ohloff. Figure 23 illustrates some of these instances. Examples 1, 4, and 6 follow Ohloff’s bifunctional rule; the remainder involve a double bond and a functional group. There are, however, many exceptions to this rule, namely those molecules for which enantiomers, isomers, or diastereomers have different intensities without there being more than one functional group capable of binding to zinc. Examples of this are muscone and Mayol. Clearly, this can have little to do with zinc binding and must be due to steric interactions within the receptor site (Fig. 24). Many of the best correlations obtained between structure and “odor” are actually done in such a way that what is being tested is the effect of structure on odor intensity rather than on odor character. For example, QSAR studies of musks (Yoshii, 1991, 1992; based on the data of Wood, 1968–1970) have been conspicuously successful in predicting odor inten-

Turin and Yoshii

Figure 24 Strong (left) and weak (right) isomers of muscone (top) and mayol (bottom), illustrating that differences in odor intensity cannot always be ascribed to the accessibility of one or more metal-coordinating groups.

sity. If one accepts the notion that a intensity is not an odor character, but reflects the ability of the odorant to bind to the receptor, then it becomes clear that what these studies are probing is the size and shape of the binding site. VIII.

SUMMARY AND CONCLUSIONS

In summary, it seems fair to say that if the ultimate goal of a theory is predictive power, then both odotopes and vibration still fall short. Neither theory, when faced with a novel molecule, is yet able to predict reliably what its odor character will be. Vibrational theory is conspicuously successful at explaining the fact that we smell functional groups even when sterically hindered and in accounting for differences in smell between isotopes. Odotope theory explains neither. By contrast, vibrational theory is intrinsically unable to explain differences in the intensity of different odorants, or which members of a set of related odorants will be odorless. We propose as a working hypothesis, to be tested by further experiment, that odor character is determined by molecular vibrations and odor intensity is determined almost entirely by molecular shape. We agree with Beets (1957) that, although the present theories may be incomplete, “we need not consider the question of whether a relationship exists between structure and odor. . . . The only question is whether it is simple enough to be detectable with our limited intellectual and technical means.” The fact that after several decades of experimental investigations, the basic mechanism by which odors are detected remains open to question shows that there is much work to be done. At the present rate of discovery, is to be expected that the answer to these questions may come in time for the next edition of this handbook.

Structure–Odor Relationships

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14 Olfactory System Cybernetics: Artificial Noses Krishna C. Persaud University of Manchester Institute of Science and Technology, Manchester, United Kingdom

I.

INTRODUCTION

the study of messages as a means of controlling machinery and society, the development of computing machines and other such automata, certain reflections upon psychology and the nervous system, and a tentative new theory of scientific method.” The developments in “electronic nose” technology now herald the birth of “olfactory system cybernetics.” The motives arise from (1) the perceived limitations of traditional analytical chemistry and instrumentation in classification of gas mixtures or odors; (2) applications where the gestalt of a defined mixture of chemical species may be important in perception, applications where it is important to separate subjective and objective assessments; and (3) the commercial drive to achieve devices capable of operating rapid, on-line process measurement and control in areas of foods, beverages, chemical industries, and waste management. Another motive driving research is the attempt to create biomimetic devices that emulate aspects of biological sensory systems. The developments described in this chapter were based on the coalescence of several evolving scientific disciplines where key developments and concepts were evolving and converging in the last four decades, leading to a veritable explosion in the last decade. Today, electronic noses abound in various configurations and are used in specific applications, but “artificial noses” do not yet exist.

The remarkable capabilities of biological chemosensory systems in detecting, recognizing, and discriminating complex mixtures of chemicals, together with rapid advances in understanding how these systems operate, has stimulated the imagination and interest of many researchers and commercial organizations. Such stimulation has led to the development of electronic analogues. An artificial sensing system that emulates the human sense of smell is of upmost need in a number of fields, including the food, flavor, beverage, and cosmetic industries, as well as environmental protection industries and governmental agencies. Although the human sense of smell has been used for centuries in applied industrial settings in quality control and other processes, it is liable to variation from illness and other factors, including subject age, gender, and training. Moreover, there are linguistic limitations in communicating odor experiences among individuals. While, as noted in other chapters of this volume, the human sense of smell is exquisitively sensitive and can provide reliable estimates of odorant intensity and quality, there can be considerable variability of response among untrained individuals to different odors and odor concentrations. An instrument that could perform simple odor discrimination and provide an accurate indication of odor intensity with less variation than that observed in human responses would be very useful in modern industry. Cybernetics was defined by Norbert Wiener (1948) as “the theory of control in engineering, whether human or animal or mechanical”, including “not only the study of language but

II.

KEY CONCEPTS

Some of the key ideas and developments inspiring the multidisciplinary evolution of olfactory cybernetics are 295

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worthwhile noting. The survival of animal life in complex, changing environments requires the use of sophisticated sensory systems to detect, classify, and interpret patterns of input stimulation. This involves the development of a coding mechanism by which a certain pattern of stimulation may be described. Such a code may be defined as a set of symbols that can be used to represent patterns of organisation and the set of rules that governs the selection and use of these symbols. Sensory coding mechanisms in biological systems would appear to project some representation of a pattern at a high level of the nervous system, the resulting neural activity being related to the previous experience with regard to this pattern or associated patterns (Uttal, 1973). The rapid evolution of research into artificial intelligence, leading to devices that classify and interpret patterns, has resulted in fundamental mathematical understanding of how patterns may be encoded and classified by biological systems. The seminal papers setting out the key requirements in the design of parallel feature detection systems with learning capabilities (e.g., Minsky and Papert, 1969; Rosenblatt, 1962; Selfridge, 1959), together with recent advances in microelectronics, have led to the employment of some of these features in “intelligent toys” for children. Some of the fundamental principles of pattern classification that seem to be common to biological and artificial systems include “template matching,” whereby the pattern to be classified is compared with a set of templates, one for each class, the closest match determining the classification, and “feature detection systems” in which a number of measurements are taken on the input pattern and the resulting data are combined to reach a decision. These systems may involve either a sequential approach, in which information from the evaluation of some features is used to decide which features to evaluate next, or a parallel approach, where information about all features are evaluated at the same time with no weight being placed on any particular feature. A characteristic of the activity of the nervous system is that it is persistent throughout the life of the organism and so is in some sense stable. However, it also appears to have a stochastic nature that is essential—a nervous system that is stereotyped would not exhibit adaptive behavior in a changing environment. Animals have goal-driven behavior that works reliably in an unpredictable world. The aforementioned methods of pattern classification can be elaborated so that stochastic behavior is achieved, and many models that may be applicable to the sensory systems of animals have been developed. Such models date from several sources, including (1) the early work of Turing (1952), who suggested that an initially chaotic or inhomogeneous state is moved by random fluctuations towards a more stable state, (2) concepts of catastrophe theory expressed by

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Thom (e.g., Thom, 1970, 1972), and (3) concepts of neural mechanisms based on a model of the brain as a spatio-temporal lattice of nonlinear processing elements (e.g., Freeman, 1974, 1991). Data from a number of sources, including electrophysiology, chemical structure-activity studies, psychophysics, and molecular genetics, support the view that a finite number of olfactory receptor classes exists and that a given class of receptor cells responds to a range of molecular entities, with some overlap among classes (e.g., Amoore, 1962; Beets, 1978; Boelens, 1974; Buck, 1997a, Chess et al., 1992; Mombaerts et al., 1996) Molecular parameters important for determining an olfactory response are varied but likely include the adsorption and desorption energies of the molecule from air to a receptor interface, partition coefficients, electron donor/acceptor interactions (depending on the polarizability of the molecule), and molecular size and shape. The rapid advances in neurobiology, electrophysiology, and biochemistry that were occurring concurrently in the 1960s, leading to an understanding of the convergent nature of the olfactory system (Shepherd, 1998; see Chapters 4–10). This allowed models of the sensory systems for the instinctive recognition of patterns, including olfaction and taste, to be constructed, as exemplified by Deutsch (1967) based on an idealized organization of the cortex of the brain. It is useful to recapitulate such an approach. A compound is taken to contain primary elements or stimuli to which the system can respond, e.g., compound X contains the elements A, B, and C. A complex matrix of neurons make the system concentration-independent. These “amplitude-matching neurons” consist of a set of neurons, each of which is set to trigger at a different concentration. The output of these neurons converge to the common gate of an output neuron, so that the output from this neuron is concentration independent. Another matrix of neurons is proposed that finds the ratios of the components A/B and B/C. These produce an output to the sensory pattern output neurons, where the output of the stimulus ratio neurons are recognised as unique to compound X. The system is inherently flexible and has room for excitatory and inhibitory control at each stage, making it an attractive proposition for olfactory cybernetic development. This understanding that receptors with partial specificity could in principle be used to achieve reliable discrimination was later exploited by Persaud and Dodd (1982) to achieve a functional device using just three broad specificity sensors. From the discussion above, it may be recognized that four main problem areas could be early identified in the design of a ‘model’ olfactory system: 1.

The selection of an effective set of parameters for the description of the patterns in question. Where an odor is concerned, the problems to be addressed

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are which characteristics of the molecule can be measured, and how to encode the resulting pattern. 2. The selection of the decision procedure—how is the receptor output to be categorized? 3. The selection of a procedure for processing the parameters chosen to represent the pattern so as to optimize the parameter values. This increases the resolution of the system so that certain representative pattern classes may be differentiated from others. 4. The selection of “hardware” required for the simulation of the transduction, coding and pattern recognition activities of the olfactory system.

III.

ARRAY-BASED ODOR SENSORS

The discriminatory power of a small sensor array lies in the utilization of cross-sensitivities between sensor elements. The responses of the individual sensors, each possessing a slightly different response towards the sample odors, when combined by suitable mathematical methods, can provide enough information to discriminate between sample odors. These systems have been given the terminology “electronic nose” and consist of an array of chemical sensors possessing broad specificity, coupled to electronics and software that allow feature extraction of salient data for further analysis, together with pattern recognition giving identification of sample odor. Software techniques and material science are important aspects of the development of the system. Advancement in software signal processing techniques, coupled with pattern recognition, enable optimum usage of sensor responses. The specificity and sensitivity of existing chemical sensors are constantly being developed, as well as new materials. Typically an electronic nose consists of three elements: a sensor array which is exposed to the volatiles, conversion of the sensor signals to a readable format, and software analysis of the data to produce characteristic outputs related to the odour encountered (Fig. 1). In order to discriminate between samples, the output from the sensor array may be interpreted via a variety of methods such as pattern recognition algorithms, principal component analysis, discriminant function analysis, cluster analysis, and artificial neural networks. A.

Sensor Technology

Early research in the field was hampered by the lack of suitable sensor materials capable of emulating the functional characteristics of the olfactory system, as well as by the limited understanding of the transduction and information

Figure 1 Elements of an electronic nose system. Odor is sampled and presented to individual elements of a sensor array simultaneously. Signals transduced from the sensors are converted to digital format, normalized, and presented to a pattern-recognition engine (PARC) that outputs some parameter that is a determining characteristic of the incoming odor. This may be discrimination from other known odor classes or a mapping to some sensory quality determined by a human panel.

processing systems of biological systems. Tanyolaç and Eaton (1950) explored the surface tension changes of a liquid when volatile molecules were adsorbed, while the advent of the transistor stimulated interest in utilizing germanium as a chemically sensitive transducer material. Among the pioneers who attempted to emulate functional properties of the olfactory system electrically were Dravnieks and Trotter (1965), who measured contact surface potentials of various materials in the presence of adsorbed volatiles, Wilkens and Hartmann (1964), who measured redox potentials when various chemicals were adsorbed onto electrodes, and Buck et al. (1965), who investigated conductivity changes in solids due to interactions between adsorbed odorant molecules and charge carriers in the solid. However, it was not until the commercial availability of gas sensors based on metal oxides in the 1970s (Figaro Inc., Japan) that it become practical to test some of the conceptual ideas, leading, for example, to the work of Persaud and Dodd (1982). The general architecture of this early device, although based on an array of only three sensors and the somewhat limited knowledge of the time of how olfactory processing occurs, forms the basis for most of the instrumentation used today. Such devices have become known as electronic noses (Gardner and Bartlett, 1994) and consist of “an instrument, which comprises of an array of electronic-chemical sensors with partial specificity, and an appropriate pattern recognition system, capable of recognising simple or complex odours” such as shown in Figure 1. A large number of sensor technologies are now available that are applicable to construction of sensor arrays for

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Figure 2 Common sensor technologies used in electronic noses: quartz crystal microbalance, metal oxide sensor, conducting polymer.

electronic nose applications. Figure 2 illustrates three common sensor types: quartz crystal microbalance, metal oxide, and conducting polymer. The sensors must meet key design parameters for the system. These include sensitivity, speed of operation, cost, size, manufacturability, the ability to operate in diverse environments, and immunity to contamination or deactivation (i.e., “poisoning”). The sensors must adsorb large numbers of molecules of a particular species to produce a measurable effect on the sensor that can be transduced into a signal. 1. Metal Oxide Sensors Many researchers have chosen commercially available metal oxide sensors such as Taguchi Gas Sensors (TGS) (Figaro Inc., Japan) or Capteur Sensors (Capteur, UK) as the core sensing element in their investigation of arraybased odor detectors. These devices consist of an electrically heated ceramic pellet onto which a thin porous film of SnO2 doped with various precious metals has been deposited. The doped SnO2 behaves as an n-type semiconductor and the chemisorption of oxygen at the surface results in the removal of electrons from the conduction band. Gases interact with the surface adsorbed oxygen and thereby affect the conductivity of the SnO2 film. The devices are run at elevated temperatures (typically 300–400°C) to achieve rapid response/recovery times and to avoid interference from water. This results in relatively high power consumption. The response characteristics can be tailored by varying the operating temperature and the

doping agent. The physical and chemical mechanisms by which gases can transduce signals are relatively well understood (Kohl, 1989, 1991, 1996). Sensors have been developed for the detection (down to the ppm level) of a range of target molecules including H2, CO, NH3, H2S, NOx, SOx, ethanol, and hydrocarbons. As an alternative to the commercially available metal oxide sensors, several research groups have fabricated thin film SnO2 arrays using planar microelectronic technology. Potentially these could have a number of advantages including a reduction in size and lower power consumption. An elegant variation on this device technology is a sensor array with a gradient of temperature across the surface as well as a membrane sputtered on the surface (Menzel and Goschnick, 2000). These arrays are now commercially manufactured and incorporated into electronic nose devices (Ehrmann, 1998). 2.

Quartz Resonator and Surface Acoustic Wave Devices

Quartz resonator gas sensors consist of a piezoelectric quartz crystal oscillator coated with a sensing membrane. Typically a quartz disk is sandwiched between two electrodes. The adsorption of volatile molecules onto the membrane results in a decrease in the resonant frequency due to the increased mass. This frequency shift can be used as the sensor output, and the device response can be varied by using different membrane materials (Nanto, 1997).

Olfactory System Cybernetics

Surface acoustic wave (SAW) and bulk acoustic wave (BAW) devices consist of interdigitated electrodes fabricated onto a piezoelectric substrate (e.g., quartz) onto which a thin film coating of a selective material is deposited. An applied radio frequency voltage produces a Rayleigh surface acoustic wave (i.e., a surface oscillation). Adsorption of odors onto the coating increases its mass and perturbs the wave, leading to a shift in frequency. To compensate for pressure and temperature effects the sample sensor is usually connected to a reference SAW device and the frequency difference is detected. As with quartz resonator devices the coating material determines the selectivity. SAW devices, however, can be operated at higher frequencies, which reportedly results in improved sensitivity (Rapp and Reibel, 1996; Yang, et al. 2000). The advantages of SAWs and BAWs include high selectivity, high sensitivity, stability over wide temperature ranges, low response to humidity, and good reproducibility. The disadvantage is the complexity in the interface electronics. 3. Electrochemical Sensors Electrochemical sensors are widely used for detection of specific chemical analytes. Typically a redox reaction is induced to occur at the anode or cathode of an electrochemical cell when a specific gas is dissolved in the electrolyte. Commercial sensors are made by a variety of companies such as City Technology Ltd, UK. A variety of amperometric or coulometric devices are applicable to sensor arrays for electronic noses (Stetter, 1995).

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advantages include high sensitivity (ppm), high selectivity, and ease of integration with other electronics. A potential disadvantage is that the odorant molecules must penetrate the transistor gate, and the devices are not yet available commercially. Variations incorporating conducting polymers as chemical sensing surfaces have been devised (Hatfield et al., 2000). 6.

Optical Sensors

A fiber optic sensor for gas sensing is a conventional optical fiber typically coated with a coating, which interacts with the odorant molecules. The coating may be a fluorescent dye. An optical pulse is applied to the sensor and is adsorbed by the coating. The interaction of the odorant molecules and fluorescent dyes produces a frequency shift in the returned fluorescent signal. The returned signal is then analyzed to determine the properties of the odorant molecules. Porphyrins and other materials may also be used as the active adsorbent material in optical sensing (Di Natale et al., 2000). More recently, microscopic polymer beads impregnated with a fluorescent dye have been incorporated into the ends of optical fibers, and these have proven to be extremely sensitive to vapors, and “optical electronic noses” have been developed. Imaging optical fibers in conjunction with twodimensional detectors such as CCD cameras have been used to fabricate array sensors. These sensors contain spatially separated photopolymers containing analyte-sensitive fluorescent indicators on an imaging fiber tip. Spatial resolution of the indicators is maintained through the imaging fiber array and projected onto a CCD detector (Dickinson et al., 1996, 1998, 1999; Walt et al., 1995).

4. MOSFET Sensors MOSFET’s (metal oxide semiconductor field effect transistors) have been used as gas detectors, the vapor producing a shift in the capacitance-voltage characteristic. MOSFET sensor behavior can be modified with coatings of zeolite of various pore sizes or by careful attention to a gas-sensing layer in close proximity to the gate of the MOSFET, resulting in a change of capacitance on exposure to the gas. Using arrays, patterns can be generated for a range of solvent volatiles and chemicals such as ammonia and hydrogen (Gardner, 1998; Stetter et al., 2000). Some “Electronic Nose” companies now commercially use such sensors. 5. ChemFETS A chemical field effect transistor (ChemFET) is a transistor with the gate electrode coated with a selective coating. This coating adsorbs odorant molecules, which changes the conductivity across the transistor’s gate. The

7.

Mass Spectrometry–Based Devices

“Virtual” chemical sensors based on a mass spectrometric approach have been developed for multicomponent analysis of organic vapors. The sensing principle is based on the injection of a complex sample headspace into a mass spectrometer, creating a mass spectrometric pattern of the unresolved gaseous mixtures. After selecting particular fragment ions, the resulting reduced mass spectrum of the sample is treated with pattern recognition (software) typically based on principal components analysis or neural networks (Dittmann and Nitz, 2000; Dittmann et al., 1998, 1999, 2000). This methodology has been commercialized by a number of companies such as Agilent Inc., USA HKR Sensor Systeme (Germany), and Alpha MOS (France). 8.

Carbon Black/Polymer Composite Materials

Cyrano Sciences Inc. USA have commercialized portable instruments based on 32 carbon black/polymer composite

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1.

The sensors show rapid adsorption and desorption kinetics at room temperature. 2. The sensor elements feature low power consumption (in the order of microwatts) as no heater element is required. 3. The structure of the polymer can be closely correlated to specificity towards particular classes of chemical compounds. 4. The sensors are resilient to poisoning by compounds that would normally inactivate some inorganic semiconductor type sensors.

materials. The carbon black forms the conducting phase of the sensor and is dispersed into an insulating organic polymer. When the polymer comes into contact with an organic vapor it swells, causing a change in the electrical resistance of the sensor. An electrical potential is applied across each sensor so that the resistances may be recorded. The use of different polymers, such as those used in gas chromatography columns, gives the sensors a range of sensitivities to a variety of chemicals (Lewis and Freund, 1996; Lewis and Severin, 1997). 9.

Organic Materials

Much research has been carried out on sensors based on metal-substituted phthalocyanines, each acting as a simple chemoresistor. Their applications have been limited to detection and measurement of gases such as NO2 and H2S (Cranny and Atkinson, 1992), and they have suffered from poor individual sensor response and reproducibility. On the other hand, the unique electrical properties of organic conducting polymers, derived from aromatic and heteroaromatic materials, have led to a large amount of research and the application of these materials in different areas. Since 1979, when Diaz et al. (1979) first prepared polypyrrole as a freestanding film, thousands of publications have appeared and many researchers have studied conducting polymer gas sensors based on resistance changes in thin film structures. Configurations have been used to measure the shifts in the work function caused by the adsorption of a range of organic volatiles. The response of the polypyrrole film (i.e., the magnitude and the sign of the work function shift) is determined by the electrochemical deposition conditions and, in particular, the electrolyte/solvent system used. Measurements have also been made of the change in the optical absorption spectra resulting from exposure to organic vapors. These data, together with the work function shifts, suggest a small but reversible charge transfer (either donor or acceptor) when a gas is adsorbed at the polymer surface. Gas sensors using polypyrrole films deposited as an overlayer onto quartz resonator devices have been investigated by Slater and coworkers (Slater and Watt, 1991). In addition to monitoring the mass loading affect of adsorbed volatiles, a simultaneous measurement of conductivity changes was made on a separate device. A range of volatiles has been studied including NH3, methanol, cyclohexane, acetone, and H2S. Since 1985, we have concentrated on development of conducting polymers as odor-sensing devices, and many materials have been synthesized and characterized for odor transduction (Persaud and Pelosi, 1985; Persaud et al., 1996b). The reasons for choosing conducting polymers as odor sensor elements are as follows:

10.

Electronic Nose Hybrid Technology

Neither the manufacturer nor the user is restricted to using only one type of technology, and no one technology is yet generally applicable to generic odour sensing. Hence the use of modular sensor systems, or integrated sensor systems containing different sensor technologies, is a growing trend (Kohl, 1997; Stetter et al., 2000). B.

Sensor Output and Data Processing

Figure 3a shows a commercial instrument, the Aromascan A32S (Osmetech plc), based on a conducting polymer sensor array (Fig. 3b). This was launched in 1994, and several hundred units were sold. No matter which sensor technology is utilized, the raw data response of each sensor in the array to a volatile sample such as shown in Figure 4a is normalized as shown in Figure 4b, and the resulting pattern is used as descriptor for discrimination between different samples. Sensing systems have been developed where individual elements in an array show broad and overlapping selectivities to chemical species, each sensor element responding more selectively to certain groups of chemicals. This approach has the advantage that the array can respond to many thousands of chemical species due to the broad selectivity of the adsorbent surfaces. On the other hand, extremely selective information for discrimination between adsorbed chemical species or mixtures can be obtained by analysis of the cross-sensitivities between sensor elements. The relative responses between sensor elements produce patterns that serve as unique fingerprints that may be used as odor descriptors. Algorithms to eliminate noise are needed in some systems, since there are often uncontrolled factors present in measurement data due to effects such as day-today variations in ambient temperature and instrument signal drift, which can introduce systematic error into the data. The aim of many pattern recognition techniques is to identify similarities and regularities present in the data. One method is cluster analysis, which attempts to find natural

Olfactory System Cybernetics

(a)

(b) Figure 3 (a) Commercial electronic nose system—Aromascan A32S. (Courtesy Osmetech plc). (b) Conducting polymer sensor array (32 sensors) used in the system.

classifications in data. Computers are usually used as an aid in the cluster analysis of data of more than three dimensions (since it is difficult for humans to visualize such vector spaces).The centers of clusters are represented by a set of coordinates, and these are called codebook vectors. A typical chemometric goal in electronic nose applications is to find variables to separate known groups and, in particular, to be able to classify a gas or mixture of gases according to various properties. A primary aim of chemometrics is to reduce the number of dimensions used to

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represent the characteristics of the data set. Various methods are available to accomplish this, either by considering only a subset of the original variables or by creating more efficient representative set of new variables. The creation of new variables can be approached in a number of ways; two of these are projection and mapping. Projection is more common and involves using a weighted linear combination of the original variables to derive a new, more compact data set that contains nearly the same informational content as the original variables. Clearly this involves a certain amount of loss of data, and this is not ideal since key features may be discarded. Thus, techniques have been developed to minimize information loss. A commonly used projection technique is principal components analysis (PCA) (Hotelling, 1933). The purpose of the PCA transformation is to rotate the old coordinate system in a direction so that the new system will have most of the relevant information aligned along a few new axes. The majority of the new axes will carry a very small proportion of the total information and could be disregarded without too much loss. It attempts to maximize the variance information present in data in the minimum number of mutually orthogonal dimensions. Graphically, PCA distorts the axes to conform to axes that contain a maximum of variance information. One way to describe this would be to consider looking at the data from a different direction in space. Algebraically, this can be performed as a simple linear transformation in any number of dimensions (Fig. 5). Mapping is a similar technique to projection, but the data transformations are nonlinear and attempt to preserve key properties of the data (such as the distances between points), while reducing the number of dimensions. Other chemometric techniques applied include partial least squares (PLS), discriminant analysis (DA), and discriminant factorial analysis (DFA). DFA is a multivariate technique that determines a set of variables that best discriminates one group of objects from another. For statistical pattern recognition algorithms to be successful, there must be some criteria for them to base the allocation of different classes, and this is usually based on cluster analysis. A given cluster of points representing a class has a center of gravity and a standard deviation distance that specifies the location and spread of the cluster. These values are obtained during the training phase of the algorithm, with replicate examples that are known to be members of a given class. Artificial neural networks (ANNs), which have been used to analyze complex data and to recognize patterns in many other disciplines (Rumelhart et al., 1986), have shown promising results in recognition of volatile compounds and odors in electronic nose applications. When an ANN is combined with a sensor array, the number of

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Figure 4 (a) Raw data response from a sensor array made from conducting polymers. Each sensor responds to the same concentration of analyte with different sensitivity. By normalizing the response across the entire array, a pattern (b) is obtained that is a descriptor of the analyte presented to the sensor array.

detectable chemicals is generally greater than the number of unique sensor types. The advantages of ANNs over other methods of pattern recognition are numerous. Their performance is less affected by noisy or incomplete data. Another advantage is that they do not require linearly separable data sets. Many ANN configurations and training algorithms have been used to build electronic noses, including (1) backpropagation-trained feed-forward networks, (2) self-organizing maps (SOMs) (Kohonen, 1989), (3) learning vector quantizers, (4) fuzzy ARTmaps, (5) Hamming networks, (6) Boltzmann machines, and (7) Hopfield networks. Classification systems applicable to electronic noses are reviewed by Horner (1995). Modern neural networks may be either realized in hardware or a simulation program running on a computer. The software simulation, although generally slower, is less expensive and more flexible than

the hardware counterpart. In both cases the network consists of a series of processing nodes arranged into different layers and, as such, mimics the neurons in the brain. Each element is relatively simple, and is typically two function blocks, as shown in Figure 6a. All neural networks consist of many interconnected nodes, each having several inputs xi and a single output y. Weights wi are associated with each input, and the weighted sum S is calculated according to the formula: S


n

兺 wi xi i
0

(1)

The weighted sum passes through some thresholding function, which is a nonlinear in-output transfer function. In some systems, if the value of S exceeds the threshold function, then the output is 1. If S is less, then the output is 0. This is termed hard limiting.

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Figure 5 A principal component analysis of replicate samples of different analytes. Each point represents a multidimensional pattern from 32 sensors projected onto two dimensions.

The Perceptron was invented in the 1950s. It has an input and an output layer and contains multiple processing elements (PEs). Perceptrons can solve pattern recognition problems only if a hyperplane can separate the classes. A feedforward network has the ability to separate complex data. Such a network is termed a multilayer perceptron (MLP) if it uses the gradient descent method to learn. The multilayer perceptron is an extended version of the perceptron (see Fig. 6b). It contains inner (or hidden) layers, and the structure can be related to the neuron/synapse structure of the brain. It is these elements that store the learned responses. The choice of the number of layers and the number of elements in each layer is crucial, since too few elements can cause slow learning and inaccurate responses, but too many elements can cause redundant copying of the input layers. We can define the following terms. A single unit will be referred to by the indices i and j attached to the symbols listed below. Unit j may be on any layer of the network, but where both i and j are used, unit j will be closer to the output layer than unit i. The values n and m denote the number of units in the current layer. wji denotes the strength of the weight from unit i to unit j on the next layer. The weight change to be made is denoted by Wji. oj denotes the output from unit j. vj is the input to unit j. dj is the desired output from unit j. ej is the error derivative on unit j. f(•) is the network activation function, and f ’(•) is the

derivative of the activation function. is the learning parameter,  is the momentum parameter. If one considers the activation function from Eq. (1) and generalizes it, then it sums the product of the output from each unit below and the weight with which it is connected to the current unit. oi
f

冢兺w o 冣 m

ji

i

(2)

i
1

The simplest and most common output measure is the difference between the output from a unit oi and the desired value di for that unit. The error derivative for the output unit is calculated as: ei
f ' (oi) (di  oi)

(3)

so that the error is now expressed as the derivative of the activation function. Errors may now be backpropagated to the hidden layer, where the error on each hidden unit in layer i is calculated as the summed product of the error derivatives ej of the units in row j above and the strengths of the weights connected to them. ei
f '(oi)

n

兺 ejwji j
1

(4)

The weights may then be updated over the whole network shown in Figure 6b, the change in weight from unit i

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Figure 6 (a) Functional block of a node in a neural network comprising a summation of several inputs followed by a nonlinear transform. (b) Architecture of a multiplayer perceptron, a commonly used neural network architecture.

to unit j is calculated as the product of the learning rate, the error derivative for unit j and the output from unit i: wji
 ej oi

(5)

where unit i is in the layer below unit j. Hence by presenting exemplar patterns to the input nodes of a neural network, it may be trained so that only specified output nodes are activated for a given class of pattern. The two most commonly used approaches for electronic noses are backpropagation-trained feed-forward networks and SOMs (Ziegler et al., 1998). Backpropagation is a supervised algorithm that must be trained with labeled odors. It learns the relationship between the sensor values and the given odor labels. A SOM is an unsupervised algorithm that does not require predetermined odor classes for training. It essentially performs clustering of the data into similar groups based on the measured attributes or features that serve as inputs to the algorithm. It can be thought of as way of projecting multiple dimensions (often each dimension represents a different sensor output or a feature extracted from the sensor array) onto a two-dimensional output allowing the user to visualize the groupings and relationships of the odors or chemical volatile compounds.

IV.

APPLICATIONS

Electronic noses are in commercial use for a wide variety of odor and volatile compound applications. Their most popular applications currently are in food processing, environmental monitoring, medical diagnostics, process control, and fragrance development. A.

Food Industry Applications

Traditionally, food quality was assessed by panels of human experts and through application of analytical chemistry methodology. These are costly and time-consuming and can be difficult to calibrate and establish sustained reliability. Electronic noses do not replace sensory panels, but they have useful roles in the food processing industry, especially where simple comparisons need to be made. A host of applications tested include quality assessment in food production, inspection of food quality by odor, control of food cooking processes, inspection of fish, monitoring fermentation processes, checking for rancidity and spoilage, establishing fruit ripeness, verifying sources of juice concentrates, and inspecting packaging material for malodors (Ali and O’Hare, 1997;

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Figure 7 Use of a neural network to predict aging of biscuit samples. As the biscuits age, the fats oxidize and the measured odor pattern changes. The neural network is then used to map these changes so that the lifetime of the product can be predicted in terms of weeks of storage life. Each point corresponds to an odor-measurement pattern, and these are mapped to time from the fresh control sample by the neural network.

Aparicio et al., 2000; Boerjesson et al., 1996; Brezmes et al., 2000). Figure 7 illustrates an application using conducting polymer sensor arrays in investigating the shelf life of biscuits where oxidation of fats cause changes in smell and taste of the product prior to the onset of rancidity. In this case a neural network–based prediction system was developed using radial basis function neural network architecture.

an electronic nose. Odors coming from body fluids can indicate metabolic problems as well as infections. Other extremely interesting biological applications are now being developed. These include monitoring mammalian cell growth (Bachinger et al., 2000), DNA detection (Clausen-Schaumann, et al., 2000), and growth of bacterial cell cultures (Gardner et al., 1998).

B.

C.

Medical Diagnostic Applications

Smell used to be a common diagnostic tool in medicine, and physicians were trained to use their sense of smell during their medical training. In modern times, odor diagnostics have been relegated to secondary status as a diagnostic method only applicable in primative settings. Electronic noses now offer the potential of a robust analytical approach to odor measurement for medical diagnostics (Gardner et al., 2000; Gibson et al., 1997). Electronic nose technology has been used to examine odors emitted from the body such as from breath, wounds, and body fluids, and to identify possible problems, such as bacterial vaginosis (Chandiok et al., 1997). Breath analysis can be used to diagnose gastrointestinal probems, sinus problems, infections, diabetes, and liver problems. Infected wounds and tissues emit distinctive odors that can be detected by

Environmental Applications

Environmental applications of electronic noses increasingly include agricultural malodor applications. Malodors emanating from cow and pig slurries are an increasing source of environmental pollution, as well as an odor nuisance to human populations in the vicinity. Many substances produced during the anaerobic digestion of feces have very low human olfactory thresholds and so are perceived as odor nuisances at very low concentrations in air. These include volatile fatty acids, p-cresol, amines, sulfhides, disulfide, mercaptans, and many heterocyclic compounds. It is now possible to correlate sensor responses to odor measurements derived from olfactometry using a human panel (Misselbrook 1997; Persaud et al., 1996 a, b). Monitoring of indoor air quality is also an important application (Hathcock, Jr., 1999). A hybrid system was

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used aboard the Russian space station MIR to monitor air quality within the station (Persaud et al., 1999). The problems of monitoring industrial chemical hazards may be addressed by array sensing technology (Huby, 1999; Khopkar, 1998; Stetter et al., 1984). Contaminating residues of insecticides (lindane and synthetic pyrethroids) and products from the manufacture of leather and plastic products (phenols, nitrobenzene, anilines) are often offloaded into streams or rivers, despite legislation and in disregard of the danger they pose to the health of the population and the survival of fish and flora. Electronic nose devices may prove promising in such monitoring applications (Baby et al., 2000). D.

Other Applications

Many interesting applications are being developed, and the state of the art is reviewed in Gardner and Persaud (2000). They include fruit ripening, determining the origin of olive oil, spoilage of meat and fish, grain quality, fermentation monitoring, city pollution, and thousands of others. It is clear that the perceived usefulness of such technology is high.

V.

THE FUTURE

With any new technology, there are many unanticipated problems. The field of odor sensing is a multidisciplinary area that attracts academic and industrial scientists from a wide range of fields. Perceived problems that need further development include sampling methodology, transferability of databases from one sensor array to another, repeatability and reproducibility over the long term, the need for standardization of test protocols, and improved sensor technology. All of these areas are in active development, and it would appear that there is a long-term future for this technology. At this point, many devices exist that are applicable to specific tasks. The electronic nose does not yet emulate a human nose, and the development of an artificial nose, where the output code from the sensor array can be easily correlated to human sensory perception, is still some time away.

REFERENCES Ali, Z., and O’Hare, L. (1997). Analyzing food flavors, Chem. Rev. (Deddington, U.K.) 6:2–7. Amoore, J. E. (1962a). The stereochemical theory of olfaction. 1. Identification of the seven primary odours. Proc. Sci. Sec. Toilet Goods Assoc. 37:1–12. Amoore, J. E. (1962b). The stereochemical theory of olfaction. 2. elucidation of the stereochemical properties of the olfactory receptor sites. Proc. Sci. Sec. Toilet Goods Assoc. 37:13–23.

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15 Olfaction and the Development of Social Behavior in Neonatal Mammals Richard H. Porter Institut National de la Recherche Agronomique/Centre National de la Recherche Scientifique, Nouzilly, France

Benoist Schaal Centre Européen des Sciences du Goût, Dijon, France

I.

INTRODUCTION

than unfamiliar or unrelated animals. As discussed in the present chapter, volatile chemicals play an important role in the mediation of early interactions among conspecifics, as well as in the development of social discrimination and preferences (see also Chapter 17). Although the primary focus of this chapter is the influence of biological odors on adaptive behavioral responses and social preferences of human infants, studies of other species are cited when appropriate to illustrate or clarify particular points. The term “olfaction” is used to refer to the perception of odorants even when the underlying chemosensory systems have not been identified precisely. Thus, the possible involvement of chemical senses other than the olfactory system per se (e.g., trigeminal nerve, vomeronasal organ, nervus terminalis) is subsumed under this label.

The survival and development of newborn mammals depends upon complex coordinated interactions with the mother and, at least in some species, other conspecifics, including the father and siblings. Although neonates cannot live without the resources provided by their mother, they are not simply passive recipients of maternal care. Beginning shortly after birth, mammalian young exhibit species-typical behaviors that contribute to their own wellbeing. Mothers in particular are highly sensitive to signals that communicate the physiological and motivational state of their infants. For example, when removed from their nest, mouse and rat pups emit ultrasonic “distress” cries that attract their mother and thereby enable her to retrieve them (Allin and Banks, 1972; Noirot, 1966; Zippelius and Schleidt, 1956). Isolated lambs bleat, similarily facilitating parent-offspring reunion and nursing (Walser et al., 1984). In a reciprocal manner, adults communicate via various sensory modalities with their newborns, who, in turn, display overt responses to their parents (or cues that they produce) that are critical for survival. The ability of mammals to discriminate between individuals or members of different social categories is evident at an early age (Colgan, 1983; Hepper, 1991; Holmes, 1991). Suckling young of many species rapidly develop the ability to recognize their own mother and respond preferentially to her as compared to other females. Moreover, littermates—or other classes of close kin —interact differently

II.

INFLUENCE OF CONSPECIFIC CHEMICAL SIGNALS ON THE BEHAVIOR OF NEONATES

A.

Nonhuman Mammals

Empirical studies of the responses of newborn mammals to naturally occurring olfactory cues have been conducted primarily with Norway rats and a few other easy to maintain and breed species of rodents and lagomorphs. Prior to weaning, pups of such species are continuously exposed to an array of odors emanating from urine, feces, and specialized glands of their mother and littermates, which permeate the nest area. 309

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1. Arousal When young rodents are deprived of maternal odors, an increase in distress calling, as well as exploratory or locomotive behaviors, typically occurs (e.g., Schapiro and Salas, 1970). Such behaviors are mitigated by reintroduction of the mother or by contact with air that has been passed over the mother or her soiled bedding, although in rats soiled bedding is effective only in young pups (ages 5–13 days) (Hofer and Shair, 1980; Oswalt and Meier, 1975; Randall and Campbell, 1976; Shair et al., 1997). Rat pups rendered anosmic by destruction of the olfactory receptors with nasal infusions of zinc sulfate solution continue to display heightened rates of ambulation even when exposed to their mother (Hofer, 1975, 1976), reiterating the importance of olfactory cues in suppressing such behavior. 2. Physical Attraction/Orientation Lactating female rats produce chemical signals that are attractive to suckling young. The pioneering research by Leon established that the emission of such signals by nursing females, as well as their pups’ physical orientation in the direction of those cues, are strongest from the time that the offspring become capable of independent locomotion until they are weaned (Leon and Moltz, 1971, 1972). Pups of that age move preferentially toward the odor of their own mother or the odor of a strange lactating female, rather than an empty goal box or the scent of a nulliparous female. The mother therefore appears to emit an “olfactory tether” that guides pups to her or the nest area and keeps them from wandering away. Subsequent studies have found evidence of similar olfactory attractants produced by lactating females of other species, including house mice (Breen and Leshner, 1977) and spiny mice (Acomys cahirinus) (Porter and Doane, 1976). 3. Nipple Localization and Sucking As early as the first nursing bout, neonates are active participants in the feeding process. Successful ingestion of mother’s milk requires locating, grasping, and sucking a nipple. Olfactory signals are critical in this process. Thus, nipple localization and attachment and maintenance of weight are seriously disturbed in rat and mouse pups following olfactory bulbectomy or zinc sulfate–induced anosmia (Cooper and Cowley, 1976; Risser and Slotnick, 1987; Singh et al., 1976; Teicher et al., 1978). Because mothers displayed no observable differences in their responses to anosmic versus normal offspring, problems in finding the nipple appear to reflect alterations in the pups’ capacities rather than inadequate maternal behavior (Cooper and Cowley, 1976; Singh and Tobach, 1975).

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The role of maternal olfactory cues in neonates’ feeding behavior can also be assessed by directly manipulating the odorant source. Thorough cleansing of the nipples of anesthetized lactating female rats results in reduced sucking efficiency (Hofer et al., 1976; Teicher and Blass, 1976). When thermal, tactile, or olfactory characteristics of the nipple region are altered, only the latter treatment adversely effects nipple attachment (Blass et al., 1977). Initial nipple attachment by rat pups is guided by the odor of saliva or amniotic fluid spread by the mother on her ventrum while grooming during parturition (Blass and Teicher, 1980). Nipple localization or attachment by suckling rabbits also depends upon maternal olfactory cues. In contrast to rodents, female rabbits return to their nest only once a day for a single brief nursing period (typically lasting several minutes) (Hudson and Distel, 1982; Zarrow et al., 1965). Once the mother positions herself over the litter, the pups must quickly find a nipple and begin to suck, or risk starvation. Anosmic rabbit pups exhibit anomalies in nipple attachment and feeding behavior during this critical time (Schley, 1977). Related research has revealed that rabbit pups are attracted to an odorous substance carried in the mothers milk and concentrated at the base of the nipples (Coureaud et al., 2000; Hudson and Distel, 1983; Keil et al., 1990; Schley, 1981). 4.

Huddling

In litter-bearing mammals, huddling by littermates is a conspicuous form of early social interaction. Altricial rat pups reduce heat loss and oxygen consumption by maintaining bodily contact with their siblings and actively exchanging positions within the huddle (Alberts, 1978b). The sensory control of huddling behavior changes over the first 2 weeks after birth. During the first week, huddling preferences are determined primarily by the temperature of the target stimulus. In one study, 5-day-old pups spent more time huddled with a warm, fur-covered tube than with an anesthetized rat (Alberts and Brunjes, 1978). However, the reverse preference was observed from day 10 onwards. An anesthetized gerbil was as effective as another rat in eliciting huddling responses by 5- to 10-day-old pups, but there was a clear conspecific preference beginning on day 15. As pups become older, olfaction plays an increasingly important role in huddling choices. Whereas zinc sulfate–induced anosmia appears to have no effect on younger pups’ huddling with an anesthetized cagemate, the same treatment markedly disrupts such huddling on day 10 (Alberts, 1978a). Contrary to untreated control pups, 2-week-old anosmic animals spend as much time in

Olfaction and Social Behavior in Neonatal Mammals

contact with a heated inanimate object as with an anesthetized target rat (Alberts and Brunjes, 1978). Egyptian spiny mouse (Acomys cahirinus) neonates likewise select huddling partners based upon their odor phenotypes, but congregate in indiscriminate clumps after their nares are flushed with zinc-sulfate solution (Porter et al., 1978).

B.

Human Neonates

1. Early Development and Responsiveness of the Chemoreceptive Systems At least four chemoreceptor subsystems are located within the human nose, all of which differentiate very early during prenatal ontogeny—the main olfactory system (CN I), trigeminal system (CN V), vomeronasal organ, and the terminal nerve (CN O). The major developmental events of these systems are summarized below to highlight their potential functions from the fetal period onwards (for reviews, see Doty, 1991, 2001; Schaal, 1988a; Schaal and Orgeur, 1992) (see also Chapters 46–48). The main olfactory system develops during the first trimester of gestation (see Chapter 6). At the most peripheral level, mature-appearing ciliated olfactory neuroreceptors are seen as early as week 11 of gestation (Pyatkina, 1982), and histological studies between gestational months 5–9 reveal that the fetal olfactory epithelium is well developed by that time (Nakashima et al., 1984). Olfactory neuroreceptors grow clusters of axons toward the forebrain, forming a visible olfactory nerve by 7–8 weeks. The shape of the olfactory bulb is evident around 6–8 weeks (Bossy, 1980), and its stratified internal structure is seen from week 10 onwards (Humphrey, 1940). Development of more central structures involved in olfactory processing has not been well described, but it is clear from the behavioral and psychophysical data presented below that they are functional, at least in a primitive sense, prenatally. The trigeminal system, the free nerve endings of which mediate somesthesic and common chemical sensations, is visible in 4-week-old embryos and has been demonstrated to respond to tactile stimulation between 7.5 and 10 gestational weeks (Brown, 1974). The vomeronasal or Jacobson’s organ is well developed in the human fetus, appearing as small symmetrical invaginations in the nasal septum near the nare opening (Moran et al., 1995). Sensory-like cells in this structure are detected at 5–13 gestational weeks (Bossy, 1980; Ortmann, 1989). The secondary central structures associated with the vomeronasal organ, the accessory olfactory bulbs, can be clearly localised between 5 and 18 weeks. After this time, regression or reorganization of the accessory olfactory bulbs occurs in some individuals. Humphrey (1940) suggests

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that only a rudimentary structure subsists in the older fetus or newborn. However, much more anatomical investigation is needed before we have a clear picture of the status of the vomeronasal organ in the perinatal period (see Chapter 46). A final potential chemoreceptive system is present in the fetal nasal passages, the terminal nerve, which sends free nerve endings to the anterior nasal septum and to the olfactory mucosa (Brown, 1987; Oelschlager et al., 1987). The chemosensory functions of this structure remain unexplored in humans (but see Chapter 48 for such functions in other forms). The relative contributions of these four different chemoreceptive systems to smell sensation is unclear. However, it is generally accepted that the olfactory system is tuned to detect low concentrations of volatile odorants, whereas the trigeminal system is mainly sensitive to the irritative effects of higher intensity chemical stimulation. It is questionable whether the vomeronasal and terminal systems are functional in humans. The reader is referred to Chapters 46–48 for a more detailed discussion of vertebrate studies of these systems. Nasal chemoreceptors develop in a prenatal environment that carries a wealth of potential chemosensory stimuli (Schaal and Orgeur, 1992; Schaal et al., 1995d). Odorous compounds enter the amniotic fluid via transcutaneous water transfer of tracheal and gut wastes, especially the ever-increasing urination of the fetus. The volume and composition of these substances fluctuate throughout gestation and even display daily cycles (Abramowich, 1981). Additional sources of such olfactory stimuli include the mother’s metabolic activity, immunogenetic constitution, and diet (Hauser et al., 1985; Mennella et al., 1995). Ultrasonography allows direct visualization of the flow of fluids through the fetal respiratory tract (Logvinenko, 1990). Swallowing and inhalation movements observed by such methods increase the volume of amniotic fluid that comes into direct contact with fetal chemoreceptors. It is interesting to note that fetuses inhale much more fluid than they swallow (Duenholter and Pritchard, 1976), suggesting that nasal chemoreceptors may be intensely stimulated. Thus, before birth, each neonate is likely to be exposed to a unique profile of dietary aromas and other compounds related to feto-maternal metabolism. The manner in which these early environmental conditions contribute to the shaping of chemosensory function (sensitivity, preferences) is detailed below. The functional onset and abilities of the nasal structures are difficult to investigate directly in the human fetus for obvious ethical and practical reasons. However, data from two indirect research strategies suggest that they are involved in early sensory processing by the fetal and neonatal brain. One of these strategies assesses the

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responsiveness of premature human infants shortly after birth to gain insight into functional chemosensory abilities of fetuses of equivalent gestational age. Limited data from a study by Sarnat (1978) suggest that infants born after 24 (or fewer) weeks of gestation are only weakly responsive to nasally administered chemical stimuli, whereas those of at least 28 weeks gestational age exhibit reliable behavioral responses to odors. However, since menthol was the odorant used in this study, both trigeminal and olfactory afferents may have been activated. More recent experiments suggest that at 31–37 weeks gestation, premature infants detect and discriminate among lower intensity odorants that activate primarily either the olfactory or the trigeminal system (nonanoic acid and cineole, respectively), but not both (Pihet et al., 1996, 1997). A second research strategy examines responsiveness of the fetus to chemical stimuli. This approach involves animal models and complements related experiments with human premature infants, in that it addresses the issue of whether chemosensory systems can function in a liquid medium. Fetal rats receiving intra-oral infusions of citral (lemon scent) during their last day of gestation display sharp increases in gross motor activity and changes in heart rate (Smotherman et al., 1991), possibly mediated via stimulation of the trigeminal nerve (Allen, 1929; Doty, 1995). Likewise, intranasal infusions of odorous solutions of citral or methylthiazoline elicit differential heart-rate variations in near-term ovine fetuses; while control saline does not trigger significant changes, citral induces a weak accelerative effect and foulsmelling methylthiazoline a sharp decelerative response (Schaal et al., 1991). Very similar results were obtained with mouse fetuses (Coppola and Millar, 1997). Although the specific sensory system(s) mediating such prenatal responses to distinct chemical cues remains to be elucidated, these results indicate that shortly before birth murine and ovine fetuses are able to detect and discriminate between odorants even when they are presented in aqueous solutions. There is no biological basis for assuming that the same physicochemical processes do not apply to the human case.

Porter and Schaal

had a noxious odor should treat this problem by consuming “fragrant wine and sweet food.” More recently, Charles Darwin (1877) remarked that his one-month-old son behaved as if he “perceived his mother’s bosom when three or four inches from it.” Darwin expressed doubt that the baby’s response was based upon visual cues, but speculated that he may have been “guided through smell.” It is somewhat surprising that experimental evidence in support of Darwin’s hypothesis was published almost a full century after the anecdotal description of his son’s perception of his mother’s “bosom.” While observing mothers and infants during breastfeeding, Macfarlane (1975) also noticed that neonates turned their head towards the mother’s breast before there was any physical contact with it and made no obvious eye movement in that direction. Based upon these preliminary observations, he developed a method for systematically testing infants’ orientation responses to various combinations of paired odor stimuli. Infants were placed individually on their back in a cot, with two stimulus pads suspended next to the baby’s face, one touching each cheek (Fig. 1). Two successive tests were conducted using the same pair of odor stimuli, with their left-right positions reversed after the first trial to control for any directional bias. When presented simultaneously with a pad that had been worn on their mother’s breast and a clean control pad, 17 of 20 breast-fed infants (2–7 days old) spent more time oriented to the former stimulus, thereby indicating that they perceived and were attracted to the maternal breast odor.

2. Nipple Localization/Sucking Discussions of the presumed significance of breast and milk odors produced by nursing mothers can be found in documents dating to antiquity (reviewed by Fildes, 1986). For example, an Egyptian papyrus manuscript written in the sixteenth century B.C. suggests that bad breast milk smells like fish, while good milk can be recognized by its manna-like odor. Seventh century A.D. writings by Paul of Aegina recommended that nursing mothers whose milk

Figure 1 Apparatus for testing neonates’ directional choice response to two simultaneously presented odorized pads.

Olfaction and Social Behavior in Neonatal Mammals

Following Macfarlane’s pioneering research, a number of studies using similar testing procedures found that olfactory cues emanating from the breasts of lactating women are particularly attractive to young infants. For example, 2-week-old bottle-fed infants oriented longer to a breast pad from an unfamiliar nursing mother than to the axillary odor of this same woman or to breast odors from a nonparturient woman (Makin and Porter, 1989). Responses to an axillary pad from a lactating woman did not differ from those to an odorless control pad. Another study found that neonates of this same age, who had been exclusively bottle fed since birth, spent more time turned towards a pad containing a lactating female’s breast odor than a pad scented with their familiar formula (Porter et al., 1991). Thus, even though the infants had no prior direct contact with such breast odor and had repeatedly been exposed to the odor of their familiar formula in the reinforcing context of feeding, the breast odor elicited more interest. Further observations of newborn infants reveal that they are active participants in the nursing process and that maternal odors contribute to successful early nipple attachment and sucking. Widstrom and colleagues (1987) described the spontaneous activity of infants placed on their mother’s bare chest immediately after delivery. A recurring sequence of behavior was observed, including hand-to-mouth movements, sucking movements of the mouth and tongue, rooting, nipple attachment, and effective sucking. Heightened nonnutritive sucking movements were similarly observed in other studies when

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babies were exposed to the odors of their mother’s breast (Meza et al., 1998; Russell, 1976). To assess whether chemical cues associated with the maternal nipple/areola region are implicated in spontaneous feeding behavior in the natural context, Varendi et al. (1994) laid infants in a prone position between the mother’s breasts after one breast had been thoroughly washed to eliminate (or at least reduce) naturally occurring odors. Observations commenced 5–13 minutes postpartum and continued until the infant found a nipple and began to suck vigorously—with no assistance from the mother. From the total sample of 30 infants, 22 selected the unwashed breast for their initial feeding bout (Fig. 2). Since the washing procedure had no effect on the surface temperature of the nipple/areola, the infants had most likely responded to olfactory differences between the washed vs. unwashed breasts. This experiment was later replicated with a sample of 2- to 3-day-olds with similar results; significantly more babies spontaneously grasped and sucked from the mother’s unwashed nipple/areola rather than the alternative breast that had been cleansed of its natural scent (Varendi et al., 1997) (see Fig. 2). Additional evidence of infants’ sensitivity to odors when breastfeeding is found in accounts of neonates’ reactions to strong odorants applied directly to the mothers’ nipples. In experiments conducted during the nineteenth century, neonates refused breasts treated with strongsmelling substances such as diluted petroleum, Asa foetida, and succinic acid (Kroner, 1882; Preyer, 1885).

Figure 2 Influence of breast and amniotic fluid (AF) odors on neonates’ spontaneous nipple preferences. ** p  0.01; *p  0.05. (Adapted from Varendi et al., 1994, 1996, 1997.)

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When similar odors were presented at their nostrils, sucking infants responded by releasing the nipple or withdrawing (Garbini, 1896). The salience of odors in this context is also suggested by anecdotal reports of disturbed sucking patterns by neonates whose mothers had recently consumed highly spiced/flavored foods or beverages. A survey of French infant-care manuals found that mothers are commonly advised to refrain from consuming highly odorous (or odor-producing) dietary items such as garlic, onions, cabbage, leeks, asparagus, spices, alcohol, and coffee (De Perceval and Lallemand, 1980). More recent scientific investigations have substantiated the influence of maternal diet on the odor of breast milk. Members of an adult sensory panel judged the perceived intensity of the odor of mothers’ milk as being “most strong” or “more like garlic” 2 hours after the donor had ingested garlic capsules (Mennella and Beauchamp, 1991). Nursing infants likewise appeared to detect the changes in their mother’s milk since they remained attached to the nipple for a longer period and displayed higher sucking rates per feed when it smelled of garlic. The flavor of garlic transferred to breast milk did not seem to be strongly aversive to these infants since there was no consistent effect on the amount of milk that they consumed. Additional common substances that have been found to flavor breast milk after being consumed by lactating women include vanilla, mint, cheese, and alcohol (Mennella, 1995). 3. Arousal Aside from their influence on nipple localization and sucking, maternal odors also appear to affect more generally newborns’ motor activity and arousal. In one study, exposing 2- to 10-day-old infants to either their mother’s breast or neck odor resulted in a reduction of arm and head movements (relative to the responses to a clean control pad) (Schaal, 1986). The effect of odors on autonomic activation was assessed in another study by recording infants’ respiratory rates (Soussignan et al., 1997). When a cotton swab moistened with milk from an unfamiliar lactating woman was placed near their nostrils, breast-fed infants evinced greater changes in their breathing rate (relative to control trials) than did formula-fed babies of the same age. Human neonates that are separated from their mother (or caregiver) typically show high rates of crying that are abruptly curtailed when maternal bodily contact is reestablished (Christensson et al., 1995). Crying in this context appears to be functionally analogous to the separation distress vocalizations described in nonhuman mammalian young. One-day-old crying infants more rapidly stopped crying when they had access to the odor of a hospital gown that had been worn by their mother—or another woman

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who had recently given birth—relative to a clean control gown (Sullivan and Toubas, 1998). In another study, the effects of different naturally occurring odors on early spontaneous crying were compared (Varendi et al., 1998). During a 60-minute test session commencing 30 minutes after birth, babies who were exposed to the odor of their own amniotic fluid spent significantly less time crying than did babies with no odor exposure. In contrast, within each of the 15-minute test intervals, babies who were presented with their mother’s breast odor cried more than those in the control condition. Although a clear explanation of the opposite effects of amniotic fluid and breast odors cannot be given at present, the calming effect of amniotic fluid may reflect prenatal familiarization with that substance, as discussed below. On the other hand (as seen above), odors emanating from the breasts of lactating females are attractive to newborn infants and help guide them to an appropriate food source. Therefore, since the babies in the latter experiment had continual exposure to breast odors but were not able to locate a nipple and suck, they may have become disturbed or aroused. Macfarlane (1975) similarly reported that 2- to 10-day-old breastfed babies displayed increasing activity and crying after their nose remained in contact with a maternal breast pad. C.

Ontogenetic Mechanisms

Because neonates of a wide range of mammalian species respond preferentially to particular conspecific odors shortly after birth (before they have the opportunity to gain significant postnatal chemosensory experience), such discriminative responsiveness may be termed “inborn.” Although the underlying bases of such olfactory preferences have not been well elucidated, genetic, maturational, and experiential factors have all been implicated. For example, selective responsiveness to odors could reflect genetically mediated expression of olfactory receptors, as illustrated by specific hyposmia in mice or in our own species (Pourtier and Sicard, 1990; Whissel-Buechy and Amoore, 1973) (see also Chapter 16). Moreover, maturational changes in olfactory detection thresholds have been described over the first several days after birth (Alberts and May, 1980; Lipsitt et al., 1963). As noted below, investigations of the role of experience in the development of early odor preferences have often focused on whether such responses are dependent on exposure or learning. The biological substrates bearing odor cues attractive to newborns may be placed into two broad categories: those to which the neonate’s chemoreceptors had obvious contact in utero, and those to which they were apparently not directly exposed prior to postnatal testing. Fetal fluids examplify the first category of odor mixtures that are

Olfaction and Social Behavior in Neonatal Mammals

alluring to newborns. As discussed previously, rat pups are initially attracted to amniotic fluid that the parturient dam had spread on her own abdomen (Teicher and Blass, 1977). Whereas washed nipples were not attractive to pups, they became so when painted with amniotic fluid from unfamiliar parturient females, thereby indicating that neonatal rats respond positively to any sample of such fluid, regardless of the donor female. Nonetheless, newborn rats display a preference for their own amniotic fluid over that from another litter, which is evidence that they can discriminate between these two olfactory substrates (Hepper, 1987). Related studies with precocial newborn ungulates found significant preferences for amniotic fluid over water [pigs (Parfet and Gonyou, 1991) and lambs (Schaal et al., 1995b)] and for own versus alien amniotic fluid [lambs (Schaal et al., 1995b)]. Early postnatal attraction to the odor of amniotic fluid suggests that the fetus becomes familiar with that smell and retains a memory of it after birth. Evidence that odor learning, in fact, can occur in utero has been obtained using a conditioned aversion paradigm involving prenatal exposure to an artificial odorant (injected in the amniotic fluid) paired with a noxious stimulus (intraperitoneal injection of lithium chloride) (Smotherman, 1982; Stickrod et al., 1982). In choice tests conducted after birth, rat pups in the prenatal treatment condition, but not control pups, avoided the training odor. The aversions acquired in this manner appear to be stimulus-specific since they did not generalize to other odorants. Furthermore, as the postnatal test ensured that the stimulus was presented exclusively in a gaseous phase, the conditioned aversion was likely mediated, at least in part, by olfaction. Pedersen and Blass (1982) employed a less intrusive manipulation in another study, whereby fetuses were merely exposed to an artificial odorant injected into the amniotic sac (see also Chotro and Molina, 1990; Smotherman, 1982). At birth these pups were fostered onto untreated females to eliminate any possibility of odor transfer through the biological mother’s milk. One hour after birth, pups that had been exposed to citral prenatally attached rapidly to unwashed nipples in a citral-odorized atmosphere but did not seize a nipple in the absence of that scent. The expression of this early preference was dependent, however, on a short period of postnatal exposure to citral, contingent with tactile or pharmacological activation of the pups. Positive results have also been reported when the fetal environment of lambs (Nolte et al., 1995; Schaal et al., 1995b, 1995c), rats (Hepper, 1988a), and rabbits (Bilko et al., 1995) was manipulated by feeding females aromas throughout pregnancy. The structural, functional, and ecological requisites for prenatal perception of chemosensory stimuli thus seem to be verified during the late stages of gestation. Recent

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research also indicates that newborns of nonmammalian, amniote animals (including species of amphibians, reptiles and birds; e.g., Burghardt, 1971; Hepper and Waldman, 1992; Porter and Picard, 1998; Sneddon et al., 1998) likewise retain some “olfactory images” from their larval or embryonic chemical environment. Biological substrates to which there has been no obvious prior exposure are more varied. Newly born lambs display heightened autonomic and behavioral activation when first exposed to the intense odor of ewe’s inguinal gland secretions (Vince and Ward, 1984). This arousal response was greater for inguinal wax than for maternal wool or milk and for the mother’s wax as compared with that from an unfamiliar ewe. During their initial sucking attempts, neonatal piglets’ attraction to the nipple line is mediated to a great extent by “specific ventral substances” produced by the sow (Morrow-Tesch and McGlone, 1990). Another example of such attractive cues is the odor that releases the stereotyped nipple-searching pattern in newborn rabbits. This stimulus, which is emitted on the lactating doe’s abdomen in the vicinity of the nipples (Coureaud and Schaal, 2000; Coureaud et al., 2000; Hudson and Distel, 1983), directs localization and oral grasping of the nipple. A functionally similar volatile compound or mixture is also present in the doe’s milk, since pups respond to this substance with immediate orientation, searching head movements, and attempts to seize the object bearing the milk sample (Keil et al., 1990). Because the neonates in the latter series of studies had typically been separated from their mother at birth, the reported olfactory preferences are unlikely to be the result of prolonged postnatal experience. Nonetheless, there are several alternative developmental mechanisms that could, in theory, underlie the manifestation of early attraction to specific chemical stimuli by these newborn organisms. Neonatal chemoreceptors might be attuned by prenatal experience to detect somewhat similar cues involved in the guidance of behavioral patterns crucial for survival (see below). Furthermore, brief postnatal exposure, often in association with the reinforcing effects of the mother’s first nurturing activities (e.g., licking, nursing, warm and soft contact, vocalizations), could be sufficient to bias later hedonic responsiveness to those scents. What evidence is there that human neonates remember and make use of olfactory information that they acquired during their amniotic life? First, human newborns turn their head preferentially towards the odor of their familiar amniotic fluid when given a choice between that substance and distilled water (Schaal et al., 1995a). Moreover, like young rats and lambs, neonates of our own species demonstrate a head-orientation preference for the odor of their own amniotic fluid when it is contrasted with unfamiliar

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fluid in a two-choice test (Schaal et al., 1998). The fact that the infants in these experiments had their nasal passages cleared and were washed within minutes after birth and that even bottle-fed infants (who were not reexposed to putative amniotic-like compounds that may be carried in breast milk) preferentially responded to their own amniotic fluid, supports the conclusion that they had become familiar with that odor within the uterus or during the birth process. Infants in a second series of studies were tested with the odor that they had encountered in their amniotic environment simultaneously paired with a highly salient postnatal odor: colostrum or milk. Facing such a choice, 2-day-old breast-fed infants did not display differential head orientation or mouthing movements (Marlier et al., 1997, 1998a) (Fig. 3). By 4 days of age, however, infants spent more time oriented towards the odor of their mother’s milk than to the scent of amniotic fluid (Marlier et al., 1998a). It can be concluded from these results that (1) breast-fed infants remain attracted during a limited postnatal period toward odor stimuli from the uterine environment, (2) amniotic and colostral odours initially have similar hedonic properties, and (3) after repeated exposure to milk during the first 3–5 days, its odor is preferred over that of amniotic fluid. Finally, a pattern of results similar to those outlined above was obtained in a study in which human infants

Figure 3 Mean relative duration (SEM) of head orientation in five groups of breast-fed infants in a series of two-choice tests pairing the odor of their own amniotic fluid (open bars) and the odor of their mother’s lacteal secretions (black bars). For age groups 1–5, the number of infants is 9, 20, 12, 16, and 9, respectively. *p  0.01. (Adapted from Marlier et al., 1997.)

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were observed in the biologically relevant context of breastfeeding. When one of a mother’s breasts was moistened with amniotic fluid, and the other not, significantly more babies selected, minutes after birth, the moistened breast over the non-moistened breast (Varendi et al., 1996) (Fig. 2). Such early orientation to the odor of amniotic fluid may have been adaptive thoroughout the evolutionary history of our species, since women presumably handled their babies during and immediately after expulsion from the birth canal. The mother’s hands would then have been soiled with the birth fluids, which in turn would have been transferred to her breasts when she first attempted to nurse her newborn infant. Attraction to the odor of amniotic fluid would therefore have facilitated nipple localization. In a subsequent experiment, there was no reliable difference in the number of babies who spontaneously sucked from a naturally scented breast, versus one treated with amniotic fluid, at 1–4 days of age (Varendi et al., 1997). However, babies who had selected the AF-treated breast at that time showed a strong preference for the untreated breast when given the same choice several days later (Fig. 2). This developmental shift from an initial orientation to a prenatal odor followed by a relative preference for a postnatal odor likely reflects differential exposure to those cues. At birth, amniotic fluid was more familiar than breast or colostrum/milk odors and therefore elicited preferential responses. Over the first several days postpartum, however, babies had increasing reinforced contact with breast odors but were no longer exposed to amniotic fluid (see also Marlier et al., 1997, 1998a,b; Schaal, 1988b; Schaal and Orgeur, 1992.). Neonatal attraction to amniotic fluid may therefore be explained by the continuity of odor information acquired prenatally. But the means by which odorous compounds not previously directly encountered (e.g., colostrum, milk, exocrine secretions) acquire positive hedonic properties that are evident within minutes or hours after birth remains unknown. Amniotic fluid, colostrum, milk, and exocrine secretions originate to at least some extent from the maternal blood stream, and might therefore share a degree of chemical resemblance reflecting maternal diet, genotype, or metabolism. The aromas of specific food items ingested by the pregnant and lactating mother are indeed known to be transferred to her amniotic fluid and lacteal secretions. Hepper (1988a) and Schaal et al. (1995b) observed preferential—or less aversive—responses to odors by rat pups or lambs born to females whose diet contained those same aromas during gestation. Moreover, food preferences of weanling rats can be traced back to the flavor composition of their mother’s diet while she was nursing them (Capretta and Rawls, 1974; Galef and Henderson, 1972; Mainardi et al., 1989).

Olfaction and Social Behavior in Neonatal Mammals

The adaptive significance of transnatal olfactory continuity was assessed further in rabbits. Pregnant females were fed flavored fodder and their pups were fostered at birth to females fed either the “familiar” maternal diet or a novel diet (Coureaud et al., 1998a,b). Those pups that experienced prenatal-postnatal chemosensory discontinuity were less successful in obtaining colostrum than were agemates exposed to transnatal continuity. These findings are particularly significant when the natural history of the species is considered; female rabbits typically nurse their offspring only during a single brief period (3–5 min) every 24 hours. Therefore, continuity of chemical signals before and after birth enhances the likelihood that the pups will obtain the nutrients necessary for survival. From the data reviewed above, it appears that newborn mammals are able to detect olfactory similarity between chemicals present in their amniotic fluid and those externalized by the mother following parturition. The chemosensory overlap between amniotic and lacteal fluids is less well documented in our own species than in other mammals; however, there is evidence of transfer of the flavors carried in the pregnant and lactating human mother’s diet into her amniotic fluid (Hauser et al., 1985; Mennella et al., 1995) and milk (e.g., Mennella and Beauchamp, 1996).

III. THE ROLE OF OLFACTORY CUES IN INDIVIDUAL RECOGNITION Animals do not interact in an indiscriminate or random manner with other members of their own species. This is readily apparent in the exclusive relationship that develops between mammalian infants and their nursing mothers and in the encounters between mates, or members of the same subgroup, that often differ noticeably from those involving unfamiliar individuals. Following parturition, females of many species quickly restrict their caregiving behavior to their own newborn offspring and reject or even attack alien young that approach them and attempt to suck. Young likewise tend to respond preferentially to their mother and littermates as compared to other adult females or agemates. Based upon such discriminative social interactions, it can be inferred that animals are capable of recognizing individuals or distinct classes of conspecifics. The age at which mother and sibling recognition first becomes evident varies across species and is correlated with their natural history and the rate of development of sensory and locomotor capabilities of the young. For example, precocial young that live in groups composed of a large number of females and their offspring (e.g., sheep and other ungulates) typically begin to respond in a selective manner

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to their own mother within the first day or two after birth. In contrast, individual recognition of mother and littermate siblings may develop more gradually in altricial neonates that are confined to a nest until weaning (e.g., ground squirrels). Across taxonomic groups, social recognition tends to be mediated by the same sensory systems that are otherwise most salient for communication. Thus, terrestrial mammals such as rodents, ungulates, and carnivores rely to a great extent on olfactory phenotypes to distinguish between individuals of their own species (reviewed by Halpin, 1986; Hepper, 1991). Although the sense of smell of primates is presumed to be less salient than vision and audition for the regulation and coordination of social interactions, odor cues have been found to provide a sufficient basis for individual recognition in a number of primate species (Colgan, 1983; Halpin, 1986), including humans (discussed further below). A. Neonates’ Discriminative Responses to Maternal Olfactory Signatures After initially establishing that neonates are attracted to their mother’s breast odor in the absence of alternative olfactory cues, Macfarlane (1975) conducted a further series of two-choice preference tests to determine whether breastfeeding infants recognize their own mother’s unique olfactory signature. The subject babies were exposed simultaneously to a breast pad that had been worn by their mother and a breast pad from another woman who was also nursing her own infant of the same age. Six- and 10-day-old neonates spent significantly more time oriented towards their own mother’s breast odor, thereby indicating that they were able to discriminate between the two odor stimuli and preferred that of their own mother. Odors of secretions collected from the nipple and areola region of lactating females are nonirritating and of weak intensity, and therefore unlikely to stimulate the trigeminal nerve (Beauchamp et al., 1991; Doty, 1991; Schaal, 1988a). Additional data support the conclusion that the infants in Macfarlane’s study displayed a positive hedonic response to their mother’s breast odor rather than avoiding the unfamiliar woman’s scent. Babies who had been breast-fed since birth oriented preferentially to an unfamiliar lactating woman’s breast odor when paired with that same female’s axillary odor or a clean control pad (Porter et al., 1992). It will also be recalled that bottle-fed infants similarly spent more time turned towards a breast pad from a lactating woman than in the direction of various alternative scents, including that of their familiar formula (Porter et al., 1991). Moreover, in Macfarlane’s original experiment,

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maternal breast pads elicited longer head orientation than did odorless control pads. A subsequent experiment employing a variation of Macfarlane’s olfactory discrimination apparatus found evidence that breast-fed infants can discriminate their mother’s individually unique breast odor at 3 days postpartum (Schaal et al., 1980). Reduced movements of the head and arms were observed when their nose came into contact with their mother’s breast pad compared to either a clean control pad or one that had been worn by an unfamiliar lactating female. Russell (1976) likewise noted differences in the sucking and orienting responses of 6-week-olds when their nursing mother’s breast pad was held under their nostrils, rather than a strange mother’s pad or one moistened with cow’s milk. Infants at an even younger age (~24 hours) increased their rate of mouthing movements to a greater extent when exposed to the scent of their mother’s hospital gown (area that had been in contact with her breast/axillary region) than during trials with another mother’s gown (Sullivan and Toubas, 1998). In the above research on maternal-odor recognition, babies consistently showed discriminative responses to olfactory cues emanating from their own mother’s breasts. To determine whether individually distinctive maternal odors are produced at other bodily sites, 2-week-old infants were tested for their head-turning responses to their mother’s axillary odors (Cernoch and Porter, 1985). Gauze pads that had been taped in the armpit of their nursing mother before the tests elicited longer directional head orientation than did axillary pads from either nonparturient women or unfamiliar lactating mothers. It therefore appears that the olfactory signatures that enable infants to recognize their own mother are not restricted solely to the nipple/areola region. B.

Underlying Basis of Individual Olfactory Signatures

An individual’s characteristic body odor (“olfactory signature”) ultimately reflects a complex interaction between genetically mediated factors (e.g., metabolic and endocrine processes, density and distribution of skin glands) and various environmental influences (Schaal and Porter, 1991). It is common knowledge that the consumption of strongly flavored or highly spiced substances may alter the scent of the skin surface, faeces, urine, or breath. Micro-organisms also play an important role in the production of bodily scents; many secretions and excretions are odorless unless they are processed by resident microflora. Perhaps the clearest evidence that minor genetic differences may result in discernible differences in olfactory phenotypes is provided by research with house mice of

known genotypes. Females derived from wild populations can discriminate between odors of soiled bedding material from males heterozygous for one of the recessive t alleles (/t) and the scent of males homozygous for the wild-type allele (/) (Lenington and Egid, 1985). Urinary and whole-body odors of inbred mice that are genetically identical except for genes of the major histocompatibility complex (MHC) can also be distinguished by conspecifics (Yamaguchi et al., 1981; Yamazaki et al., 1999) as well as human subjects (Gilbert et al., 1986). In addition, (unidentified) genes from other autosome and sex chromosome loci are known to contribute to the individual odor types of mice and rats (Boyse et al., 1991; Schellinck et al., 1993). Given the polygenic contributions to olfactory signatures, one would expect a positive correlation between the degree of genetic relatedness of individuals and the similarity of their odor types. In accordance with this hypothesis, sheep (Romeyer et al., 1993), Turkish hamsters (Heth et al., 1999), and rats (Hepper, 1983) appear capable of detecting olfactory resemblance between individual conspecifics that are closely related to one another (kin). A similar relationship between genotype and human body odor is evident from several lines of research (Porter, 1999). Over 4 decades ago, Kalmus (1955) reported that highly trained tracking dogs did not distinguish between the scents of identical twins when the samples were presented successively rather than simultaneously. Related experiments have since determined that dogs could learn to respond differentially to the odors of dizygotic (fraternal twins) fed identical diets and between monozygotic twins whose diets were distinctly different (Hepper, 1988b). On the other hand, when monozygotic twins had been eating the same diet, the dogs showed no signs of discriminating between their scents. Likewise, human subjects more accurately discriminated between the (hand) odors of two unrelated individuals of the same sex than the odors of identical twins or full siblings (Wallace, 1977). Once again, odors of monozygotic twins were easier to discriminate when the paired stimulus individuals were eating different diets. Moreover, closer matches were found in the chromatography patterns of sweat samples obtained from identical twins than in those from nonkin pairs (Mc Cormick et al., 1995; Sommerville et al., 1990). In a series of studies concerning olfactory recognition of newborn infants, several parents commented that the scent of their baby reminded them of other family members (i.e., the neonate’s other parent or an older sibling) (Porter et al., 1983, 1986). These anecdotal claims of detectable “familial odors” served as the basis for an experiment in which adult subjects attempted to match the body odors of mothers and their 3 to 8-year-old offspring

Olfaction and Social Behavior in Neonatal Mammals

(Porter et al., 1985). Stimulus mothers and children wore a t-shirt for 3 consecutive nights; they were also given the same brand of soap to use during that period and instructed to avoid using deodorants, perfumes, or other scented toilet articles. During the tests, subjects sniffed a t-shirt that had been worn by one of the stimulus children (standard) and then attempted to identify by scent alone the shirt that had been worn by that child’s mother (included in an array of shirts worn by four different women). The same subjects were given the opposite task in the other test trial, i.e., asked to select the child’s shirt whose odor most closely resembled that of a standard mother. In both series of tests, odors of mothers and children were correctly matched significantly more often than expected by chance alone. While these data indicate that mothers and offspring bear a detectable olfactory resemblance to one another, their overlapping odors could reflect shared environmental factors (e.g., similar diets, identical ambient household odors) rather than genetic determinants. Therefore, to assess these alternative hypotheses, additional odor-matching tests were conducted with t-shirts worn by individuals who were not genetically related (husbands and wives) but lived in the same house and shared meals. Because the accuracy rate of matching the odors of husbands and wives did not differ from chance expectations, environmental similarity was not sufficient for the spouses to acquire similar body odors. Taken together, these data suggest that the perceived olfactory resemblance of mothers and their offspring was mediated at least partially by the shared portion of their genomes. Little is known regarding the identity of specific gene loci that may affect human olfactory signatures. Nonetheless, as discussed above for mice, preliminary data indicate that human body and urinary odors are also influenced by the MHC and both human and rat subjects are capable of detecting olfactory resemblance among adults with similar MHCs (Eggert et al., 1999a; Wedekind and Furi, 1997). Moreover, a number of genetically based metabolic disorders, including phenylketonuria and sweaty feet syndrome, are characterized by strong odors that can be valuable diagnostic aids (Cone, 1968; Liddell, 1976). C.

Ontogenetic Mechanisms of Individual Olfactory Recognition

Converging lines of empirical evidence indicate that infants develop the ability to recognize their mother’s olfactory signature through a process of familiarization and learning. Earlier in this chapter, we remarked that breast-fed infants discriminate between axillary odors from their own mother versus another lactating woman at

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2 weeks postpartum (Cernoch and Porter, 1985). These results differ markedly from those of bottle-fed infants of the same age, who spent no more time oriented to their own mother’s axillary pad than to an axillary pad from either a nonparturient woman or an unfamiliar (nonlactating) mother. Thus, whereas breast-fed infants recognized their mother’s axillary odor, there was no indication that the bottle-feeders did so. It follows, therefore, that prenatal learning sensus stricto cannot account for 2-weekold’s discriminative responses to their nursing mothers’ olfactory signatures. That is, if the fetus becomes familiar with the unique odor of its mother and subsequently remembers that chemical cue at 2 weeks postpartum, bottle-feeders should also show evidence of recognizing their mother’s axillary odor. Nevertheless, a possible influence of prenatal experience on the development of maternalodor recognition cannot be completely excluded; fetal familiarization could facilitate postnatal learning or perhaps even provide a sufficient basis for neonates to discriminate their mother’s olfactory signatures within a brief period after birth. Although the differing reactions of the bottle- and breastfed infants to maternal scents cannot be fully explained at present, they most likely reflect differential patterns of mother-infant interactions in these two feeding categories. When sucking at the breast, an infant’s nostrils are kept in close physical proximity to the mother’s skin surface and concomitant olfactory cues for an extended period of time. On the other hand, because bottle-feeders do not necessarily experience the same degree of routine exposure to the mother’s bare flesh, they have less opportunity to become familiarized with her characteristic odor phenotype. Thus, one would expect maternal odor recognition to develop more rapidly in breastfeeders. A similar lack of exposure and opportunity for learning could account for (2-week-old) breast-fed infants’ failure to respond discriminatively to axillary odors from their father (Cernoch and Porter, 1985). Experiments in which mothers wore artificial scents provide further evidence of learned recognition and hedonic preference for odors associated with breastfeeding. At 1 and 2 weeks postpartum, infants responded preferentially to the familar perfume scent that their mothers had applied to their breasts before each nursing bout (Schleidt and Genzel, 1990). In a similar manner, exposure to a pad previously taped to their mother’s neck, and bearing an “obvious” scent of her perfume, resulted in reduced motor activity by neonates (Schaal, 1986). Research with rodents demonstrates that specific odorants may acquire a positive hedonic value during early infancy as a function of mere exposure (Caza and Spear, 1984; Porter and Etscorn, 1974). These animal data served as the impetus for an analogous study of the effects of early odor exposure

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on newborn human infants (Balogh and Porter, 1986). On the day of birth, a pad treated with the essential oil of ginger or cherry was taped inside each infant’s cot and remained there throughout a familiarization period lasting approximately 23 hours. During that time there was no readily identifiable conventional reinforcement associated with the odor exposure. Following the familiarization period, the scented pad was removed from the cot and all babies were tested (~45 minutes later) for their head-turning reponses to a cherry versus a ginger odor pad. Thus, in each instance one of these odors was “familiar” (the one to which the infant had been previously exposed) and the other novel. A reliable preference for the familiar odor was displayed by female infants in this test, but males demonstrated a marked right-turning bias that may have overridden any odor-mediated response. In later experiments using a similar odor-exposure paradigm, however, both male and female infants oriented preferentially in the direction of a familiar exposure odor (Davis and Porter, 1991). Since mere exposure is sufficient for babies to recognize subsequently an artificial odorant, it is likely that this same process plays a role in the development of breast-fed neonates’ recognition of the maternal olfactory signature. Furthermore, in the nursing context, the characteristic scent of the mother is associated with reinforcers such as milk intake, physical contact, and warmth, which should enhance the learning process. Indeed, one-day-old infants more readily developed discriminative behavioral responsiveness to an artificial odorant when it was paired with reinforcing tactile stimulation (Sullivan et al., 1991). Colostrum ingestion may be a particularly reinforcing component of breastfeeding. In lambs, gastric infusion of colostrum is as effective as a normal sucking bout in mediating the development of recognition and preference for their mother (Goursaud and Nowak, 1999). During the first hours following birth, babies appear to be physiologically prepared to learn rapidly their mother’s olfactory signature (Porter and Winberg, 1999). The locus ceruleus has been reported to be highly active during the perinatal period (Lagercrantz and Slotkin, 1986), and plasma levels of norepinephrine (NE) are 20–30 times greater within the first few postnatal hours than afterwards (Lagercrantz, 1996). NE and associated arousal of the locus ceruleus have recently been implicated in olfactory learning in nonhuman mammals (Brennan et al., 1990; Kendrick et al., 1992; Leon, 1992).

IV. INFLUENCE OF EARLY OLFACTORY EXPERIENCE ON ADULT SOCIAL PREFERENCES Numerous studies have documented long-term effects of infantile olfactory experience on the behavior of mammals,

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including social interactions among adults (see also Chapter 17). An early example involved treating rat pups and their mothers with cologne for the first 30-days postpartum (Marr and Gardner, 1965). In adult breeding tests, rats from the early cologne-exposure group were less sexually responsive than controls when paired with a normalodor individual of the opposite sex. Similar results were obtained in a series of experiments by Mainardi and colleagues (1965); as adults, female mice that had been raised by perfumed parents preferred males bearing that same scent, while control females spent more time with untreated males. In a more recent experiment, male rat pups sucked from females that had lemon scent (citral) applied directly to their nipples and vagina (Fillion and Blass, 1986). When sexually mature, these males ejaculated more quickly when paired with a receptive female treated with the familiar lemon odor as compared to tests with an unscented partner. Social experience in the nest has been found to have enduring effects on the behavioral responses of golden hamsters to conspecific odors (Todrank et al., 1999). Adult males demonstrated that they remembered the odors of their individual littermates following a 9month separation period beginning at weaning. Fostering of newborn young onto substitute parents is a common technique for assessing the role of the early rearing environment in the development of social preferences. Rodents that are reared by females of a different species often respond more positively to natural odors from their foster species than do control animals (McCarty and Southwick, 1977; Porter et al., 1977). In some instances, cross-species fostering results in heightened adult preferences for the foster species’ odor along with reduced responsiveness to conspecific chemical cues (Huck and Banks, 1980; Quadagno and Banks, 1970). Mice and rats discriminate between the odors of conspecifics that are genetically identical except for difference in the major histocompatibility complex (MHC) (Brown et al., 1987; Eggert et al., 1999b). In choice experiments, male mice of at least some inbred strains tend to prefer females whose MHC differs from their own (Yamazaki et al., 1976). Such preferences for nonself MHC appear to result from early familial (olfactory) “imprinting.” Males that had been raised until weaning by foster parents with a different MHC mated preferentially with females that shared their own MHC rather than that of their parents (Yamazaki et al., 1988). These males therefore avoided mating with females whose olfactory phenotype resembled the familiar foster parent odor. However, a somewhat different pattern of results was obtained in subsequent experiments with additional inbred strains of mice; the effect of the rearing environment on MHC-based mating preferences was greater for females than males (Arcaro and Eklund, 1999). Early

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familial odor imprinting also appears to account for the avoidance of males with the foster parent MHC genotype that is shown by wild-derived female mice maintained under semi-natural conditions (Penn and Potts, 1998). As discussed above for newborn infants, olfactory signals have also been implicated in social discrimination and preferences by older humans. Recognition of individuals by their odor signature is well documented in children and adults. Despite considerable intersubject variability, tests of self-identification by olfactory cues have generally yielded positive results (Hold and Schleidt, 1977; Lord and Kasprzak, 1989; Mallet and Schaal, 1998; Russell, 1976). Beginning within hours after delivery, mothers reliably identify the odor of their own neonate when asked to sniff the soiled t-shirts or heads of an array of stimulus babies (Kaitz et al., 1987; Porter et al., 1983; Russell et al., 1983; Schaal et al., 1980). Moreover, mothers and fathers distinguish between the distinctive olfactory signatures of their two (full sibling) offspring, and children (3–8 years old) accurately identified their sibling’s t-shirt by odor cues alone (Porter and Moore, 1981). Schaal et al., (1980) reported that a significant majority of 45- to 58-month-old children preferred the odor of a t-shirt that had been worn by their own mother over that of an unfamiliar woman. Overall, adults of both sexes did better than expected by chance in tests for their ability to recognize the body odor of their sexual partner (Hold and Schleidt, 1977). At present, there is no clear evidence that human males or females produce odor signals that function as general sexual attractants in a manner analogous to what is seen in a number of other mammals. Nonetheless, particular odors (e.g., perfumes, scented soaps, the characteristic body odor of individuals) may be arousing or attractive because of their association with a sexual context (Kirk-Smith and Booth, 1987). Several research teams have been investigating hedonic responses to MHC-correlated odors and their possible influence on mating preferences in our own species. Female university students rated the body odors of MHC-dissimilar men more positively than the odors of men whose MHC was similar to their own and also claimed that the former scents reminded them more of their own mate (Wedekind et al., 1995). Although it is not known whether humans indeed select sexual partners based upon MHC-based odors, preferences for mates whose MHC differs from one’s own would presumably be adaptive. The rate of spontaneous abortions has been reported to be positively correlated with MHC homozygosity between mates (Beer et al., 1981). Furthermore, because of their MHC polymorphism, offspring of parents with dissimilar MHCs might be able to resist a wider range of pathogens (Apanius et al., 1997).

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The extent to which early (pre- or postnatal) exposure to odors per se may continue to affect subsequent hedonic responses to chemical stimuli by human adults—or even older children—has yet to be elucidated. In practice, this question would be very difficult to assess since it would be necessary to restrict pretest experience with particular scents to a relatively brief period of perinatal development. This type of long-term longitudinal research design might be possible with laboratory animals (see above), but it is less appropriate for human subjects. Despite these practical limitations, recent experiments provide some initial insight into the durability of the memory of perinatal chemosensory experience. Infants show behavioral evidence of remembering odors after a 2-week interval. Within the first 2–3 days postpartum, babies were exposed to an artifical odorant (cherry or ginger) for approximately 22 hours (Davis and Porter, 1991). The odorant was removed at the end of the exposure session, and subjects had no further contact with that scent until tests conducted 2 weeks later. At that time, the infants spent reliably more time oriented towards a pad treated with the exposure odor than in the direction of a novel scent, thereby indicating that they remembered the familiar odorant. Contrary to these data, however, Schleidt and Genzel (1990) reported that 4-week-old infants did not respond discriminatively to a perfume (rose oil) that their mothers had worn on their breasts during the first 2 weeks after delivery—even though strong preferences had been exhibited at 1 and 2 weeks. These contradictory results could reflect procedural differences. Schleidt and Genzel used a conditioning paradigm in which the mother’s perfume was initially associated with breastfeeding. Thus, the subsequent 2 weeks when mothers no longer perfumed themselves may have served as an extinction period resulting in reduced responsiveness to the training odor. In contrast, the odorant in the mere-exposure experiment was not associated with any obvious reinforcer, nor was there any extinction phase. Other methodological differences were the type of odor stimuli, the length of pretest exposure, the age at testing, and the age during the 2-week interval without odor exposure. There are few reports concerning hedonic responses to odors by infants older than 2–3 weeks. This is due, at least in part, to procedural difficulties that arise when attempting to assess olfactory perception and preferences at this age. The head-orientation test described above is less adequate as babies become more active and their motor capacities increase. Physiological measures and patterns of nonnutritive sucking have been used to assess early olfactory sensitivity and discrimination, but their utility for investigating odor hedonics has not been clearly demonstrated (Beauchamp et al., 1991). Within the first months after birth,

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babies begin to explore and manipulate toys, and recent research indicates that this is a fruitful context for studying the influence of odors on behavioral responses (Schmidt and Beauchamp, 1989). A sample of 6- to 13-month-old infants reacted differently to toys scented with ethanol or vanilla as compared to an unscented toy (Mennella and Beauchamp, 1998). Interestingly, babies’ responses to the scented toys were correlated with maternal consumption of products with similar odors. Infants who lived with one or two alcoholic parents spent more time mouthing an alcohol-scented toy than did babys of nonalcoholic parents, and infants whose mothers frequently ate vanilla-flavored food spent more time looking at a toy treated with that odorant. It is likely that the infants became familiar with vanilla or alcohol scents/flavors in the home, possibly in their mothers’ milk. However, the authors of this study were careful to point out that clear causal effects cannot be deduced from their correlational data and that any long-term consequences of such early flavor experience remain unknown. REFERENCES Abramovich, D. R. (1981). Interrelation of fetus and amniotic fluid. In Obstetrics and Gynecology Annual, R. D. Wynn (Ed.). Appleton-Century-Crofts, New York. Alberts, J. R. (1978a). Huddling by rat pups: multisensory control of contact behavior. J. Comp. Physiol. Psychol. 92:220–230. Alberts, J. R. (1978b). Huddling by rat pups: group behavioral mechanisms of temperature regulation and energy conservation. J. Comp. Physiol. Psychol. 92:231–245. Alberts, J. R., and Brunjes, P. C. (1978). Ontogeny of thermal and olfactory determinants of huddling in rats. J. Comp. Physiol. Psychol. 92:897–906. Alberts, J. R., and May, B. (1980). Ontogeny of olfaction: development of the rat’s sensitivity to urine and amyl acetate. Physiol. Behav. 24:965–970. Allen, W. F. (1929). Effect on respiration, blood pressure, and carotid pulse of various inhaled and insufflated vapors when stimulating one cranial nerve and various combinations of cranial nerves. Am. J. Physiol. 87:319–325. Allin, J. T., and Banks, E. M. (1972). Functional aspects of ultrasound production by infant albino rats, Rattus norvegicus. Anim. Behav. 20:175–185. Apanius, V., Penn, D., Slev, P. R., Ruff, L. R., and Potts, W. K. (1997). The nature of selection on the major histocompatibility complex. Crit. Rev. Immunol. 17:179–224. Arcaro, K. F., and Eklund, A. (1999). A review of MHC-based mate preferences and fostering experiments in two congenic strains of mice. Genetica 104:241–244. Balogh, R. D., and Porter, R. H. (1986). Olfactory preferences resulting from mere exposure in human neonates. Infant Behav. Dev. 9:395–401.

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Porter and Schaal Singh, P. J., Tucker, A. M., and Hofer, M. A. (1976). Effects of nasal ZnSO4 irrigation and olfactory bulbectomy on rat pups. Physiol. Behav. 17:373–382. Smotherman, W. P. (1982). Odor aversion learning by the rat fetus. Physiol. Behav. 29:769–771. Smotherman, W. P., Robinson, S. R., Ronca, A. E., Alberts, J. R., and Hepper, P. G. (1991). Heart rate response of the rat fetus and neonate to a chemosensory stimulus. Physiol. Behav. 50:47–52. Sneddon, H., Hadden, R., and Hepper, P. G. (1998). Chemosensory learning in the chicken embryo. Physiol. Behav. 64:133–139. Sommerville, B. A., Green, M. A., and Gee, D. J. (1990). Using chromatography and a dog to identify some of the compounds in human sweat which are under genetic control. In Chemical Signals in Vertebrates, V. D. W. MacDonald, D. MullerSchwarze, and S. E. Natynczuk (Eds.). Oxford University Press, Oxford, pp. 634–639. Soussignan, R., Schaal, B., Marlier, L., and Jiang, T. (1997). Facial and autonomic responses to biological and artificial olfactory stimuli in human neonates: re-examining early hedonic discrimination of odors. Physiol. Behav. 62:745–758. Stickrod, G., Kimble, D. P., and Smotherman W. P. (1982). In utero taste odor aversion conditioning of the rat. Physiol. Behav. 28:5–7. Sullivan, R. M., and Toubas, P. (1998). Clinical usefulness of maternal odor in newborns: soothing and feeding preparatory responses. Biol. Neonate 74:402–408. Sullivan, R. M., Taborsky-Barba, S., Mendoza, R., Itano, A., Leon, M., Cotman, C. W., Payne, T. F., and Lott, I. (1991). Olfactory classical conditioning in neonates. Pediatrics 87:511–518. Teicher, M. H., and Blass, E. M. (1976). Suckling in newborn rats: eliminated by nipple lavage, reinstated by pup saliva. Science 193:422–425. Teicher, M. H., and Blass, E. M. (1977). First suckling response in the newborn albino rat: the roles of olfaction and amniotic fluid. Science 198:635–636. Teicher, M. H., Flaum, L. E., Williams, M., Eckhert, S. J., and Lumia, A.R. (1978). Survival, growth and suckling behavior of neonatally bulbectomized rats. Physiol. Behav. 21: 553–561. Todrank, J., Heth, G., and Johnston, R. E. (1999). Social interaction is necessary for discrimination between and memory for odours of close relatives in golden hamsters. Ethology 105:771–782. Varendi, H., Porter, R. H., and Winberg, J. (1994). Does the newborn baby find the nipple by smell? Lancet 344:989–990. Varendi, H., Porter, R. H., and Winberg, J. (1996). Attractiveness of amniotic fluid odor:evidence of prenatal olfactory learning? Acta Paediatr. 85:1223–1227. Varendi, H., Porter, R. H., and Winberg, J. (1997). Natural odour preferences of newborn infants change over time. Acta Paediatr. 86:985–990. Varendi, H., Christensson, K., Porter, R. H., and Winberg, J. (1998). Soothing effect of amniotic fluid smell in newborn infants. Early Human Dev. 51:47–55.

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16 Genetics of Olfactory Perception Nancy L. Segal California State University, Fullerton, California, U.S.A.

Tari D. Topolski University of Washington, Seattle, Washington, U.S.A.

I.

BEHAVIORAL-GENETIC APPROACH TO OLFACTORY CHARACTERISTICS

The present chapter endeavors to fulfill the following objectives: (1) present an overview of behavioral-genetic methods available for examining genetic and environmental contributions to olfactory characteristics, (2) review selected findings from nonhuman animal studies that have considered genetic influences on olfactory behavior, and (3) survey findings from twin and family studies at the juncture of olfactory perception and kin recognition. Twin and family studies are emphasized given the research interests of the authors.

Interest in the nature and bases of olfactory function in human and nonhuman populations has stimulated considerable research activity (Kohl and Francoeur, 1995; Griff and Reed, 1995; Kodis, 1998; Laurent, 1999). Studies of individual differences in olfactory characteristics have focused on measures related to experience (Castle, Van Toller and Milligan, 2000), age (Murphy et al., 2000; Kline et al., 2000), health (Wszolek and Markopoulou, 1998) (see Chapter 22), smoking (de Jong et al., 1999; Davies and Davies, 1999), and sex (Gangestad and Thornhill, 1998; Yousem et al., 1999). Genetic influences on olfactory measures have, however, received relatively less attention. For example, overviews by Wysocki and Beauchamp (1991) and Segal and Topolski (1995) referenced only a few twin and family studies of olfaction, in contrast with the larger accumulation of nonhuman research. The ensuing years have witnessed some additional twin and family studies in this area, yet they remain few in number. Neglect of a behavioral-genetic perspective in olfactory research is unfortunate because this approach has demonstrated genetic effects across a wide range of human psychological and physiological characteristics (Segal, 1999a). A behavioral-genetic approach to the study of odor identification and preference can contribute substantially to our understanding of variation in normal and abnormal olfactory perception.

II. BEHAVIORAL-GENETIC RESEARCH METHODS AND DESIGNS Behavioral genetics is concerned with identifying genetic and environmental factors underlying individual differences in behavior. The three major designs used in human research are twin, family, and adoption studies. Nonhuman research includes strain studies (studies of animals inbred for at least 20 generations) and selection studies (studies of animals selected and bred for a trait of interest). Molecular biological techniques promise to identify DNA sequences associated with phenotypic variation (Plomin et al., 2002). A.

Biology of Twinning

1.

Types of Twins

Monozygotic (MZ or identical) twins result when a fertilized egg (zygote) divides during the first 2 weeks postconception 329

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(Bryan, 1992). MZ co-twins are essentially genetic duplicates, yet various sources of influence can interfere with this plan (see Segal, 1999a and references therein). Nondisjunction (failure of chromosomes to separate properly during cell division) can produce MZ twins discordant for Turner’s syndrome (X0) or Down syndrome (trisomy 21). Differential X chromosome inactivation can result in MZ female twins discordant for X-linked traits, such as colorblindness and fragile X syndrome. Zygotic division delayed until after day 8 is thought to underlie physical and anatomical reversals (e.g., handedness or hair whorl) in approximately 25% of MZ twins. Splitting of the fertilized egg is also associated with MZ twins’ increased frequency of midline abnormalities (e.g., spina bifida and symmelia); in 80% of these cases only one twin is affected (Bomsel-Helmreich and Mufti, 1995). Physical and behavioral differences between MZ twins are also associated with chorion type (Prescott et al., 1999), unequal fetal blood supply (Bryan, 1992), and birth order (Boggess and Chisholm, 1997). Dizygotic (DZ or fraternal) twins are the product of the separate fertilization of two eggs by two spermatozoa. DZ twins, consequently, share the same genetic relationship as nontwin siblings or 50% of their genes, on average, by descent. DZ twin pairs may be same-sex or opposite-sex. These two types of DZ twins have been assumed to occur with equal frequency, but an excess of same-sex male pairs has been suggested (Boklage, 1985). DZ twins’ genetic and environmental differences are responsible for behavioral and physical differences between them. Unusual variations of DZ twinning include superfecundation (conception of DZ twins following separate coital acts during the same menstrual cycle) and superfetation (multiple conceptions separated by three to 4 weeks) (see Segal, 1999a). Some superfecundated twins have different fathers, situations eventuating in extraordinary custody suits (Ambach et al., 2000). These processes are presumed to be rare, but this may reflect lack of medical detection and documentation. Twinning rates have risen considerably in recent years. Between 1980 and 1997, the number of twins born to women between the ages of 40 and 44 years increased by 63% (National Center for Health Statistics, 1999). This change is tied largely to new assisted reproductive technologies (ART) involving hormonal treatments and/or transfer of multiple embryos to women’s uteruses (Hecht and Magoon, 1998). A surprise finding is that ART has also increased the MZ twinning rate (albeit, less dramatically), possibly by altering the early environment of the developing embryo (Hecht, 1995). 2. Determination of Twin Type Correct classification of twin type is an essential step in the research process. This is because misclassification yields

Segal and Topolski

misleading estimates of genetic and environmental influences on phenotypes (Segal, 1999a). Comparative analysis of co-twins’ multiple blood group systems provides accurate results, especially when combined with physical measures (Lykken, 1978). Researchers are now relying increasingly on DNA profile analysis as a more accurate and less expensive procedure. Some laboratories analyze twins’ DNA patterns from buccal smears, cells obtained by gently scraping the inner cheek (Richards et al., 1993). A recent, noninvasive “swish and spit” technique obtains buccal cells by having subjects rinse with mouthwash and expectorate into a saline solution (Hayney et al., 1996). Samples can be prepared at home and forwarded to laboratories in special kits. Physical resemblance questionnaires can be reliably substituted for laboratory methods if the latter are precluded (Segal, 1999a). B.

Twin Study Logic and Methods

The logic of the classic twin design was first described by Sir Francis Galton (1875): “It is, that their history affords means of distinguishing between the effects of tendencies received at birth and of those that were imposed by the circumstances of their after lives; in other words, between the effects of nature and nurture.” The embryological bases of twinning were not firmly established until the early 1900s, yet Galton correctly reasoned that meaningful interpretations of twin data required reference to the distinction between twin types. It is, therefore, surprising that some investigators fail to use standard methods for zygosity diagnosis or omit discussion of their procedures in published work (see Segal, 1986, 1999b). Sources of influence on twins’ similarities and differences include genetic factors, shared environmental factors, and nonshared environmental factors. Greater trait resemblance between MZ co-twins, relative to DZ cotwins, demonstrates a genetic contribution to individual differences in that trait. Greater trait resemblance between MZ twins reared together, relative to MZ twins reared apart, indicates shared environmental influence on that trait. Case studies can also be useful for associating environmental effects and MZ within-pair differences, leading to more systematic studies. A fundamental assumption in twin research is that traitrelevant environmental influences function similarly for MZ and DZ twins. This concept is revisited periodically, yet there is slim evidence of meaningful associations between twins’ treatment and their behavioral outcomes (Hettema, Neale and Kendler, 1995; LaBuda, Svikis and Pickens, 1997). Other criticisms of twin studies include primary biases (twins’ unique prenatal events) and recruitment biases (excess MZ and female twin representation in

Genetics of Olfactory Perception

volunteer studies. Discussion and resolution of these issues have been addressed in several sources (see Plomin et al., 1997; Prescott, Johnson and McArdle, 1999; Segal, 1999a). Twin resemblance for continuous traits (e.g., height or IQ) is expressed as an intraclass correlation. Twin similarity for age and sex can spuriously inflate estimates of genetic effects, so data are age- and sex-corrected prior to analysis (McGue and Bouchard, 1984). Twin resemblance for discrete characteristics (e.g., blood type or mental disorder) is expressed as a concordance rate (Gottesman, 1991; McGue, 1992). A phi coefficient which incorporates population incidence and familial resemblance for “either-or traits” is also available (Plomin, 1990). Recent advances in analysis of twin data include discriminant function (DF) multiple regression (DeFries and Fulker, 1985) and biometrical modeling procedures (Neale and Cardon, 1992; Nance et al., 1998). III. TWIN RESEARCH ON OLFACTORY SENSITIVITY A.

Scientific Studies

Hubert et al. (1980, 1981) compared detection sensitivity thresholds for acetic acid, isobutyric acid, and 2-sec-butylcyclohexanone in a sample of 51 MZ male twin pairs and 46 DZ male twin pairs. Evidence of heritable variation in odor sensitivity was not detected for any of the three substances. Interestingly, concordance for a specific anosmia was not detected among any of the twin pairs. Relatively reduced sensitivity to isobutyric acid was shown by individuals who smoked, were lighter in weight, or who infrequently consumed alcoholic beverages. However, these correlates of odor sensitivity accounted for only a very modest portion of the variance. Forrai et al. (1981) assessed genetic influence on ketone-smelling ability (acetone and methylethylketone, or MEK) using 87 MZ twin pairs and 61 DZ twin pairs. Genetic effects for acetone, but not for MEK, were suggested. Ward et al. (1983) administered odor detection and odor discrimination tests to 14 MZ twin pairs and 6 DZ twin pairs who were discordant for Parkinson’s disease. Olfactory function was inferior in affected twins, relative to their unaffected co-twins, in 13 of the 14 MZ twin pairs. It was suggested that the observed olfactory impairment was acquired, rather than inherited. Wysocki and Beauchamp (1984) compared resemblance for sensitivity to androstenone and pyridine in 17 MZ twin pairs and 21 DZ twin pairs. An ascending concentration, two-sample (odorant vs. blank) forced-choice

331

procedure was used. All the MZ twin pairs, but only 61% of the DZ twin pairs, were concordant for sensitivity/insensitivity to androstenone, demonstrating genetic influence. In contrast, genetic influence on sensitivity to pyridine was not detected. The authors commented that the experimental method employed in their study may have been unable to identify a genetic component associated with pyridine sensitivity. Gross-Isseroff et al. (1992) detected genetic influence on androstenone threshold in a study of 17 MZ and 15 DZ twin pairs. A significant genetic effect was additionally found for sensitivity to isoamyl acetate, but not for sensitivity to citral or eugenol. These investigators suggested that Hubert et al.’s (1980) failure to detect a genetic component may reflect choice of odorants and/or the relatively older age of the participants. Segal et al. (1995) administered an odor-detection threshold test (perfume-grade phenyl ethyl alcohol, or PEA) to MZ and DZ twin pairs in California. Genetic influence on PEA sensitivity was not detected. Twins also completed the University of Pennsylvania Smell Identification Test (UPSIT), a self-administered, standardized test of odor identification (Doty, 1995). A genetic effect on this measure was indicated by the somewhat higher MZ (MZ r1
0.31; N
45 pairs) than DZ intraclass correlation (r1
0.15; N
37 pairs). A larger genetic effect for males than for females was also observed. Similarity in odor preference ratings for the 40 UPSIT items were compared for MZ and DZ twins (Topolski, 1993). Genetic influence was observed on the weak-strong dimension for items classified as spicy, flowery, and burned and on the unpleasant-pleasant dimension for items classified as flowery. Both MZ and DZ twins showed generally low correlations in ratings for foul and resinous items. The latter result supports the Schleidt et al. (1988) “preprogrammed survival” theory, which asserts that detection of environmental hazards fosters survival. Kopala et al. (1998) compared UPSIT scores for 12 MZ twin pairs discordant for schizophrenia and 12 healthy controls. The score for the combined twin group was significantly lower than that of the control group, and affected and unaffected MZ co-twins did not differ from one another. The researchers suggested that genetic factors contribute to cerebral dysfunction as assessed by odor identification ability. Finkel (2000) studied odor identification and cognitive functioning in 86 MZ twin pairs (31 reared apart and 55 reared together) and 141 DZ twin pairs (72 reared apart and 69 reared together). Moderate heritabilities were derived for four odor functioning measures, although only those for odor identification (0.29) and intensity (0.25) were significant; heritabilities for odor detection and

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pleasantness were not. A verbal component was found to be highly correlated with odor identification. Age did not contribute substantially to associations between olfactory measures and cognitive abilities. B. Selected Case Studies: Kallmann’s Syndrome Individuals affected with Kallmann’s syndrome show hypogonadotropic hypogonadism, eunuchoidal features, and anosmia or hyposmia. This disorder is associated with a defect in the synthesis and / or release of lutenizing hormone-releasing hormone (LHRH) (Dark, 1997–1998). Hipkin et al. (1990) compared olfactory function in MZ male twins discordant for Kallmann’s syndrome. The affected twin was anosmic, while the unaffected twin was hyposmic. Reasons for the incomplete expression of this disorder in these twins were unknown. The twins’ parents and sister showed normal olfactory function. Gasztony et al. (1997) detected hyposmia with hypogonadotropic hypogonadism in a DZ female twin and diagnosed it as Kallmann’s syndrome. Kallmann’s syndrome underlines the significance of smell in sexual development through progenitor cells in the olfactory placode. This is because cells of the hypothalamus that secrete luteinizing hormone–releasing hormone arise from these cells. C. Other Twin Research Designs and Applications in Olfactory Research There are approximately 10 variants of the classic twin design reviewed in Segal (1990, 1999a). Several are summarized below because of their potential relevance to olfactory researchers. 1. Longitudinal Twin Study Longitudinal studies sample behavior at selected periods during the life span to record age-related developmental changes. Longitudinal twin designs offer additional opportunities to examine relative genetic and environmental contributions to the timing and expression of characteristics. Greater similarity in the level and contour of MZ than DZ twins’ profiles suggests genetic influence on the developmental progress of the trait(s) under study. This area of research, now termed chronogenetics, was anticipated in Galton’s 1875 paper. Incorporating both twins and their singleton siblings into a longitudinal study improves the efficiency of this research design (see Wilson, 1983). New sophisticated methods for analyzing longitudinal twin data have been applied (Nance et al., 1998).

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Doty et al. (1984) documented age differences in olfactory sensitivity, as measured by cross-sectional studies of performance on the UPSIT. Longitudinal investigations using MZ and DZ twin pairs would provide new perspectives on this behavior and other age-related changes and continuities (Plomin et al., 2001). 2.

Twin-Family Design

Families composed of MZ twins, their spouses, and children are referred to as MZ half-sibling families, a model derived from nonhuman studies (Gottesman and Bertelsen, 1989). The children of MZ twins are genetically equivalent to half-siblings because they share a genetically identical parent (twin mother or twin father). Furthermore, twin parents share the same genetic relationship (50%) with their nieces and nephews as with their own children. This unique family constellation enables behavioral and physical comparisons between co-twins, nontwin spouses, twins and spouses, parents and their own children (who share an environment), parents and nieces/nephews (who do not share an environment), siblings, “half-siblings,” “half-brothers,” and “half-sisters.” This informative twin design has never been used in human olfactory research. The many types of relationships that can be generated offer numerous opportunities to explore hypotheses and predictions concerning the transmission of olfactory characteristics. Twin-family designs also promise to highlight the role of olfactory cues in human kin recognition (Segal, 1999a) (also see below). 3.

Twins Reared Apart

MZ twins reared apart (MZA) offer a direct estimate of genetic effects when twins are separated early in infancy and raised in separate homes chosen at random (McGue and Bouchard, 1998). The study of DZ twins reared apart (DZA) enables additional tests of possible genetic interactions. Information on olfaction is unavailable in three early studies of twins reared apart (Newman et al., 1937; Shields, 1962; Juel-Nielsen, 1965). Reared apart twins were, however, studied in conjunction with reared together twins in Finkel et al.’s study cited above. A small subset of participants in the Minnesota Study of Twins Reared Apart, directed by Dr. Thomas J. Bouchard, Jr., at the University of Minnesota completed the UPSIT and olfactory preference questionnaire administered to twins at CSUF. Such data should reveal how different rearing environments affect twin resemblance in olfactory characteristics.

Genetics of Olfactory Perception

IV. FAMILY AND ADOPTION METHODS AND APPLICATIONS IN OLFACTORY RESEARCH According to quantitative genetic theory, the magnitude of resemblance between relatives for continuous traits (traits influenced by multiple genes) should vary as a function of their genetic relatedness. Specifically, if genetic factors influence a trait, full brothers and sisters should show greater resemblance than half-brothers and half-sisters. Quantitative traits are also influenced by environmental factors. Biological relatives living in a common environment may display similarities or differences due to either genetic or environmental effects. Genetic variance may be decomposed into several components, such as additive variance and epistatic variance. Environmental variance can be divided into shared and nonshared components. If family resemblance is not detected for a given trait, this indicates that neither common genetic nor common environmental factors are importantly influencing that trait (Plomin et al., 2001). A.

Family Designs

Family studies provide opportunities to trace the mode of genetic transmission of a trait across generations. This task is feasible in the case of single-gene traits that are highly resistant to environmental effects (e.g., color-blindness, carried on the X chromosome), but is complex in the case of continuous traits. This is because parents transmit both genes and environments to their offspring, thus confounding genetic and environmental sources of variance. Other problematic features of family studies include age differences among relatives such that the developmental levels and experiences of family members may differ substantially. Brown and Robinette (1967) tested cyanide sensitivity in 2885 European school children and 86 parent pairs by a serial dilution technique. The participants were organized into several categories of relatives (e.g., same-sex twins, same-sex siblings, same-sex parent and offspring). A simple pattern of genetic inheritance was not indicated; rather, the findings appeared to be more compatible with a familial environmental factor that differentially affected males and females. The correlations increased from siblings to same-sex siblings to same-sex twins, a finding interpreted as suggesting a genetic effect similar to that of complex physiological traits; however, the number of twin pairs was small (N
20) and zygosity was not assessed. (It also appears that MZ and DZ twin pairs were combined, thus precluding more meaningful appraisal of these data.) Other same-sex family members (e.g., father-son pairs) were more highly correlated than the same-sex sibling pairs,

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compounding the difficulty in interpretation. The roles of age and various environmental factors were emphasized as affecting olfactory sensitivity to cyanide. Whissell-Buechy and Amoore (1973) analyzed sensitivity to musk using 109 Caucasian families. Insensitivity to this odor was observed in 36 families, with males and females being equally affected. The results suggested simple recessive autosomal inheritance for odor blindness to musk. Wysocki and Beauchamp (1991) reported a study examining the mode of inheritance of androstenone and pyridine in 67 biological families. An X-linked pattern of transmission was suggested for androstenone, while genetically influenced sensitivity to pyridine was not indicated. B.

Adoption Designs

Adoption studies include biological relatives raised apart or unrelated individuals raised together. Resemblance between biological relatives raised apart is associated with shared genes in the absence of correlated, trait-relevant rearing environments. Similarities between unrelated individuals living together are explained by common environmental factors. Methodological difficulties associated with adoption designs, such as selective placement (i.e., congruence between features of the biological and nonbiological families), have been described in Segal (1997, 2000). Most adoption research has focused on IQ resemblance between pairs of adopted away children and their biological parents, adoptive parents and children, and adoptive siblings. Adoption studies have never been used to examine variation in olfactory sensitivity and preference, but are well-suited to this purpose. A new adoption design focusing on same-age unrelated siblings (UST-SAs or “virtualtwins”) may offer superior estimates of shared environmental influences. This is because these unique siblings enter their home at the same time and share more developmental experiences than siblings differing in age (Segal, 2000). Published or ongoing adoption studies of olfactory function are unavailable. Such work would uniquely contribute to the literature in this field (Segal and Topolski, 1995). Adopted individuals have been included in studies examining odor recognition and emotional closeness among siblings, reviewed below. C.

New Research Directions

The introduction of modern molecular genetic techniques has paved the way for new analyses of normal and abnormal olfactory functioning (Breer et al., 1996). Jones and Reed (1989) identified an olfactory neuron specific protein (Golf) that appears to mediate olfaction. The mRNA

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encoding Golf was expressed in olfactory neuroepithelium, but not in several other tissues. The authors noted that a variant form of the G protein had been associated with pseudohypoparathryoidism (PHP), a condition in which impaired olfactory functioning is observed. Recently, Doty et al. (1997) questioned the link between olfactory dysfunction in PHP and and Gs alpha protein deficiency, suggesting that other mehanisms may be responsible. Buck and Axel (1991) indicated that isolation of odorant receptors and understanding of their specificity, diversity, and expression will enhance knowledge of olfactory perception. These investigators cloned and characterized 18 members of a multigene family that may encode a diversity of odorant receptors. They suggested that a possible tandem arrangement of genes “provides a template for recombination events” that may be associated with increased diversity within this system. Current work suggests associations between specific genes, olfactory function, and cognitive traits. Graves et al. (1999) found that individuals who were anosmic at baseline and who had at least one APOE-epsilon4 allele were at 4.9 times the risk for cognitive decline, relative to normosmics lacking APOE-epsilon4 alleles. Clearly, findings from twin, family, and adoption studies of olfactory function will enrich, and be enriched by, ongoing and future molecular genetic analyses. Related reviews of developments in this exciting area are provided by Anholt (1991) and Mombaerts (1999) and in Chapters 4, 22, and 23. V.

NONHUMAN STUDIES

Nonhuman studies enable experimental control over genetic and environmental factors underlying behavior (Plomin et al., 2001). As indicated in Sec. II, two forms of genetic control can be achieved. In addition, selected aspects of the environment may be manipulated to identify important genotype-environment interactions. Alternatively, environments may be controlled in order to highlight genetic effects. Another advantage of nonhuman research is the relatively brief life spans of the test animals, allowing observation of behavioral transmission across many generations. A.

Drosophila

Many insects are highly dependent upon olfactory cues for facilitating the identification of food sources, mates, and other resources essential to their survival (Fuyama, 1976, 1978). Research interest in genetic variation underlying olfactory response in drosophila has been considerable, largely owing to progress in drosophila neurogenetics during the 1960s (Ayyub et al., 1990). Despite increasing

knowledge of the molecular bases of olfaction, however, genetic factors underlying olfactory variation in drosophila are not well understood (Mackay et al., 1996). Attempts to unravel the intricacies of the drosophila olfactory system include studies of flies exposed to various mutagens, as well as studies of variability present in natural populations (Alcorta and Rubio, 1989). Available research includes inter- and intrastrain comparisons, selection studies, and molecular cloning of olfactory genes. Only selected findings from this body of work are reviewed in the present chapter; more detailed reviews are provided by Griff and Reed (1995) and Carlson (1996). Five separate strains of Drosophila melanogaster, taken from different locations, were exposed to ethyl alcohol, acetic acid, lactic acid, ethyl acetate, and n-butyraldehyde (Fuyama, 1976). Interstrain differences in attraction to all substances, except acetic acid, were demonstrated. Fuyama (1978) also demonstrated variability in olfactory response to ethyl acetate among 40 lines, homozygous for the second chromosome, chosen from a natural population of Drosophila melanogaster. Olfactory response to 5% ethanol was observed among lines of D. melanogaster selected for increased knockdown resistance to ethanol (Hoffman and Cohan, 1987). Ethanol was used as a metabolic resource to a greater extent among selected lines than among unselected lines, while unselected lines showed greater attraction to ethanol. Based on these findings, the authors suggested that behavioral alteration may be “nonadaptive,” such that flies tolerating ethanol more efficiently are less attracted to it. Genetic variability in response to ethyl alcohol and acetaldehyde was detected among isofemale lines representing natural drosophila populations chosen from homogeneous and heterogeneous habitats (Alcorta and Rubio, 1989). This study enabled analysis of a link between genetic and environmental variation. It was expected that relatively increased sensitivity and reduced variability of response would characterize inhabitants of homogeneous environments and that in situations with scarce resources, a prompt response would facilitate acquisition of food, mates, etc. Observed responses to ethyl alcohol (but not to acetaldehyde) confirmed these expectations. Pruzan and Bush (1977) showed that both wild-type (B) and mutant (bB) melanogaster larvae display preferences for odors of their own strain under a variety of stimulus conditions. Greater olfactory sensitivity was displayed by the wild-type larvae. Other investigators have also documented inter- and intraspecific variation among different strains of drosophila in response to cues provided by larvae (see Hoffman and Parsons, 1986; Monte et al., 1989). Lilly and Carlson (1990) defined and characterized the smellblind (sbl) locus. They showed that two alleles (identified in independently isolated mutants) are associated

Genetics of Olfactory Perception

with a common defect in larval olfactory response, as well as similarity in chemosensory and visual behaviors. They asserted that these findings suggest specificity, rather than generality, in neural or motor dysfunction for these mutants. Studies of olfactory correlates of mutations at X-linked loci have been reported. Helfand and Carlson (1989) showed that the origin of specific anosmia to benzaldehyde can be genetically mapped to a small region of the X chromosome near the pentagon locus. Ayyub et al. (1990) demonstrated that mutations in five of six X-linked loci were associated with partial anosmias in reponse to aldehydes or acetate ester. Ayer and Carlson (1991) reported that the abnormal chemosensory jump 6 (acj6) is responsible for “both reduction in electroantennogram amplitude and diminished behavioral response, as if reduced antennal responsivesness to odorant is responsible for abnormal chemosensory behavior in the mutant.” Molecular cloning of olfactory genes offers an informative approach to understanding olfactory functioning in Drosophila melanogaster. Several genetic screens have identified olf mutants causing altered responses to odorants (Griff and Reed, 1995). Hasan (1990) describes the molecular cloning of the olfactory gene olfE, which influences response to benzaldehyde in larve and in adults. Mutations in specific X-linked genes have been shown to affect response to benzaldehyde. It is noted, however, that mutations in a specialized neural circuit that mediate adult response to this substance may be implicated. The molecular character of such genes is, therefore, of interest. The olfE gene appears to have at least two transcripts, one of which may be directly involved in olfactory function. The possibility that the gene may be associated with functions other than olfaction was also raised. The olf A, olf B, and olfF mutants are also defective in detecting aldehydes, whereas the olf C mutant is defective in detecting acetate esters and olf D mutants are defective in responding to all tested odorants (Griff and Reed, 1995). Futher efforts along these lines are needed to elucidate the roles played by specific alleles with respect to olfactory characteristics. B.

Mouse Genetics

The mouse has been used in behavioral-genetic research for many years (Plomin et al., 2001). Studies applying a genetic approach to understanding the developmental neurobiology and organization of the mouse olfactory system are, however, scarce (Greer, 1991). This may reflect the fact that only three genotypes associated with morphological anomalies of the olfactory system [neurological mutant, staggerer (also called reeler); Purkinje cell degeneration (PCD); and the inbred strain Balb/c] have been

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described (Greer, 1991). Perturbation of the olfactory bulb in the staggerer mutant has been associated with poor performance on an olfactory associative learning task, but not on an olfactory habituation task (Deiss and Baudoin, 1999). This olfactory deficit may partially explain abnormalities in the social and sexual behavoirs of these mice (Deiss and Baudoin, 1997). Mice with PCD show degenerating fibers in the lateral olfactory tract. These animals offer opportunities to study olfaction using developmental neurobiological and instrumental genetic methods. Finally, members of the Balb/c strain are missing a synapse from the olfactory receptor axon onto the periglomerular cell dendrite. The consequences of this feature for processing odors are also unresolved. Additional information about these interesting genotypes is available in Greer (1991). Several studies have documented genetic differences in olfactory characteristics between strains, although the mechanisms underlying these observations require further investigation. A sampling of these studies is discussed below. Some analyses offer models for studying specific olfactory characteristics in humans. Recent advances in molecular-genetic techniques and findings relevant to olfaction in the mouse are summarized in Taylor (1991). Wysocki et al. (1977) used a conditioned aversion technique and odors considered to be primary or complex in humans to assess relative odorant insensitivity among male mice from several inbred strains. This approach was guided by a general theory of olfaction in which specific anosmias are associated with different profiles of odor insensitivities in humans. Olfactory deficiency among C57 mice was confined to isovaleric acid; C57 mice were distinguishable from AKR mice in that a general olfactory deficit was not indicated. That the anosmia observed among the C57 mice may be analogous to a specific anosmia in humans was suggested. The olfactory deficit in C57 mice observed by Wysocki et al. (1977) was subsequently confirmed by Pourtier and Sicard (1990). The latter recognized, however, that individual variations in animal behavior may be associated with variations in motivational state or physiological functioning. Evidence that the olfactory mucosa and olfactory bulb of C57BL/6J mice are activated by high concentrations of isovaleric acid was also cited in their study. Pourtier and Sicard (1990) thus interpreted their findings as indicating a partial, rather than a complete, deficit in isovaleric acid sensitivity. Greer (1991) has commented that the response to increased concentrations of isovaleric acid suggests a role for subsets of receptors less sensitive to this substance. In contrast, an absence of response (as reported by Wysocki et al., 1977) would suggest an absence of a relevant subset of receptors. More recently, Griff and Reed (1995) assessed the genetic transmission of specific anosmia to isovaleric acid

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(concentrations of 105–106) using progeny from C57BL/6J and DBA/2J matings. An autosomal recessive pattern of inheritance for specific anosmia to this odorant was observed. Genetic differences in olfactory-mediated choice behavior were shown to occur at day 10 in rat pups (Koski, et al., 1977). (The nostrils of mouse pups are not fully formed until days 5–8 after birth). Translocation animals with a Wv marker (which has been associated with slight macrotic anemia) showed significantly less olfactorymediated choice behavior relative to nontranslocation siblings, marker animals, and nonmarker siblings. The possibility that environmental factors associated with the test situation (e.g., handling and stress) may have influenced behavior was, however, not eliminated. The effects of olfactory bulbectomy on olfactory behaviors have been of interest. Wysocki et al. (1978) assessed the impact of bulbectomy on conditioned taste aversion using different strains of housemice (C57BL/6J and AKR /J and their reciprocal F1 hybrids). Genotypic differences in preference for saccharin were displayed prior to bulbectomy. Following conditioning, bulbectomy was shown to markedly disrupt the conditionability of C57 mice, with little effect on the conditionability of AKR mice. F1 hybrids showed an intermediate response. The effects of chemically induced peripheral damage to the olfactory mucosa on exploratory behavior have also been assessed in mice (Van Abeelen and Crusio, 1985). In this study, obstruction of animals’ olfactory information-processing abilities, by means of intranasal zinc sulfate irrigation, reduced exploratory behavior due to interference with novelty detection.

VI. STUDIES OF OLFACTION AND KIN RECOGNITION The identification and recognition of conspecifics, as well as olfactory communication of characteristics such as gender and social and reproductive status by olfactory cues, are well documented in the nonhuman animal literature (Brown, 1995; Heth, et al., 1999). It is proposed that organisms possess “olfactory signatures,” or stable, salient odors cues allowing recognition by relatives (Porter, 1998–1999). Mateo and Johnston (2000) recently showed that golden hamsters use their own phenotype as a referrent for recognition of kin (see also Chapter 15). The possibility that attraction and recognition in humans may be partly mediated by olfactory cues, even if less efficiently than by visual or auditory means, has generated considerable research interest (Wells, 1987; Porter, 1998–1999; Segal, 1999c). The roots of this interest are

Segal and Topolski

largely associated with Hamilton’s (1964) theory of kin selection. His premise was that natural selection should favor alleles predisposing individuals to behave in ways that favor the transmission of those alleles into subsequent generations. Alleles influencing individuals to favor others likely to carry replicas of these alleles is an indirect means by which these alleles achieve future representation. This reasoning suggests the presence of mechanisms that facilitate recognition of close kin and identification of kin and nonkin. Inclusive fitness refers to the effects of genes on the survival and reproductive success of an individual, plus the effects of genes on the survival and reproductive success of individuals with whom alleles are shared. Inclusive fitness is greater when individuals are more closely related genetically than when they are more distantly related. A. Nonhuman Animal Studies of Kin Recognition The literature on kin recognition in animals refers to a wide variety of organisms (Sherman and Holmes, 1985; Pfennig and Sherman, 1995; Hepper and Cleland, 1998–1999). Proposed mechanisms by which kin recognition may occur include green beard alleles, recognition alleles, and phenotypic matching systems. The green beard effect refers to signs that would both indicate relatedness between individuals and predispose these individuals to act altruistically toward others possessing the same sign (Dawkins, 1989). This mechanism is not considered likely, given the low probability of the same gene being associated with the label and with the tendency toward altruistic behavior. Recognition alleles refer to the display of a phenotypic marker and the ability to detect this marker in other organisms. Phenotypic matching refers to familiarity with one’s own phenotype and recognition of this phenotype in others. Kin recognition includes not only identifying a signal, but also acting on it, posing complex questions regarding underlying mechanisms (Hepper and Cleland, 1999). The effects of rearing conditions on kin relations, as assessed by olfactory preference, have also been investigated. Males and females from two mouse species were either reared by natural parents or were cross-fostered (McCarty and Southwick, 1977). Mice who were crossfostered showed reduced attraction to homospecific odors, as shown by preference for the soiled bedding materials of adult members from the rearing species. Studies of kin recognition have also included full sibling and half-sibling pairs. Werner et al. (1987), using a side-choice paradigm, demonstrated preference by green hatchling iguanas for unfamiliar siblings over familiar nonsiblings, following physical contact with pure kin groups.

Genetics of Olfactory Perception

A compelling series of studies using lambs offer additional insights into processes of kin recognition. Porter et al. (1991) showed that ewes preferred their own familiar lamb to the co-twin of this lamb who was raised apart in isolation. Nevertheless, the ewe showed significant preference for the twin lamb that had been taken from her, as compared with an isolated alien lamb or an alien lamb raised by its own mother until the time of testing. It was suggested that the fraternal co-twin lambs’ odors, while different, may have been somewhat similar, enabling discrimination and identification of these siblings by the mother. Of course, the source of the first-borns’ odors could have reflected genetically based scents, acquired scents from sucking or being licked by its mother, or both. Furthermore, some twins were opposite-sex, so co-twin distinctions may have been facilitated by the sex difference. Subsequent experimental manipulations, using 9–29 mother-twin pairs, separated out these various influences (Romeyer et al., 1993). These experiments are described below: 1.

Same-sex fraternal twins (full contact with firstborn twin): First-born lambs remained with their mothers, while second-born lambs were cleansed of amniotic fluid and reared in isolation from birth. Ewes preferred their familiar first-born twins to their second-born twins and preferred their second-born twins to unfamiliar unrelated (alien) lambs. These findings suggested that twin lambs shared genetic and environmental features allowing their mothers to find their separated second-borns. 2. Identical twins (full contact with first-born twin): Identical twin lambs were created by artificially cleaving fertilized eggs and implanting them in an unrelated ewe. As in the fraternal twin experiment, first-born lambs remained with their mothers, while second-born lambs were reared in isolation. Ewes showed greater acceptance of their first-born than second-born twin lambs, possibly because firstborns had acquired their mothers’ odors from her urine, saliva, or milk during their interactions. Mothers also favored their own isolated lamb over an alien lamb. 3. Same-sex fraternal twins (partial contact with firstborn twin): Following delivery, first-born twins were immediately placed in separate chambers in their mothers’ presence, allowing maternal access to infants’ odors but preventing physical contact. Second-born twins were housed in isolation. Several hours later, ewes preferred their first-born twins over their second-born twins, as well as their second-born twins over alien lambs. These findings supported the researchers’ earlier conclusion that fraternal twin

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lambs share genetically influenced odors, enabling their mothers to identify them. 4. Identical twins (partial contact with first-born twin): As in the fraternal twin experiment, firstborn twins were placed in separate chambers in their mothers’ presence, while second-born twins were isolated. Several hours later, ewes favored the identical twins much more than alien lambs, but, more importantly, they treated first-born and second-born identical twins alike. Ewes bearing identical twins apparently recognized the secondborn’s odor, based on their exposure to the firstborn’s odor. Since these lambs share identical genes they would be expected to exude similar scents. Of course, lack of discrimination between twins in this experiment may have partly reflected the mother’s inability to apply her own scent to first-born twins because physical contact was denied. Twin lambs were preferred over alien lambs, as expected. In these experiments, first-borns were preferred because they remained with mothers while second-borns were isolated. There was nothing intrinsic to being a first-born twin that would have affected ewes’ preferences. Finally, we can only wonder how well the ewe whose cell led to Dolly, the cloned lamb, would have identified her daughter—the ewe was no longer alive when Dolly was born. Their common genetically based scents may have helped this process, but the absence of acquired odors from the birth process or from sucking or licking may have impeded it. The possibility that anatomical and physiological variation associated with genetic variation in the major histocompatibility complex (MHC) explains distinctive odor compounds has been raised (Boyse et al., 1991; Eggert et al., 1998–1999a). Lenington and Egid (1985) observed the ability of wild-type female mice to distinguish between males with differing T-locus genotypes by means of olfactory cues. A subsequent study by these investigators demonstrated that males also rely on olfactory cues to distinguish between genotypes at the T locus, but that the genetic basis differs between males and females (Egid and Lenington, 1985). The role of the X and Y chromosomes in the chemosensory identity of mice of differing genotypes has also been examined (Yamazak et al., 1987). X and Y chromosomes each conferred an individual odor related to the genotype, but were less salient than the MHC. Olfactory cues associated with the MHC have also been shown to affect mate choice and frequency of pregnancy blocks in inbred strains of mice, thus promoting heterozygosity in the MHC (Egger et al., 1996). Brown (1995) observed that congenic mouse and rat strains differing in

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Class I and Class II regions of the MHC produce different urinary odors contributing to mate selection and parental recognition. However, it was also found that dietary differences affect individual odors more significantly than differences at one MHC locus. These results suggest that urinary odors reflect an interaction among the MHC, commensal bacteria, and dietary substances. A recent study of gerbils showed that the behaviors of individuals toward kin and nonkin vary with the situation and age and sex of the interactants. These findings raise the possibility that the MHC may differentially affect kin recognition and kincorrelated behavior (Hepper and Cleland, 1999). How the immune system affects body odors and ultimately social relations remain important questions. Future studies assessing the mediation of social preferences by olfactory cues among genetically related and unrelated animals, reared together and apart in diverse settings, will, hopefully, clarify the underlying mechanisms. The role of prenatal learning of olfactory cues also requires more serious consideration (Hepper, 1999). Recent advances in gene mapping should assist in the identification of genes relevant to these processes. B.

Twin Studies of Kin Recognition

Porter (1987) suggested that if “humans actually have genetically-determined biochemical ‘fingerprints’ that serve as a basis for individual odors . . . one would expect a correlation between genetic similarity and odor similarity.” Several twin studies have addressed this interesting issue. Wallace (1977) conducted a series of experiments involving exposure of research participants to the palm odors of two unrelated females, MZ female co-twins on the same diet, and MZ female co-twins on different diets. Subjects were more successful in distinguishing between the unrelated females than between the twins. Subjects could, however, more easily discriminate between twins eating different diets than between twins eating similar diets. Galton (1875) was the first investigator to question whether dogs could distinguish between MZ twins on the basis of odor cues. It was some time later that Kalmus (1955) demonstrated failure by dogs to discriminate between MZ cotwins when presented with twins’ odors as part of a retrieval task. Dogs could, however, differentiate between MZ cotwins’ odors in a tracking task. Hepper (1988) showed that dogs could discriminate between the odors of both MZ and DZ co-twins in a matching-to-sample experiment; MZ cotwins were living apart and followed different dietary practices. Dogs were unable to distinguish between infant MZ co-twins living in the same home and fed identical diets. A twin study of olfaction and kin recognition was completed in our laboratory at CSUF (Segal et al., 1995). MZ and

DZ twin adolescents and young adults wore t-shirts for three consecutive nights while refraining from using perfume or other cosmetics. Judges sniffed a target t-shirt, then attempted to identify the “relative” from an array of three tshirts. (These three shirts belonged to the co-twin and to two unrelated twins of the same age and sex.) Contrary to expectation, the proportion of correct identifications for MZ twins did not significantly exceed that of DZ twins. Judges’ lack of relatedness and/or familiarity with the twins may be associated with the nonsignificant findings. Most previous studies demonstrating accuracy in identification of individuals by odor cues from garments have used genetically related or socially familiar judges. Twins’ dietary practices may have also affected the findings. For example, older twins showed significantly greater differences in spicy food intake than younger twins and received fewer correct ratings. Porter (1987) noted that to “assess the influence of familiarization on the development of recognition of particular kin, one would ideally like to test individuals with the signatures of relatives with whom there has been no prior contact.” MZA and DZA twins are well suited to such analyses. Spouses and children (who have lived with one twin, but not the other) and unrelated individuals could be requested to distinguish between the odors of reared apart twins. In addition, reunited co-twins might be asked to identify the odor of the co-twin from an array of odors (Segal, 1999a). Similar analyses could be conducted using various other pairs of biological and adoptive relatives. Practical aspects of conducting these studies may, however, be difficult to resolve. C.

Family Studies of Kin Recognition

An increasing number of studies are demonstrating olfactory recognition by relatives living together. Macfarlane (1975) found that infants as young as 6 days of age preferred the breast pad worn by their own mother to that of another mother, as measured by the amount of time the infant turned toward the pad. Cernoch and Porter (1985) found that 4-day-old breast-fed infants, but not bottle-fed infants, were sensitive to differences between the underarm odors of their mothers and unfamiliar females. Makin and Porter (1989) observed that bottle-fed female infants display a nonspecific preference for the breast odors of lactating females. The role of olfactory cues in the motherinfant relationship is further summarized in Porter and Schaal (1995) and Porter (1998–1999) and in Chapter 15. Porter and Moore (1981) found that children were correctly identified by siblings and by mothers from odors on their clothing. Other studies have demonstrated that new mothers can correctly distinguish their own infants from unrelated infants by odor (Russell et al., 1983; Porter et al., 1983) and that fathers, grandmothers, and aunts can identify

Genetics of Olfactory Perception

the odors of garments worn by newborn relatives (Porter et al., 1986). Porter et al. (1986) also showed that adults discriminated between the odors of garments worn by siblings from whom they had been separated for 1–30 months and an age- and sex-matched stranger. In contrast, research participants could not match the odors of spouses from the odors of t-shirts worn for 3 consecutive nights (Porter, Cernoch and Balogh, 1985). Individuals can, however, recognize their own odor (Schleidt, 1980; Lord and Kasprzak, 1989) and the odor of their spouse (Schleidt, 1980). Recent work extends these themes. A small sample of full siblings, half-siblings, step-siblings, and some of their mothers participated in a study of odor recognition and emotional closeness (Gall, 1999). Full siblings and biological mothers correctly chose the t-shirt worn by their sibling or child, respectively, while half-siblings, step-siblings, and their parents were less able to make these discriminations. Biological mothers correctly identifying their offspring indicated a significant preference for their children’s olfactory cues. In contrast, correct identification was not associated with preference for the co-sibling’s odor in the three sibling groups. It was suggested that this finding may reflect reduced reliability among the young respondents. Contrary to expectation, warmth/closeness ratings were not elevated among either full or half-siblings who correctly identified their co-sibling. Self-reports, supplemented by parental assessments and naturalistic observations, may offer a more complete picture of siblings’ social relations. Variations on the studies reviewed above would further refine knowledge of the role of olfactory cues in human kin recognition. The unique relationships generated by MZ half-sibling families would help disentangle genetic and environmental effects on olfaction in the context of kin recognition. In addition, the comparative accuracy with which mothers are able to identify biological children whom they have reared versus biological children given away for adoption and / or children they have adopted would clarify the contribution of shared genes and environments to kin recognition, based upon olfactory cues. Individuals might also be requested to discriminate between biological and nonbiological siblings. An intriguing research design would request MZA twins to identify the reared apart twin and an adoptive sibling (with whom the rearing environment was shared), although practical considerations may render such studies unfeasible. VII. A.

SUMMARY Conclusions and Future Research Directions

Twin studies have not been fully exploited as research tools for investigating genetic and environmental influences

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on olfactory characteristics. The classic MZ-DZ twin comparison and its variants offer a wealth of new ways for considering the nature and bases of olfactory traits and their role in human social relationships. Future research should address the role of genetic factors in age-related changes in olfactory sensitivity. Alteration of the nuclei of the supporting and sensory cells of the olfactory epithelium has been associated with aging (Naessen, 1971), although marked individual differences have been reported (Doty and Snow, 1988). Olfactory neurons are unique in that they are continually being replenished during the life span of the organism. Fully mature neurons maintain their juvenile nature, a process believed to be under genetic control. However, evidence that rearing in a filtered air environment increased the life span of olfactory cells in mice (Hinds et al., 1984) led to increased interest in environmental influences on olfactory neurogenesis. Current evidence supports the view that mitosis in the olfactory epithelium is influenced by genetic factors but may be modified by environmental events (see also Chapter 5). Twin and adoption studies might help resolve these issues. Future studies focusing on human and nonhuman genotypes associated with olfactory impairment would be welcome. Such work may improve understanding of olfactory dysfunction (as in the case of Kallmann’s syndrome) and direct us toward better treatments for olfactory difficulties. New olfactory studies of kin recognition are also needed. Nicolaides (1974) has noted the large number of factors affecting the composition of fatty acids produced by the skin and the consequent rarity of two individuals producing the same substances in identical proportions. More recent findings are defining intriguing topics for further study. Identical twins’ sweat samples showed greater resemblance than those of unrelated pairs as revealed by gas chromatography (McCormick et al., 1995). In addition, Eggert et al. (1998–1999b) found that human urine odors and the profile of volatile components of urine are associated with the MHC. Most importantly, the profile of some specific components and volatiles constitutes MHC-related odor signals in humans. Wedekind et al. (1995) reported that the MHC affects both human body odors and preferences and that female preferences are influenced by their hormonal status. Interestingly, females preferred the odors of males whose MHC differed from theirs. Consistent with this work, Ober et al. (1997) and Genin et al. (2000) found fewer HLA haplotype matches than expected among Hutterite couples. Significant negative assortative mating was confirmed after considering parental origin of shared haplotypes. The conclusion was that mate choice was influenced by HLA haplotypes with avoidance of spouses whose haplotypes match

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one’s own. Preference for mates differing in selected genetic factors may help avoid the unfavorable behavioral and physical consequences associated with inbreeding. In contrast with Ober et al.’s results, Hedrick and Black (1997) found no evidence of negative assortative mating for MHC alleles using South Amerindian couples, given that HLA-sharing proportions were close to random mating expectations. In a related study, Wedekind et al. (1997) found that males and females’ pleasantness ratings of six different shirts correlated negatively with the MHC similarity between smeller and t-shirt wearer. More revealing, perhaps, raters were reminded of their own current or former partners when sniffing t-shirts whose wearers’ MHC alleles generally differed from theirs. The general consistency of findings across studies (albeit a small number) suggests that MHC similarity and dissimilarity contributes to body odor preferences. Further work along these lines would add depth and dimension to current understanding of mate choice. Advances in decoding the human genome will no doubt also contribute to what we know and can know about human olfaction and related characteristics. This information could be used to prevent loss of olfactory sensitivity with aging. It may also highlight the relevant genetic factors mediating social attraction, yielding novel insights into the fabric of human relations. ACKNOWLEDGMENTS Studies by the authors that are referenced in this chapter were supported by awards from the Fragrance Research Fund, Ltd. (now Olfactory Research Fund, Ltd.), New York, NY, and California State University, Fullerton (Dr. Nancy L. Segal), and by a grant from NIDCD, PO1DC00161 (Dr. Richard L. Doty). Research assistance was provided by Kathleen W. Brown, Ph.D., Linda Araki, M.A., Dinah G. Gitlin, B.A., Steven M. Wilson, Ph.D., Jennifer Trevitt, M.A., and Cesar Gasca. REFERENCES Alcorta, E., and Rubio, J. (1989). Intrapopulational variation of olfactory responses in Drosophila melanogaster. Behav. Genet. 19:285–299. Ambach, E., Parson, W, and Brezinka, C. (2000). Superfecundation and dual paternity in a twin pregnancy ending with placental abruption. J. Foren. Sci. 45: 181–183. Anholt, R. R. H. (1991). Odor recognition and olfactory transduction: The new frontier. Chem. Senses 16:421–427. Ayer, R. K., and Carlson, J. (1991). acj6: A gene affecting olfactory physiology and behavior in Drosophila. Proc. Natl. Acad. Sci. 88:5467–5471.

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343 Segal, N. L. (1990). The importance of twin studies for individual differences research. J. Counsel. Dev. 68:612–622. Segal, N. L. (1997). Same-age unrelated siblings: a unique test of within-family environmental influences on IQ similarity. J. Ed. Psychol. 89:381–390. Segal, N. L. (1999a). Entwined Lives: Twins and What They Tell Us About Human Behavior. Dutton, New York. Segal, N. L. (1999b). Silent partners: twins with selective mutism. Twin Res. 22:235–239. Segal, N. L. (1999c). Kin recognition by olfactory cues: What can twins tell us? Twin Res. 2:293–298. Segal, N. L. (2000). Virtual twins: new findings on within-family environmental influences on intelligence. J. Ed. Psychol. 92, 442–448. Segal, N. L., and Topolski, T. D. (1995). The genetics of olfactory perception. In Handbook of Clinical Olfaction and Gustation. R. L. Doty (Ed.). Marcel Dekker, Inc., New York, pp. 323–343. Segal, N. L., Grimes-Hillman, M., and Topolski, T. D.(1995a). Twin study of genetic relatedness and odor similarity. Aromachol. Rev. 4:2, 10–12. Segal, N. L., Topolski, T. D., Wilson, S. M., Brown, K. W., and Araki, L.(1995b). Twin analysis of odor identification and perception. Physiol. Behav. 57:605–609. Sherman, P. W., and Holmes, W. G. (1985). Kin recognition: issues and evidence. In Experimental Behavioral Ecology and Sociobiology. In Memoriam Karl von Frisch 1886–1982, B. Holldobler and M. Lindauer (Eds.). Sinauer, Sutherland, pp. 437–460. Shields, J. (1962). Monozygotic Twins: Brought Up Apart and Together. Oxford University Press, London. Siddiqi, O. (1991). Olfaction in drosophila. In Chem. Senses, Vol. 3, Genetics of Perception and Communications, C. J. Wysocki and M. R. Kare (Eds.). Marcel Dekker, Inc., New York, pp. 79–96. Taylor, B. A. (1991). Current methods in mouse genetics. In Chem. Senses, Vol. 3, Genetics of Perception and Communications, C. J. Wysocki and M. R. Kare (Eds.). Marcel Dekker, Inc., New York, pp. 1–11. Topolski, T. D. (1993). A twin study of olfactory perception. M. A. thesis, California State University, Fullerton. Van Abeelen, J. H. F., and Crusio, W. E. (1985). Zinc-induced peripheral anosmia and mouse exploratory behavior: A quantitative-genetic analysis. Behav. Genet. 15:583. Wallace P. (1977). Individual discrimination of humans by odor. Physiol. Behav. 19:577–579. Ward, C. D., Hess, W. A., and Calne, D. B. (1983). Olfactory impairment in Parkinson’s disease. Neurology 33:943–946. Wedekind, C., and Furi, S. (1997). Body odour preferences in men and women; do they aim for specific MHC combinations or simply heterozygosity? Proc. R. Soc. Lond. B Biol Sci. 264: 1471–1779. Wedekind, C., Seebeck, T., Bettens, F., and Paepke, A. J. (1995). MHC-dependent mate preferences in humans. Proc. R. Soc. Lond. B Biol. Sci. 260:245–249. Wells, P. (1987). Kin recognition in humans. In Kin recognition in animals, D. J. C. Fletcher and C. D. Michener (Eds.). Wiley, New York, pp. 395–415.

344 Werner, D. I., Baker, E. M., Gonzales, E., and Sosa, I. R. (1987). Kinship recognition and grouping in hatchling green iguanas. Behav. Ecol. Sociobiol. 21:83–89. Whissell-Buechy, D., and Amoore, J. E. (1973). Odour-blindness to musk: simple recessive inheritance. Nature 242:271–273. Wilson, R. S. (1979). Analysis of longitudinal twin data. Acta Genet Med. Gemellol. 28:93–105. Wysocki, C. J., and Beauchamp, K. (1984). Ability to smell androstenone is genetically determined. Proc. Natl. Acad. Sci. 81:4899–4902. Wysocki, C. J., and Beauchamp, G. K. (1991). Individual differences in human olfaction. In Chem. Senses, Vol. 3, Genetics of Perception and Communications, C. J. Wysocki and M. R. Kare (Eds.). Marcel Dekker, Inc., New York, pp. 353–373.

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17 Mammalian Pheromones: Fact or Fantasy? Richard L. Doty University of Pennsylvania, Philadelphia, Pennsylvania, U.S.A.

I.

INTRODUCTION

in a more comprehensive paper to appear in the primary literature (Doty, 2003).†

The focus of this chapter is on a putative class of stimuli commonly termed “pheromones.” Pheromones are said to differ from other chemical stimuli in having been evolved to transfer specific information among conspecifics critical for sexual, agonistic, and other forms of social behavior, as well as for altering the reproductive processes of the recipient (e.g., the age of puberty, the timing of estrus, and ova implantation). Although nearly a half-century has elapsed since the pheromone term was coined, few substances considered to be pheromones have been chemically identified and there is still a legitimate question as to the utility of the pheromone concept in describing or explaining chemically mediated social behaviors and endocrine responses of vertebrates. How is one to identify a pheromone? Are there generally accepted criteria for making such an identification? Does evoking the pheromone concept add to our understanding of the behaviors or endocrine responses of interest? The goal of this chapter is to explore these issues. Largely because of space limitations, this review is not inclusive. A broader set of examples, as well as an exploration of the question as to whether humans possess pheromones,* is presented

II.

HISTORY OF THE PHEROMONE TERM

In 1932, the entomologist Bethe distinguished between hormones secreted within the body (“endohormones”) and hormones excreted outside the body (“ectohormones”), dividing the latter chemicals into those with intraspecific and those with interspecific effects (termed homoiohormones and alloiohormones, respectively) (Bethe, 1932). A quarter century later, Karlson and Lüscher (1959) replaced the term homoiohormone with the term pheromone, defining pheromones as “substances which are secreted to the outside by an individual of the same species, in which they release a specific reaction, for example, a definite behavior or a developmental process.” These authors distinguished between pheromones acting via olfaction and those acting via oral or ingestive routes, with the former producing immediate “releasing” responses (e.g., initiating and guiding the flight of the male silk worm moth, Bombyx mori, to the female) and the latter producing delayed endocrine or reproductive responses (e.g., the caste-determining and reproduction-inhibiting substances

*The

reader is referred to a number of recent critiques of studies of menstrual synchrony and the concept of human pheromones (Arden and Dye, 1998; Schank, 2000, 2001; Strassmann, 1999; Whitten, 1999; Wilson, 1992; Wöhrmannj-Repenning, 2000).

†Many

of the general principles described here could be applied to nonmammalian species as well, including, in some cases, a number of invertebrates. 345

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of many social insects). Although numerous concerns and qualifications subsequently arose regarding the usefulness of this term within insects and other arthropods, especially given their remarkable capability to learn and respond to a wide range of odors, the pheromone term nonetheless was found useful in describing many chemically mediated social and endocrine responses in invertebrates. The first use of the term pheromone in describing mammalian—indeed vertebrate—behavior was in the 1960s (Parkes and Bruce, 1961; Whitten, 1966; Wilson and Bossert, 1963). In an influential review appearing in Science, Parkes and Bruce (1961) reiterated Bethe’s general dichotomy and noted that some “chemical messengers” act within an individual (e.g., hormones and “other excitatory substances,” such as CO2), whereas others (i.e., “pheromones”) act between individuals via ingestion, absorption, or sensory receptors. These writers concluded that “Endocrinology has flowered magnificently in the last 40 years; exocrinology is now about to blossom.” The generalized pheromone concept was further popularized by the entomologist E. O. Wilson in a 1963 Scientific American article on the topic (Wilson, 1963). In this paper, Wilson explicitly set the tone for conceptualizing the nature of both priming and releasing mammalian pheromones. In the case of mammalian releasing pheromones, he focused on musks and noted: Pheromones that produce a simple releaser effect—a single specific response mediated directly by the central nervous system—are widespread in the animal kingdom and serve a great many functions....Although two of the six—the mammalian scents muskone and civetone—have been known for some 40 years and are generally assumed to serve a sexual function, their exact role has never been rigorously established by experiments with living animals. In fact, mammals seem to employ musklike compounds, alone or in combination with other substances, to serve several functions: to mark territories, to assist in territorial defense and to identify the sexes.* Up to that time, no reports of the chemical identification of putative mammalian pheromones had appeared, *The caption of the table in which the chemical structures of civetone and muskone were shown read as follows: “Six sex pheromones including the identified sex attractants of four insect species as well as two mammalian musks generally believed to be sex attractants. The molecular weight of most sex pheromones accounts for their narrow specificity and high potency.” Note that Wilson was classifying agents as pheromones without even knowing what effects, if any, they have on behavior or endocrine function.

Doty

although three phenomena seemed to be candidates for primer pheromonal mediation in mammals: the blockage of implantation in recently mated mice as a result of exposure to unfamiliar males or their urine (Bruce, 1960a), the tendency towards anestrous or pseudopregnancy in grouped female mice separated from males or their odors (Lee and Boot, 1955), and the release from anestrous among grouped mice induced by noncastrate male mice or their odors (Whitten, 1956). It was not until the late 1960s or early 1970s, however, that claims for the chemical isolation of any type of mammalian pheromone were made. The initial pheromones reported to be isolated were all of the releasing type, and included (1) substances within the vaginal secretions of rhesus monkeys that were said to elicit copulatory behaviors in males (Curtis et al., 1971; Keverne and Michael, 1971; Michael and Keverne, 1968, 1970a,b; Michael et al., 1971), (2) agents within the tarsal scent glands of male black-tailed deer that elicited licking by females (Brownlee et al., 1969; MüllerSchwarze, 1971; Müller-Schwarze et al., 1974), (3) a material from the midventral scent gland of Mongolian gerbils that received investigation from other gerbils (Thiessen et al., 1974), and (4) two steroids from the submaxillary salivary glands of boars that lowered the threshold for pressure-induced lordosis in female pigs (namely, 5-androsten-16-en-3-one and its related alcohol) (Melrose et al., 1971). III. DEFINITIONS AND REDEFINTIONS OF PHEROMONES While Karlson and Lüscher (1959) provided clear-cut criteria for establishing whether a chemical is a pheromone, it is instructive to see how dictionaries, textbooks, and scientists working in the field of animal and human odor communication have defined pheromones and the degree to which such definitions are congruent with one another and with Karlson and Lüscher’s original definition. For a term to have scientific meaning, it must be defined in a consistent and operational manner, as exemplified by such terms as hormones, neurotransmitters, and quarks. As will be seen in this section, there is considerable variation as to what authoritative sources view as pheromones. A.

Dictionary Definitions

The Random House College Dictionary (1975) defines the term pheromone as follows: “n. Biochem. any of a class of hormonal substances secreted by an individual and stimulating a physiological or behavioral response from an individual of the same species [ Gk phér(ein) (to) bear

Mammalian Pheromones

-0- (HOR)MONE].” By this definition, pheromones are equivalent to hormones and produce physiological or behavioral responses in the conspecific recipient. Webster’s New Collegiate Dictionary (1999) and related Webster dictionaries, on the other hand, do not specifically require a pheromone to be a hormone and confine the elicited response to a behavioral one: “n. a chemical substance that is produced by an animal and serves esp. as a stimulus to other individuals of the same species for one or more behavioral responses.” By this definition, the animal must produce the chemical, not simply serve as an intermediary in its transport. Dorland’s Illustrated Medical Dictionary (1981) similarly uses a less restrictive definition: “a substance secreted to the outside of the body by an individual and perceived (as by smell) by a second individual of the same species, releasing a specific reaction or behavior in the percipient.” Again, an endocrine influence is not specifically noted, although perhaps evoking the term perception implies mental awareness on the part of the recipient. In contrast, Stedman’s Medical Dictionary (1999) employs a more traditional hormone-based definition, although (1) perception is again required, (2) a pheromone is viewed as a subclass of ectohormones, and (3) the general nature of the response is defined: “A type of ectohormone secreted by an individual and perceived by a second individual of the same species, thereby producing a change in the sexual or social behavior of that individual.” Oxford University Press’s Dictionary of Science (1999) indicates that pheromones are externally secreted hormones (i.e., ectohomones), although the door is left open in terms of species specificity. Rather specific mention is made of the nature of the typical chemicals involved and their explicit presence in mammals: “pheromone (ectohormone). A chemical substance emitted by an organism into the environment as a specific signal to another organism, usually of the same species. Pheromones play an important role in the social behavior of certain animals, especially insects and mammals. They are used to attract mates, to mark trails, and to promote social cohesion and coordination in colonies. Pheromones are usually highly volatile organic acids or alcohols and can be effective at minute concentrations.” It is noteworthy that none of these definitions restrict the application to invertebrates and that, by implication, the term is to be applied to a broad spectrum of animals, typically implying externally secreted hormones. Only one of these definitions indicates that pheromones are mediated by the olfactory system, albeit nonexclusively, although the need for the pheromones to be perceived may imply this implicitly. Species specificity seems to be the most common generally held requirement. It is obvious that among dictionaries there is considerable variation as to what, in fact, is a pheromone.

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B.

Textbook Definitions

Unlike dictionaries, textbook definitions seem to be more likely to explictly imply that a pheromone is a type of olfactory stimulus. As with dictionaries, some texts have a brief and simple definition of pheromones, whereas others provide more elaboration. At one extreme is the simple definition provided by Bear et al.’s basic neuroscience textbook (1996), where a pheromone is defined as “an olfactory stimulus used for chemical communication among individuals.” This definition is quite similar to that of Konrad Lorenz’s student, Eibl-Eibesfeldt, who defines phermones in his classic 1970 textbook, Ethology (EiblEibesfeldt, 1970), without the requirement of olfaction being involved: “[Chemical] substances that are effective in intraspecific communication are called pheromones.” In both of these cases, pheromones are not explicity equated to hormones. Groves and Schlesinger, in their Introduction to Biological Psychology (1979), also do not equate pheromones with hormones, although they, like Bear et al., assume pheromones require olfactory mediation. Furthermore, they imply their existence in humans: “Many animals and insects use odors to mark territories, to attract mates, to signal sexual receptivity, and for a variety of other communications. In many instances, the receptors for these chemical communicators are highly specific and respond to remarkably low concentrations of them. These chemical messengers are termed pheromones and are used by many organisms including human beings . . . .” The hormone-like nature of pheromones is emphasized by Campbell in his popular general biology textbook (1996), although he is more tentative regarding human pheromones: Pheromones are chemical signals that function much like hormones, with one important exception; instead of coordinating the parts of a single animal’s body, pheromones are communication signals between animals of the same species. Pheromones are often classified according to their functions as mate attractants, territorial markers, or alarm substances, to name a few. Pheromones are small, volatile molecules that disperse easily into the environment and, like hormones, are active in minute amounts . . . . Compared to most animals, humans do not have well developed olfactory senses, but it is interesting to speculate whether we use pheromones to communicate. Some indirect evidence suggests we do—for example, cases of women living together for several months, as in dormitories, convents, or prisons, who begin to have synchronous menstrual cycles. Clearly, the latter definition—similar to that employed by Oxford University Press’s Dictionary of Science noted in

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the previous section—implies that pheromones mediate their effects through the olfactory system and are relatively small volatile molecules, precluding the possibility that some compounds believed by some to be pheromones, such as musks, may have relatively low volatility and could work through the vomeronasal organ or through taste. Although in this case pheromones are not considered equivalent to hormones, they “function like hormones.” Some authors seem to feel that pheromones come primarily from exocrine glands, although they do not exclude the possibility that they may come from—or are equivalent to—urine or urine-based chemicals. For example, Dember and Jenkins (1970), in their introductory psychology textbook, note the following: One special class of odorants has been of especial interest recently to biologists and animal psychologists. These are odorous substances secreted by an animal from special glands and left as an “odor trail” wherever the animal goes. Such substances are called pheromones; they are known to be secreted by a wide variety of species, ranging from ants to gerbils. Analogous to pheromones in function, if not exactly like them physiologically, are odorous substances, such as urine, excreted by animals and left at various places in the territory over which they travel. One function of pheromones and similar odorants is to “mark off” an animal’s territory. . . . Another function of such odorants is that of a trail-maker. . . . C. Definitions Employed by Scientists Working in the Chemical Senses Martin (1980) defined pheromones as “isolated chemicals shown to be relatively species-specific which elicit a clear and obvious behavioral or endocrinological function and which produce effects involving a large degree of genetic programming, influenced little by experience.” This definition, which is reasonably scientific and operational, embodies the key elements of the original definition of Karlson and Lüscher (1959), but is atypical of many of the definitions used by working scientists. Indeed, it appears that most workers, particularly those studying mammals, have not confined the pheromone term to its implicit, if not explicit, meaning, often redefining the term to meet their own specific conceptions. Shorey, in his 1976 book Animal Communication by Pheromones, defined pheromones as follows: “Pheromones are either odors or taste substances that are released by organisms into the environment, where they serve as messages to others of the same species.” Signoret (1976) noted, “The term ‘pheromone’ has been widely used for any substance produced by an individual

Doty

which, on contact with a member of the same species, evokes behavioral and/or physiological responses. Theoretically, such substances may act not only by olfaction but also by ingestion. In mammals, however, they are believed to act mainly via the sense of smell.” Aron (1979) follows the original definition of Karlson and Lüscher (1959), but further states, “A sensory stimulus whose action is prevented by either olfactory bulb removal or peripheral anosmia should be considered a pheromone in nature until we obtain further information on the compounds involved.” Izard and Vandenbergh (1982) employ a simple definition of pheromone that maintains the concept of species specificity but is rather indescript: “Pheromones are chemical messages secreted by one animal that cause a specific reaction in another individual of the same species.” Meredith (1998), moving far afield from traditional definitions, defines pheromones as “chemicals used for mutually beneficial chemosensory communication between members of a species.” This redefinition, which is reminiscent of a proposal by Rutowski (1981), has some attractive attributes, although it lacks specificity and a number of phenomena traditionally assumed to be mediated by pheromones may not necessarily provide mutual benefit, which itself is difficult to establish operationally (e.g., the elicitation of agonistic responses that may decrease the mating success of one participant, blocking of the pregnancy of an earlier male, delaying puberty). In an introduction to a biblography on mammalian pheromones, Whitten and Champlin (1972) provided a state-of-the-art description of what pheromones were believed to be at that time and added the observation that chemicals that are truly pheromones are most likely found in only one of the sexes in a given species. They also suggested that some chemicals are “co-pheromones.” They stated: [Pheromones are] substances, produced by one member of a species, which influence other members of the same species. They may be behavioural pheromones and evoke rapid behavioural reactions through nervous pathways or they may be primer pheromones and induce relatively slow endocrine responses. There will, of course, be some overlap in this classification because behavioural responses may eventually follow endocrine changes and some responses such as alarm and stress will involve neuroendocrine pathways and may be intermediate in time sequence . . . . Identification of an odorous substance from a glandular or other secretion does not prove that the substance is a pheromone. If it is limited to one sex as for example, muscone in the pod of the male musk deer, then the case is considerably

Mammalian Pheromones

stronger. If, however, as is reported for civetone it is produced by both sexes and by related species then one must look for another function. Civetone has been used for centuries as a fixative in perfume manufacture. For this purpose it intensifies and prolongs more subtle and ephemeral components of a perfume. Civetone may perform this same function for the civet and perhaps it could be considered a co-pheromone. Pheromones are important in animal reproduction and some evidence has been provided to show that they may also function in humans. If they do function in man, they could produce more regular cycles and thus aid in delineating the safe period. Alternatively if perception of sex odors is dependent on ovarian status this too could be used to define the safe period (Vierling and Rock, 1967). Although Whitten and Champlin maintained a distinction between the two basic classes of pheromones, with the responses to a behavioral pheromone being rapid and the responses to a primer pheromone being slow, they argued that the pathways through which pheromones exerted themselves could be quite variable: It is probable that most mammalian pheromones will act through the olfactory system which includes the vomeronasal organ (Jacobson, 1811) and the accessory olfactory bulbs. It is, however, possible that other pathways such as taste may be used or they may even be absorbed through the respiratory or alimentary mucosae. The pheromones may be produced in secretions of the genital organs, the skin glands or occur in the urine, faeces, or the expired air. These substances should be volatile but we do not know what type of sensation they produce in the recipient animal. Nor do we know if primer pheromones are perceived as odors. The pheromones concerned with reproduction will most likely be under endocrine control and the sensitivity to the pheromone may be influenced by gonadal status (LeMagnen, 1951). In an insightful early review, Bronson (1971) specifically stated that no mammalian pheromone had yet been isolated and expressed the need to use the pheromone term only in situations where, in fact, it was likely that some chemical agent could be identified.* He noted, “It would *There is a contradiction in this review regarding the isolation of pheromones, as he states that “ . . . the reasonably well isolated mammalian pheromone, that of the tarsal gland of black-tailed deer, is apparently a decided mixture of compounds.”

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seem most profitable to restrict the use of this term [pheromone] to situations where there seems to be a reasonable probability of isolating one or at least a restricted mixture of compounds that could, in turn, be synthesized and whose actions could then be reconfirmed experimentally,” a task that could be quite difficult a priori. Bronson also stressed that any response to a pheromone “should serve a reasonable biological function in a natural population.” Importantly, he noted, “The probability of a high degree of specificity in pheromones, however, argues against the widespread use of the more typical androgen metabolites as pheromones.” It is noteworthy that one of the pioneers of the pheromone field, Hilda Bruce, indicated in 1970 that “the meaning of the word ‘pheromone’ has been extended to include chemical communication in a broader sense and in all species.” She stated that “The term now connotes secretions which convey information of many kinds from one individual to others and evoke specific behavioural and physiological reactions in the recipients.” Nonetheless, she operationally differentiated between priming and releasing pheromones. To her, releaser pheromones were to be recognized by “An immediate and reversible response [that] operated directly through the central nervous system, e.g. recognition, or through rapidly acting neurochemical channels, as exemplified by the milk-ejection reflex (Cross and Harris, 1952),” and primer pheromones by an “exteroceptive response implicating the anterior pituitary gland. This type is slow to develop, demanding prolonged stimulation which initiates a chain of physiological effects in the recipient.” Interestingly, she added a third pheromone class, which she termed “imprinting pheromones,” to explain modification of later adult behavior by the presence of chemical stimuli within the suckling environment.* She cited two examples of such pheromones: In mice (Mainardi et al., 1965) and in rats (Marr and Gardner, Jr., 1965), social behaviour in the adult may be modified by olfactory experience during the period of suckling. Female mice reared in the absence of the father show a loss of discrimination in sexual selection when adult, and the same deficiency develops if the olfactory atmosphere of the nest is artificially altered by spraying the parents everyday with perfume (Parma violet). Young rats reared from

*Although

in the text she indicates that “mammalian olfactory pheromones belong to all three types” and provides a separate category both in her outline and in the text for imprinting pheromones, the last sentence of her paragraph above, which has been omitted for clarity, curiously reads: “This aspect of primer pheromone activity [i.e., imprinting] merits further attention.”

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birth to about four weeks of age in the olfactorily artificial atmosphere, scented either with Yardley’s Red Roses cologne or with oil of wintergreen, also showed modifications when adult. A number of subsequent authors have assumed that certain steroids are unequivocally mammalian pheromones based upon their seemingly direct effects on sex-related behaviors. Among the most common of such steroids is androstenone, a constituent of boar saliva and other secretions, which produces the “boar taint” of meat from uncastrated boars and lowers the threshold for lordosis in sexually experienced sows. Thus, Pause et al. (1999) note that “Androstenone has been studied extensively for its pheromonal properties. In the boar, it has been proven to be a pheromone, because it is produced in the boar testes, and the sow responds to its smell with the mating stance (lordosis). Evidence for androstenone being a pheromone in humans comes from an experiment carried out by KirkSmith and Booth (1980). They sprayed androstenone onto a seat in a dentist’s waiting room and observed 840 people for their seat preferences. The authors could show that more women but fewer men used the odorized seat than expected by chance.” Clearly, Pause et al.’s criteria for a pheromone (origin in testes, influences on choice of seating in a dental office) differ from those inherent in most pheromone definitions. Several relatively recent definitions of pheromone are worthy of note. Buck (2000) reiterates the commonly held view of innateness, stating that “pheromones elicit programmed neuroendocrine changes and innate behaviors, suggesting a need for a very precise recognition process.” Stern and McClintock (1998), in a paper purportedly being the first demonstration of a human pheromone (which, in fact, was not identified chemically), added a new requirement for the pheromone definition— namely, that pheromones are airborne. These investigators state: “Pheromones are airborne chemical signals that are released by an individual into the environment and which affect the physiology or behaviour of other members of the same species.” Using this definition, the many substances that have been previously deemed pheromones found in aquatic environments or transferred into the vomeronasal organ via liquid media would not be considered pheromones, nor would secretions ingested by insects and other forms that alter cast or endocrine state. Moreover, Stern and McClintock indicated that “Here we investigate whether humans produce compounds that regulate a specific neuroendocrine mechanism in other people without being consciously detected as odours (thereby fulfilling the classic pheromone definition).” It is not clear what “classic” definition of pheromone is being referring to

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here, although some workers have suggested that vomeronasal responses may be unconscious (LloydThomas and Keverne, 1982; Meredith, 1991), and several theorists have expressed the view that pheromones are unconsciously mediated by the vomeronasal organ (Belluscio et al., 1999; Dulac and Axel, 1998). Recently, Savic et al. (2001) incorrectly noted that “The pheromones are, according to the original [i.e., Karlson and Lüscher] definition, volatile [italics mine] compounds secreted into the environment (in sweat, urine) by one individual of a species . . . .” In fact, volatility was not a part of the original definition. These authors continue, “Also, it has recently been reported that a putative pheromone receptor gene is expressed in human olfactory mucosa (Rodriguez et al., 2000). These data raise the question whether there are compounds that via the nasal mucosa activate the human hypothalamus in a sex-specific mode. If so, such compounds would fulfill one important criterium [sic] to qualify as candidates for putative pheromones in humans.” To my knowledge, neither sex specificity nor the activation of the hypothalamus are widely held criteria for pheromones, although, as noted above, Whitten et al. (1972) believed that sex specificity might add to the proof that a substance is a pheromone, and some subsequent workers have employed such specificity in their definitions of pheromones. In a thoughtful article, Johnston (2000) proposed a classification scheme that operationally distinguishes between pheromonal and nonpheromonal stimuli on the basis of chemical complexity. He suggests that the term “chemical signal” be used as a generic term for chemical compounds or mixtures released into the environment that serve intraspecific behavioral or physiological functions. Johnston reserves the term “pheromone” for chemical signals that employ a single compound and the term “pheromone blend,” which has been employed in some insect studies, for mixtures of a small number of compounds that are maximally effective when they occur in precise ratios. He further suggests that the term “mosaic signal” or “odor mosaic” be used to refer to mixtures of large numbers of compounds in which many components are important for producing the full effect. Johnston indicates that “This scheme has the advantage of preserving the traditional use of the terms ‘pheromone’ and ‘pheromone blend,’ while adding a third important category to encompass signals that have previously been given little attention or regulated to a second-class status.” While indeed Johnston’s terminology does not significantly perturb the status quo, it is debatable whether it is an advantage to maintain the use of terms that implicitly or explicitly define the nature of the interaction to most biologists as hormone-like. Moreover, his suggested distinctions are

Mammalian Pheromones

difficult to apply a priori, and no differentiation is made between learned and unlearned responses. Johnston correctly points out that single molecules are rarely used as sex attractants even in insects, presumably reflecting the need for species-specific signals that are more easily established by selected ratios of given agents. It is of interest that, by Johnston’s criteria, the term pheromone would rarely be applied even to insects. A number of molecular biologists who work in the chemical senses have recently defined the vomeronasal organ as “the pheromone receptor,” assuming that the main olfactory system is relegated to the perception of nonpheromones (Belluscio et al., 1999; Buck, 2000; Dulac et al., 1998; Tirindelli et al., 1998).* Thus, Dulac and Axel (1995) wrote that: Mammals possess an olfactory system of enormous discriminatory power. Humans, for example, are capable of recognizing thousands of discrete odors. The perception of odors in humans is often viewed as an aesthetic sense, a sense capable of evoking emotion and memory, leading, to measured thoughts and behaviors. Smell, however, is also the primal sense. In most species, odors can elicit innate and stereotyped behaviors that are likely to result from the nonconscious perception of odors. These different pathways of olfactory sensory processing are thought to be mediated by two anatomically and functionally distinct olfactory sensory organs, the main olfactory epithelium (MOE) and the vomeronasal organ (VNO). This revolutionary approach takes the definition of pheromone to an entirely new level, particularly since the receptors of main and vomeronasal systems appear to share no homology. Hence, according to this perspective, pheromone detection would seem to have evolved separately from nonpheromonal odorant detection (Dulac et al., 1998). Although other molecular biologists working in this field have maintained a less bipartite distinction, nonetheless the vomeronasal organ is typically viewed as “a *It

should be noted that the effects of the one isolated agent that seems to best meet traditional definitions of phermones, androstenone, are mediated in the sow through the main olfactory system, not the vomeronasal system. Perhaps the claim of the vomeronasal organ as the pheromone detector led Hines (1997) to provide the following definition of pheromones in a Science magazine perspectives article: “a special subset of olfactory signals [that are] not perceived consciously. . . . These molecules, often fatty acids or steroids, are secreted by animals, then detected by other animals, of the same species, where they regulate such basic functions as mating, the timing of the estrous cycle, and aggressiveness.”

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chemoreceptive organ for the processing of pheromones” (Takigami et al., 1999). Hence, “the vomeronasal organ has attracted increasing attention as it is thought to mediate the stereotyped behavioral and neuroendocrine responses to chemicals commonly known as ‘pheromones’ ” (Liman, 1996). As articulated by Matsunami and Buck (1997): Pheromones are intraspecific chemical signals found throughout the animal kingdom. They regulate populations of animals by inducing innate behaviors and stereotyped changes in physiology (Karlson et al., 1959; Wilson, 1963; Sorensen, 1996). Pheromones can serve as cues for overcrowding, impending danger, reproductive status, gender, or dominance. In rodents, a variety of pheromone effects have been reported. These include effects on estrus and the onset of puberty as well as the induction of mating and aggressive behaviors (Halpern, 1987; Wysocki and Meredith, 1987; Novotny et al., 1990; Singer, 1991) . . . . The detection of pheromones is mediated by the olfactory system. However, sensory neurons that detect pheromones are typically segregated from those that detect volatile odorants (Keverne, 1983; Wysocki et al., 1987; Novotny et al., 1990; Hildebrand and Shepherd, 1997). In mammals, sensory neurons in the nasal olfactory epithelium (OE) detect volatile odorants and some pheromones, while those in the accessory olfactory organ, called the vomeronasal organ (VNO), are thought to be specialized to detect pheromones. While it has been known for years that the VNO responds electrophysiologically to chemicals not generally viewed as “pheromones” (e.g., Hatanaka, 1992; Meredith, 1982,1991), apparently this has only recently been realized by some investigators (e.g., Sam et al., 2001). This observation, however, throws into question the aforementioned notion that pheromones can be distinguished from nonpheromones on the basis of whether the VNO is activated, decreasing the alluring clarity of this distinction and making the VNO more like the main olfactory system in reflecting a broader assessment of environmental agents. Thus, Sam et al. (2001) reported, in a recent Nature paper, that “The prevailing view of the mammalian olfactory system is that odorants are detected only in the nasal olfactory epithelium, whereas pheromones are generally detected in the vomeronasal organ. Here we show that vomeronasal neurons can actually detect both odorants and pheromones. This suggests that in mammals, as in insects, odorous compounds released from plants or other animal species may act as ‘semiochemicals’—signaling molecules that elicit stereotyped behaviours that are advantageous to the emitter or to the receiver.”

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Whatever the situation with the VNO, it is clear from the information reviewed above that the term pheromone means different things to different people and has been redefined over and over in an attempt to fit a range of chemically mediated behaviors and endocrine responses into a common mold. While the term itself is intuitive and catchy, it clearly has many of the same problems as the term instinct, with which it is closely allied, leading to significant dangers. As Lehrman pointed out in his classic 1953 paper, “A Critique of Konrad Lorenz’s Theory of Instinctive Behavior,” labeling behaviors as instinctual— while perhaps gratifying—produces an either/or dichotomy with significant associated pitfalls. For example, such labeling tends to preclude the need to study developmental or experiential factors associated in the fruition of a given behavior, oversimplifying even its genetic underpinnings (Beach, 1955; Lehrman, 1953). IV. EARLY CONCERNS WITH THE CONCEPT OF RELEASING PHEROMONES IN MAMMALS As early as the late 1960s, a number of influential biologists voiced concern about the utility of describing chemicals involved in the social behavior of mammals as “releasing pheromones,” reflecting their awareness that mammalian behavior is not reflexive in the same way as the behaviors of many invertebrates. In 1968 Bronson suggested that the term “signaling” should replace the term “releaser,” and in 1973 Whitten and Champlin suggested that “behavioral” should serve as the substitute, as employed in Whitten’s definition described above. Subsequent investigators suggested replacing the releasing pheromone term with such terms as “social odors” (Brown, 1979), “homeochemic substances” (Martin, 1980), or “semiochemicals” (Albone, 1984). Bronson (1976) clearly articulated the problem in a statement 27 years ago: It is perhaps unfortunate that interest in mammalian chemical communication blossomed at a time when the study of insect pheromones was already a sophisticated field of research. Thus Whitten (1966) introduced the primer-releaser dichotomy to mammalian workers and Bronson (1968) amended it only slightly by arguing that the term signaling was a more appropriate modifier than releaser for a nonprimer pheromone, given the variable, experience-oriented behavior of mammals.* The unfortunate side of such *To the author’s knowledge, Whitten was not the first to introduce

the pheromone term to mammals, as indicated earlier in this review.

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generalizations is the tendency to think of mammalian communication in terms of simple stimulus-response systems. For example, it is now relatively common usage to refer to “aggression-promoting” (or “eliciting”) and “aggression-inhibiting” pheromones in mice (e.g., Lee and Griffo, 1974; Mugford and Nowell, 1972). The obvious implication of this terminology is the existence of two simple urinary compounds which unequivocally either release or inhibit a stereotyped aggressive response. Mammalian social behavior simply does not work that way except at the purely reflexive level. Bronson went on to point out that the nervous system of the mouse not only contains many more neurons than that of an insect, but differs significantly in terms of degree of encephalization, the numbers of associative neurons, and the flexibility afforded to the mediated behaviors. He indicated that the releasing pheromone concept is a valuable tool for describing the “often relatively simple, stimulus-response systems of such organisms,” but is questionable for mice and other mammals. He continued: Most insect pheromones are usually single compounds or simple mixtures, typically secreted by restricted glands, and normally evoking stereotyped responses even under totally inappropriate circumstances. Thus many of the standard tests for insect attractants have relied upon copulatory behavior in response to scented filter paper, repeated exposures in many cases providing little habituation of the response (Birch, 1974). It is difficult to imagine a male mouse attempting copulation with a scented filter paper let alone doing so repeatedly, and, by extension, it is exceedingly difficult to apply the simple releaser concept to much of mammalian social behavior, whether elicited in part by odors or not. Additionally, experience is a profound modifier to mammalian social behavior. There have actually been relatively few attempts to examine the role of experience in odor-induced responses in mammals. Where investigated, however, the results usually have indicated a potent role for experience. Thus species identification apparently can be easily manipulated by odors early in the life of mammals (e.g., Carter and Marr, 1970; Mainardi et al., 1965; Marr and Lilliston, 1969) and adult sexual experience is a strong determinant of response to sex odors (e.g., Caroom and Bronson, 1971; Carr et al., 1965, 1966). One wonders at this point whether the pheromone concept, so useful in insect behavior and physiology, should be bastardized to

Mammalian Pheromones

the point where it is used to cover situations in mammalian behavior where usually complex odors evoke highly variable responses which are easily modified by experience. In the same book in which Bronson voiced his concerns about the concept of releasing pheromones, Beauchamp et al. (1976) cast doubt on the utility of the pheromone concept altogether, noting that “. . . we question the current usefulness of the term ‘pheromone’ in describing the influences of biological secretions and excretions upon mammalian reproductive behaviors and suggest that the uncritical use of this term has led to a number of misconceptions in the interpretation and conduct of mammalian behavioral research.” They listed what the implicit or explicit criteria for a chemical to be termed a pheromone seemed to be up to that time—a listing that, at first glance, provided an operational basis for determining whether a chemical was, in fact, a pheromone. Their list of criteria was as follows: Species specificity A well-defined behavioral or endocrinological function A large degree of genetic programming The involvement of only one or at most a relatively few compounds Uniqueness of the isolated compounds or small set of compounds in producing the behavioral or endocrinological response These authors examined all extant claims of isolation of mammalian pheromones up to that time and pointed out that none tested or met even half of these criteria. Indeed, only one criterion, that of assumed chemical simplicity for the isolated product, was met by all of the isolated substances. They concluded: “It would appear to us that the labeling of a compound as a pheromone, when it has not been demonstrated to meet a well-defined set of operational criteria, is problematic if the pheromone term is to have any meaning beyond that of being synonymous with a ‘chemical’.” However, even these criteria, for the most part, are difficult to employ. Thus, how many species need to be tested before species specificity can be assumed? What is meant by a “large degree” of genetic programming—i.e., how can degrees of genetic programming be operationally determined? How many chemicals must be tested before the uniqueness of the isolated stimuli can, in fact, be ascertained? Answers to such basic questions are needed, however, if one is operationally establish what, in fact, is a pheromone. Of course, if authors simply label behaviors or endocrine responses as being pheromonally mediated

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without being concerned about such matters or identifying the chemicals involved, such distinctions are academic. In an attempt to address the issue of learning, MüllerSchwarze (1977) sought to add still another class of pheromones—so-called “informer” pheromones. The genesis of this suggestion was that some chemical signals in mammals are “ . . . stored in the memory and can be recalled later in a variety of contexts.” Unlike the terms releasing and priming, this term did not catch on, however, and it was apparently not used even by Müller-Schwarze in subsequent papers. However, his point—that learning is important in odor communication—is a fundamental one that throws into question the general validity of the traditionally conceived pheromone concept, as will be pointed out later in this chapter. Sorensen and Stacey (1999), while maintaining the belief that natural selection is focused on specific chemicals, nonetheless point out the practical problems with the traditional pheromone concept in terrestrial vertebrates: Presumably, chemical stimuli are predisposed to function as social signals because they are ubiquitous and discriminated with great sensitivity and specificity. Because of this specificity, organisms detect only a portion of the myriad compounds surrounding them. Pheromonal systems have proven challenging to study because it has been difficult to predict which of the many chemicals released by organisms might have pheromonal activity. Terrestrial vertebrates appear to have evolved, repeatedly and independently, a variety of sex pheromones with no clear common precursors. Few of their sex pheromones have been identified and no general theoretical framework has emerged to systematically address either the diversity of sex pheromone systems or the evolutionary processes that might have created them. V. CHEMOSENSORY LEARNING USUALLY OVERRIDES CHEMOSENSORY GENETICS As noted in the previous sections, most adherents to the pheromone concept view pheromones as species-specific hormone-like agents that differ fundamentally from the plethora of other environmental chemicals that are sensed by organisms. In general, pheromones are viewed as being little influenced by learning and are more or less genetically fixed. However, even if some odors are, in fact, inherently more preferable to animals than others, does one have to infer that an innate pheromone is the basis for this preference or is essential for the initiation of exploration or

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sniffing of a scent?* In contrast to this perspective, a case can be made that learning is involved in establishing the meaning of most odorous chemicals to mammals. Moreover, learning seems to be a key component in such classic examples of “primer” pheromones as the strange male odor pregnancy block. As described in this section, mammals appear to learn such important information as their mother’s odor, the odor of their species, the odor of their offspring, the odor of their social group (e.g., deme), the odor of a fecund sexual partner, the odor of the dominant or subordinant conspecific, and the odor of familiar conspecifics, distinguishing them from strangers. In these cases, can the stimuli involved be considered pheromones? Evidence that rodents exhibit a remarkable capability to learn and employ odors in complex “cognitive tasks” appeared in the early 1970s. Prior to these studies, the comparative intelligence of a number of animals was assessed using “learning sets” or “learning to learn” paradigms based upon visual responses, one of several approaches for assessing animal cognition.† In general, the relative performance of various species on such tasks was found to mirror the “phylogenetic scale” or scala naturae so often employed in studies of comparative anatomy, i.e., primates  other mammals  birds  reptiles  amphibians  fish  insects (Bitterman, 1965; for critique of this concept, see Hodos and Campbell, 1969). However, when the sensory specializations of different forms were taken into account, it became apparent that rodents such as the rat could perform essentially as well as primates on such

*Of

course, it does not necessarily follow from this argument that preprogrammed specific responses cannot exist, or that such responses would necessarily be unmodifiable by experience or other factors. The point being made is that one should not assume that such preprogrammed responses are the norm. † In a learning set paradigm, the subject is given a series of discrimination problems to solve, the first of which may require many trials to learn. Over a series of sessions, however, the ability to solve new problems dramatically improves, suggesting that the animal has learned “rules” or “concepts” underlying the task. For example, in a three-item reversal task, a monkey may be given two circles and a square as the first problem, being reinforced for choosing the square. After attaining high performance on this task, the monkey is then given two squares and a circle, with reinforcement given for choosing the circle. While initially the tendency of the animal is to choose the square, at some point he chooses the circle. The next reversal would be like the first, usually with a different spatial configuation to control for spatial preferences, and so on. In this case, the animal is acquiring the concept of “oddity” and at some point learns to choose the odd stimulus on a new set without ever having been reinforced for any of the stimuli employed in the new task.

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tasks, so long as olfactory, rather than visual, stimuli were employed. Thus, Jennings and Keffer (1969) and Nigrosh et al. (1975) found excellent interproblem transfer over a series of two-odor discrimination problems in the rat, such that errorless performance on subsequent reversals was commonly attained (Slotnick, 2000). These and other studies demonstrated that rats can learn to identify and discriminate among large numbers of odors and can remember whether they were reinforced or not reinforced for each of these odors in a test series (Slotnick et al., 1991). Slotnick (2002) states: Functional studies have overcome many of the technical difficulties of controlling vapor stimuli and demonstrate that, with odor cues, rats display highly efficient learning rivaling that of primates. In short, the evidence indicates that rats can ‘think with their noses’ and have the neural machinery to do so. This evidence, combined with advances in the molecular biology of olfaction (Mombaerts et al., 1996), has resulted in a renaissance in research on olfaction and to the surprising and occasionally controversial suggestion that the rodent olfactory system could serve as a model for neurobiological studies of cognition (Reid and Morris, 1993; Slotnick, 1994). In light of such observations, a strong argument can be made that, just as mammalian photoreceptors have not evolved specifically for detecting mothers, fathers, or Ferrari automobiles, so too mammalian olfactory receptors have not evolved specifically to detect the odors of mothers, fathers, or the exhaust smells of Ferrari automobiles. This lack of specificity extends to the smells of individual conspecifics, even though their long-term identification, like the visual detection of Ferraris, can occur as long as learning at some point intervenes. Thus, while the olfactory system, like the visual system, can provide specific information about the physical nature of the environment, the specificity is largely dependent or interdependent upon experience. It is noteworthy that olfactory detection thresholds of rats for perfluorocarbons—agents never encountered during their phylogeny—are at the same level of magnitude as thresholds for many organic chemicals presumably encountered during ancestral evolution, reiterating the notion that evolution has not focused on the detection of specific chemicals, but on the provision of a sensory system that is flexible and sensitive to the detection of even de novo agents (Marshall et al., 1981). A.

Prenatal Learning

It is important to be aware that the influences of experience on establishing the social significance of some odors can

Mammalian Pheromones

occur even before birth. Thus, the olfactory system of many mammals, including humans, is functional in utero and intrauterine learning can manifest itself in postpartum life. Evidence for prenatal function includes observations that premature human infants exhibit discriminative responses among low concentrations of odorants presented to them (Pihet et al., 1997; Sarnat, 1978), and rat fetuses transferred from the abdominal cavity of their mothers into saline without interruption of the maternal blood supply exhibit increased activity, altered heart rate, and facial wiping responses to odorants (Smotherman and Robinson, 1987, 1990). Evidence that experiences with odors before birth can influence behaviors later in life comes from many sources. For example, human fetuses learn odors related to their pregnant mother’s diet (Schaal et al., 2000), and such intrauterine learning can be reinforced in the nursing situation, where flavors ingested by the mother can be transmitted via the mother’s milk (Galef and Henderson, 1972; Galef and Sherry, 1973; Mennella and Beauchamp, 1991a,b, 1996). Rat pups exposed to citral in utero attach, postpartum, to washed citral-scented nipples and not to normal unwashed nipples (Pedersen and Blass, 1982). Offspring of pregnant rats receiving an infusion of an odorant into the amniotic fluid and made sick by lithium chloride injected into the mother avoid postnatally the odor to which they had been exposed (Smotherman, 1982; Stickrod et al., 1982). If no toxic agent is administered to the mother, then a postnatal preference for the prenatally exposed odorant may appear in later life, particularly if that same odorant is present in the early perinatal period (Nishiazaka et al., 1993; Pedersen et al., 1983). In some cases postnatal odor preferences can be induced by simply feeding the pregnant mother the target odorant (Hepper, 1988; Schaal et al., 1995). For example, rabbit pups whose mothers were fed aromatic juniper berries during pregnancy (such berries are part of the rabbits’ natural diet) prefer juniper at weaning, even if raised after birth by a foster mother fed standard laboratory food (Bilko et al., 1994). This preference for juniper, which is not seen in controls, is still present months later, even without additional juniper experience (Hudson and Distel, 1999). The magnitude of the summated electrical potential at the surface of the olfactory epithelium (the electro-olfactogram) in response to juniper, obtained from the epithelia of sacrified rabbits whose mothers were fed the juniper leaves, is larger than that of rabbits whose mothers were not fed the leaves, suggesting neural changes at the level of the olfactory receptors (Hudson et al., 1999). This observation is in accord with other studies demonstrating exposure-induced alterations in peripheral olfactory physiology (Coopersmith and Leon, 1984, 1986; Wang et al., 1993; Youngentob and Kent, 1995).

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B.

Neonatal Learning

In a manner conceivably analogous to the visual and auditory imprinting processes of birds, many odors are learned during early periods of the developing mammal.* While mere exposure to odors can produce learning in some instances, odor preferences are reinforced in the suckling environment by the warmth of the mother and the licking of her pup, even independently of milk reinforcement. Indeed, simply pairing an artificial odor with a warm surface or with tactile stroking is sufficient to establish conditioned olfactory preferences in rat pups (Alberts and Brunjes, 1978; Alberts and May, 1984; Dominguez et al., 1999). In general, neonates detect and find attractive the odorous components of amniotic fluid, particularly those of their own mothers (e.g., Hepper, 1987; Schaal et al., 1998; Teicher and Blass, 1977), likely reflecting intrauterine experience, as noted above, and possibly explaining their attraction to nipple-related odors and other maternal secretions around the time of birth (Schaal et al., 1994). Such attraction aids in guidance to the nipple and alters their general motor activity and arousal (for review, see Porter and Schaal, 2000). Parturient females of many species engage in self-grooming that deposits saliva and amniotic fluid on their ventra and nipple regions, and these secretions carry, in large part, the chemical message that directs the first suckling episode of the newborn (Teicher and Blass, 1976; Teicher et al., 1977). Even human infants, who preferentially exhibit head orientations towards maternal breast odors within the first few minutes of life, are likely influenced by prior experience with amniotic fluid. As noted by Porter and Winberg (1999), “the role of maternal olfactory signals in the mediation of early breastfeeding is functionally analogous to that of nipple-search

*It should be emphasized that odors are not necessarily unique, in that early learning via all of the senses occurs in mammals and many other forms, including many insects and birds (Beach and Jaynes, 1954). Cross-fostering can even influence visual social preferences in some mammals. For example, male sheep and goats cross-fostered to the opposite species, unlike their normally reared counterparts, show a nearly exclusive preference for faces of females of their foster species; cross-fostered females also exhibit, relative to normals, an increased preference for the crossfostered species’ female faces, although their preferences are more-or-less equally divided among the faces of the genetic and cross-fostered species (Kendrick et al., 2001). These investigators concluded, “these results provide strong evidence that social and sexual preferences are primarily determined by maternal and social rather than genetic influences even in mammals and that effects are stronger and more durable in males than in females.”

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pheromone as described in nonhuman mammals. To some extent, the chemical profile of breast secretions overlaps with that of amniotic fluid. Therefore, early postnatal attraction to odors associated with the nipple/areola may reflect prenatal exposure and familarization.” Some foods ingested by the mother markedly influence the smell of the amniotic fluid and an infant’s attraction to it (Mennella et al., 1995), as well as influence the flavor of the mother’s milk. Interestingly, amniotic fluid has other important properties for both the mother and offspring. In the rat, for example, the ingestion of amniotic fluid potentiates opiaterelated analgesia (Kristal et al., 1986). A number of mammalian cross-fostering studies, including ones performed on ungulates (Müller-Schwarze and Müller-Schwarze, 1971), find that it is the odor of the species or subspecies of the cross-fostered parent, not that of the genetic parent, that largely establishes subsequent social and mating preferences. In some species or instances, such effects may be more marked in the female than in the male, as would be predicted from the female’s greater investment in proper mate selection (Doty, 1974). In a pioneering study, Mainardi (1963) found that estrous female housemice of the Mus musculus domesticus subspecies, reared by both parents since weaning, preferred the odors of M. m. domesticus to those of M. m. bactrianus, whereas analogous females reared only by their mothers, in the absence of adult males, showed no differential preferences between these two subspecies. Quadagno and Banks (1970) found that female housemice (Mus musculus) cross-fostered to pigmy mice (Baiomys taylori) preferred the odor of pigmy mice to housemice in adult preference tests. One source of the odor involved may be the preputial glands, since female mice reared with mothers whose preputial glands have been excised prefer females without preputial glands in adulthood (Hayashi, 1979). McCarty and Southwick (1977) found decreased conspecific odor preferences in grasshopper mice (Onychomys torridus) and white-footed mice (Peromyscus leucopus) that were cross-fostered to the other species’ dams; cross-fostered Peromyscus males actually switched their species preference to Onychonmys. Both Mus musculus and Peromyscus maniculatus mice reared in the presence of both species’ odors prove to be more successful in heterospecific agonistic encounters than conspecific counterparts reared only with their own species’ odors (Stark and Hazlett, 1972). Interestingly, gerbils (Meriones unguiculatus) reared with parents whose midventral sebaceous glands were surgically removed show lower preferences for such odors in adulthood and engage in less social behavior with opposite-sexed conspecifics than gerbils raised with parents having such glands (Blum et al., 1975).

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While much has been made of odor-related dissortative mating preferences in mice related to genes of the major histocompatability complex (MHC) (Beauchamp et al., 1985; Yamazaki and Boyse, 1985; Yamazaki et al., 1976, 1998), genes at other loci are also involved in establishing cues employed in individual identity, and there is strong evidence that cross-fostering and diet override or attenuate such genetic predispositions (Burger et al., 2001; Penn and Potts, 1998). For example, Yamazaki et al. (1988) demonstrated in mice whose genetic differences were only within the MHC complex, that the preference for B6-H-2k males to mate with B6-J-2b females, and the preference for B6-J-2b males to mate with B6-H-2k females, was reversed when the mouse pups were cross-fostered by the opposite H-2 haplotype. Singh et al. (1990) found that the urine of individual male rats born by Cesarian section and reared in a germ-free environment were not discriminable by Lister hooded rats using a habituation-dishabitutation test. However, when these rats were moved to a non–germ-free conventional animal house, such urine was discriminable after recolonization with commensal flora, suggesting that commensal bacteria are involved in the production of unique individual odor of the urine of even MHC-congenic rats. This observation is in accord with the fact that individual odor-mediated identity seems to be largely influenced by diet in number of mammals, as described in more detail later in this chapter (for review, see Schellinck and Brown, 1999). As alluded to earlier in this section, learned responsiveness of rodents and a number of other mammals to odorants present during the preweaning period is not confined to so-called “natural,” “biological,” or “animal” odors, but can extend to “artifical” odors as well (Cornwell, 1976; Galef and Kaner, 1980; Gregory and Bishop, 1975; Janus, 1993). Thus, any of a number of odorants (e.g., ethyl benzoate, acetophenone, methyl salicyclate, Yardley’s Red Roses cologne, citral, cinnamon, cumin, and various perfumes, such as Parma Violet perfume), placed in the rearing environment, can take on the same meaning as natural biological stimuli and alter subsequent preferences for scented situations or scented conspecifics in later life (Alleva et al., 1981; Carter, 1972; Carter et al., 1970; Fillion and Blass, 1986a; Janus, 1989, 1993). For example, rats reared on lemon-scented bedding from birth to weaning acquire a seemingly permanent preference for nesting in lemon-scented surroundings (Rodriguez-Echandia et al., 1982). Adult rats previously reared with mothers and littermates odorized by artificial odors prefer conspecifics odorized with such odors and are less responsive sexually to unodorized conspecifics (Fillion and Blass, 1986b; Marr et al., 1965, 1969). The same is true for mice. Thus, Mainardi et al. (1965) reared male and female house mice

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(SWM/Mai strain) with perfumed or nonperfumed parents until the age of 21 days, when they were weaned and separated into like-sex groups. When tested in estrus at 8 months of age, 48.2% of the perfume-reared females spent more than 60% of their time with a perfumed male, compared to 21.4% of the normally reared females. Of the normally reared females, 67.8% spent more than 60% of the test time with the nonscented males, compared to 27.6% of the perfume-reared females. Such findings reiterate the fact that the olfactory system has been designed, like the visual system, to recognize, remember, and prefer variant salient features of the general odoriferous environment. C.

Adult Learning

Even though there appear to be early “critical periods” in many mammals when exposure to odorants is particularly effective in producing long-term alterations in odor preferences and odor-mediated social behaviors (Galef et al., 1980), experience with odors in adulthood can also significantly alter later odor-mediated behavioral and sexual preferences, explaining many so-called releasing pheromone effects. For example, rats, dogs, and a number of other mammals develop preferences, or markedly increase preexisting subtle preferences, for estrous over diestrous female odor as a result of adult sexual experience (e.g., Carr et al., 1965; Doty and Dunbar, 1974; Le Magnen, 1951; Lydell and Doty, 1972). Sexual preferences, however, can also be influenced by adult experiences with artificial odors placed on sexually receptive females. In one series of studies, for example, male rats were allowed to mate with estrous females who had almond extract applied to their neck and anogenital region. In subsequent tests with almond-scented and nonscented receptive females, these males ejaculated first and more frequently with the almond-scented females (Kippin and Pfaus, 2001a). Interestingly, such males tended to preferentially mount the almond-scented females immediately prior to ejaculation, implying that the preference may be conditioned to the ejaculatory event, per se (Kippin and Pfaus, 2001b; Kippin et al., 2001), although the presence of the female during a postejaculatory period is important for producing the conditioning (Kippin et al., 2001). The effectiveness of such conditioning appears to relate to the degree of satiety and the rat’s motivational state (Kippin et al., 2001). No such preference was observed in males having no experience with almond odor or trained in the presence of almond odor in an unpaired or randomly paired manner. Mammals generally have the ability to rapidly acquire and maintain memories for many types of odors, and odors

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function to denote other individuals, as well as environmental objects. Mice (Mus musculus), rats (Rattus norvegicus), hamsters (Mesocricetus auratus), Belding’s ground squirrels (Spermophilus beldingi), and guinea pigs (Cavius porcellus), species extensively tested on this point, can remember dozens, if not hundreds, of odors of individual conspecifics encountered in adulthood, apparently in some cases for a lifetime, even after having only encountered them on a single brief occasion (Beauchamp and Wellington, 1984; Brown, 1988; Johnston, 1993; Mateo and Johnston, 2000; Mossman and Drickamer, 1996).* The association of odors with objects is obvious even in humans, where odors are nearly always identified with their source, as is apparent from their names (e.g., rose, candy, pizza, gasoline, fish, licorice, chocolate, leather, mint, seashore, perfume, etc.). Porter et al. (1983) applied artificial odors (i.e., musk oil, oil of clove, lemon/lime, and cherry) every other day on four occasions after weaning to four groups (n
4/group) of spiny mice (Acomys cahirinjus) composed of two siblings and two nonsiblings. Each group received a single odor. Later preference tests conducted with odorized kin and nonkin strangers found that the mice interacted, for all practical purposes, only with mice having the same odor to which they had been exposed as adolescents, regardless of genetic relatedness. Adult female mice of the SEC1Re/J strain (SEC), when exposed to the odor and sounds of C57BL6/J (C57) males for 7 days during the immediate postweaning period, exhibit a preference for bedding odors from C57 males over that males of their own strain when tested in estrus at 120 days of age (Albonetti and D’udine, 1986). This preference was independent of whether they were fostered by a SEC or C57 dam until weaning. The influence of odors learned in adulthood extends to the social communication of what foods are safe to eat. For example, rats alter their preferences for foods eaten by other rats with whom they socialize (for review, see Doty, 1986), a phenomenon that is accentuated in proteindeprived rats (Beck and Galef, 1989; Galef et al., 1991). In

*The importance of individual recognition for chordate evolution is obvious, as without such recognition natural selection could not operate. As noted by (Clark, 1982), “individual recognition and its effects on behavior may be the single major basis for structuring mammalian and avian social relations. Its importance is easily appreciated in dominance relations, mother-offspring recognition, kin-directed behavior, and mate recognition, as familiar examples.” Individuals from every single terrestrial mammalian that has ever been tested to date, including primates and humans, are able to recognize conspecific individuals by odor (Brown and MacDonald, 1985; Carr et al., 1976; Doty, 1986; Goyens et al., 1975).

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one study it was found that rats that encounter conspecifics that have eaten banana-flavored food pellets are more likely to enter a T-maze arm known to lead to such pellets (Galef et al., 1997). In another study, it was shown that a rat will exhibit an enhanced preference for a cinnamon- or cocoa-flavored food recently eaten by a healthy rat placed in their cage for a brief period, a preference that does not generalize to similarly scented nest materials or nest boxes (Galef et al., 1994). Moreover, rats made sick after eating a series of novel foods in succession are less likely to exhibit a conditioned aversion to those foods whose odors were previously experienced on the breath of healthy conspecifics to which they were briefly exposed (Galef, 1986). This is not simply a function of greater familiarity with the novel food odor. Rats that have learned an aversion to a flavored fluid and are allowed to briefly interact with healthy rats that have drunk that fluid without adverse consequence increase their intake of the averted fluid relative to controls that have had no such social interaction (Galef et al., 1997). Even after the meaning or health consequence of an odor becomes manifest to a rodent, however, its behavior does not need to be reflexive or invariant towards that specific stimulus. For example, a male mouse typically responds to the scent marks of another male by depositing urine on or around such marks. However, if this mouse ends up on the losing end of a fight with the other mouse, it will become subordinate to it and will cease such scent marking even when it is housed in close proximity to the marked area, its circulating testosterone is maintained at a high level by a silastic implant, and the other male is removed from the situation (Maruniak et al., 1977). Hence, in this case the mouse’s scent marking is not an invariant response to a pheromone or even to endogenous levels of testosterone (which are usually highly correlated with scent marking behavior and glandular secretions), as learning has intervened (see Sec. VII. A). Social and contextual factors also influence responses to purported pheromones. For example, dodecyl propionate, a putative maternal pheromone isolated from rat preputial glands, is said to attract adult rats and to play a key role in regulating postpartum maternal licking of the anogenital area of pups, a behavior critical for initiation of defecation (Brouette-Lahlou et al., 1992). When placed on rice, however, dodecyl propionate deters licking and ingestive responses (Arnould et al., 1994), again pointing out that context can establish the nature of responses to odorants.* Social factors also influence female mouse odor prefer-

*The human analogy may be the presence of rose oil on breakfast cereal in the morning.

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ences. For example, female mice from social groups with neighboring social groups show a stronger relative preference for the scent marks of dominant males from their own groups than do females from groups with no neighbors (Heise and Hurst, 1994). In summary, it is obvious that many behaviors said by some to be mediated by pheromones are likely learned, and that distinctions between inherent and learned responses are very difficult to make, particularly since postpartum responses can be tempered by prior intrauterine learning. While it is a truism that learning is a distinguishing feature of many organisms, it is accentuated in mammals. Chemically mediated behaviors or responses would seem not to be more primitive or less influenced by learning than behaviors or responses mediated by nonchemical stimuli. Is their any advantage in terming such chemically influenced social behaviors as being mediated by pheromones? VI. COGNITIVE PROCESSES: MICE, RATS, AND OTHER MAMMALS ARE NOT INSECTS As pointed out by Bronson, mammals have much more complex nervous systems than insects, and a number of their physiological responses to external stimuli— including overt behaviors and internal hormonal changes— often are tempered, guided, and in some cases determined by such intervening factors as stress or memory of past experiences.* As noted in Sec. V, most mammals have early developmental periods during which odorants significantly impact upon later social behavior and endocrine responses, possibly in a manner analogous to visual and auditory imprinting in birds. Moreover, in many forms ideation divorced to some degree from concurrent external stimuli can likely alter hormone levels and overt behaviors. The anatomical associations of the olfactory system provide a rich substrate for the cognitive mediation of complex odorrelated phenomena. As noted by Slotnick (2002): Recent discoveries have revealed that the olfactory system is less simple and less primitive than is generally assumed: olfactory impulses have fairly direct inputs to brain regions implicated in complex functions, including limbic structures and the prefrontal cortex . . . . The first link between olfaction and cognition was the finding that cells in the olfactory cortex project to the segment of the thalamic

*The differentiation between mammals and invertebrates is one of degree, not of kind. As alluded to earlier, many invertebrates learn and remember and exhibit behaviors that are influenced by early experiences with odors and foodstuffs.

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mediodorsal nucleus that connects to the orbital frontal cortex. Subsequent reports confirmed the existence of an ‘olfactory thalamocortical circuit,’ and delineated olfactory connections to the amygdala, entorhinal cortex and hypothalamus. Thus, it would seem that an inherent problem with the traditionally conceived pheromone concept is that, by implication and definition, it obfuscates or even excludes from consideration the possibility that cognitive processes can be involved in the mediation of behavioral and endocrine responses. This is in spite of the fact that few persons who own pets or have dealt with nonhuman mammals deny that they appear to have ideation or thoughts, such as occur during episodes of dreaming (Rasmussen et al., 1993). While there is evidence that secretion of lutenizing hormone and testosterone can occur in rats in anticipation of sexual activity (Graham and Desjardins, 1980), cognitive/endocrine processes are more easily demonstrated in humans. For example, testosterone titer increases in winners and decreases in losers of tennis, wrestling, debates, chess matches, and various games, a number of which are largely intellectual enterprises (Booth et al., 1989; Cavaggioni and Mucignat-Caretta, 2000; Elias, 1981; Gonzalez-Bono et al., 1999; Mazur et al., 1992; McCaul et al., 1992; Rejeski et al., 1989; Suay et al., 1999). Erotic dreams, expectation of sexual encounters, and vicarious identification of male sports fans with a winning team can increase testosterone levels (Anonymous, 1970; Carani et al., 1990; Hellhammer et al., 1985). Identification with a losing team, on the other hand, can decrease testosterone levels (Bernhardt et al., 1998). Chronic stress and mental concerns or conflicts are known to alter a number of human and animal endocrine functions (Beaumont, 1982; Fenster et al., 1999), and it is well established that stress, induced by a number of different procedures, activates dopaminergic, noradrenergic, GABAergic, and endorphinergic systems (D’Amato and Cabib, 1987, 1990; Nakagawa et al., 1981; Yoneda et al., 1983). Hence, in some cases, the influences of odors on hormones may well be mediated via the elicitation of a mental image or memory, and the degree of effect may depend upon the prior experience or lack of experience of the organism with the involved stimulus. Obviously, more research is needed on this very fascinating topic. VII. CASE STUDIES OF PURPORTED MAMMALIAN PHEROMONES Literally thousands of studies attribute the chemically mediated behaviors or endocrine events they describe to pheromones, yet few have attempted to chemically identify

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the agents involved or have even sought to determine whether the stimulus effects are species specific. Importantly, in perhaps most of the cases where such identification has been made, the biological activity of the isolated stimulus does not faithfully mimic that noted for the parent agents. The task of identifying sets of chemicals likely inducing the activity is theoretically daunting, as in many cases there are hundreds or even thousands of potential combinations of key components to be assessed— components that themselves are beholden to specific dietary and bacterial factors. One is reminded of visual research with pigeons, where complex visual stimuli used in discrimination tasks are fractionated or dissociated in an effort to find key components that maintain the discrimination. While elements are found that seem to serve this function, they are often idiosyncratic, suggesting that there need be no universal “essence” to the complex visual stimuli that maintain the discriminative behavior (Herrnstein, 1984; Reynolds, 1961). In this section I present five examples of behaviors said to be caused by “releasing” pheromones and two examples of endocrine responses attributed to “priming” pheromones. In these cases it would seem that the pheromone concept is found wanting in explaining their quintessence. Thus, many putative “releasing” or “signaling” pheromones seem to be dependent, largely if not entirely, upon learning during at least one stage of development. Most “primer” pheromones, for which the pheromone model seems at first glance to be a better fit, seem to be much more complex than appears on the surface and often fail to meet key criteria inherent in most definitions of pheromones (Doty, 2003). A.

Releasing Pheromones

1.

The Maternal Pheromone of the Rat

Infant rats of the Wistar and Sprague-Dawley strains have been reported, in a series of innovative studies, to be attracted to a maternal odor, labeled the “maternal pheromone,” during the second through the fourth week of age (Holinka and Carlson, 1976; Leidahl and Moltz, 1975; Leon and Moltz, 1971, 1972; Leon et al., 1972; Nyakas and Endröczi, 1970). A similar phenomenon has been reported for housemice (Breen and Leshner, 1977). The main source of the stimulus appears to be the cecotrophe portion of the maternal anal excreta. Like many sources of odor from biological secretions, the attractive agent has been shown to be dependent upon diet and cecal bacterial populations, as well as the production of bile and prolactin (Leon, 1974, 1975, 1978; Moltz and Leidahl, 1977). Thus, antibiotics that eliminate the bacterial flora eliminate the

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attractiveness of the excretia. Pups raised with mothers on a particular diet are attracted to the odor of mothers eating that specific diet (Leon, 1975). Although responses of rat pups to the “maternal pheromone” have been observed in several laboratories, the salience of the effect is often difficult to demonstrate or in some cases is likely even nonexistent, depending upon the rat strain evaluated, leading one to question its generality (Clegg and Williams, 1983; Clegg et al., 1983; Galef, 1981; Kendrick, 1975). For example, Galef (1981) reports that, in his laboratory, the attractiveness of a dam’s feces was relatively weak and that the attraction differed between animals fed two slightly different formulations of Purina Laboratory Chow No. 5001. According to the manufacturer, the two formulae differed subtly in only a few constituents. Whether such attraction to cecotrophe should be viewed as “pheromonal” seems questionable, as even the scientists who first described this phenomenon readily acknowledge that the responses are learned (Leon, 1975) and that the effectiveness of the material in attracting pups depends upon subtle dietary factors (Coopersmith et al., 1984; Leon, 1975). Coopersmith et al. (1984) state that “since there is no single maternal odor, the pups must become attracted to the odor that they will approach through postnatal experience.” Such learning is likely facilitated by tactile stimulation, such as the licking that a mother rat gives to her pups (Pedersen et al., 1982). In a well-controlled series of studies, Clegg et al. (1983) were unable to demonstrate a robust “maternal pheromone” effect in several strains of rats. These investigators approached their work under the assumption that a pheromone is an entity unique from that of simply an odor. In the first of eight experiments, the age of the pups (18 days vs. 24 days), duration of maternal deprivation (3 hours vs. 18 hours), and type of test stimuli (e.g., own mother vs. a virgin female; own mother vs. an adult male) were assessed. No evidence of a statistically meaningful preference was noted for the pup’s own lactating mother over the other stimulus animals. Unlike earlier studies, these authors eventually employed anesthetized rats as stimulus objects for the following reason: This procedure [i.e., use of unanesthetized female stimulus animals] was initially followed in the present study. Eighteen PVG/C hooded rats aged 18 days were given the choice of their own mother as opposed to a virgin female of the same age and strain. Pups underwent 3 hr of pretest maternal deprivation. All of the pups entered the goal compartment containing their own mother. But it was apparent that factors other than a pheromonal agent had helped to bring this about. As soon as a

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pup was placed in the apparatus the mother became extremely agitated, possibly as a result of ultrasonic calling by the pup (e.g., Allin and Banks, 1972; Smith, 1975) and in moving about emitted auditory cues. This was in sharp contrast to the non-lactating females, which tended to settle down in the goalbox and go to sleep. Any maternal attraction mediated by olfactory cues was thus confounded by auditory cues and maternal retrieving. Mobile live stimuli can therefore not properly be used to assess maternal pheromone phenomena. In the second study of the series, Clegg and Williams tested excrement collected from lactating vs. virgin females. The amount of the fecal material was equated across groups (3 g), since lactating females typically produce more excrement than nonlactating females, thereby potentially producing a confounding factor of stimulus quantity.* No evidence for a meaningful preference for the excrement of the lactating females was observed in either the Wistar or Sprague-Dawley rats, although this was not the case for the PVG/C strain rats. Of 151 PVG/C rats, 79 chose the goalbox containing the maternal excrement, 47 chose the side containing the virgin female excrement, and 25 remained in the arena, not moving into either compartment. While the number of PVG/C animals choosing the side of the maternal excrement was significantly higher than the number who chose the side of the virgin female excrement, the large number of pups who chose the virginal excrement, in combination with those who did not make any choice at all, led these authors to the conclusion that the effect, at best, is weak. The remaining studies examined such factors as air flow rates in the testing apparatus, maternal deprivation times, olfactory function in the pups, different types of laboratory food, and the effects of presenting cecal contents obtained from sacificed dams in an effort to observe a robust phenomena. The obtained data suggested that (1) pups learn to identify olfactory cues associated with the diet and (2) cecal contents produced an approach behavior relative to an empty goal box. In relation to the latter observation, the authors write, “so once more there is a clear suggestion of olfactory cues influencing behavior, but again without the control over that behavior that would be expected on the maternal pheromone hypothesis.” Clegg and Williams (1983) reiterate the importance of Galef’s observations and note the following about pheromones in their discussion: *The authors make the assumption that gross stimulus quantity is the involved factor, even though stimulus quality may not be correlated with quantity.

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The implications of Galef’s discovery are indeed far reaching, and it highlights the problem of deciding what does or does not constitute a pheromone. The pheromone concept is employed to go beyond the notion that animals use olfactory cues. It carries the implication that the stimulus controls behavior by eliciting a stereotyped and reliable response within any particular species (Karlson and Lüscher, 1959). If certain strains within a species fail to demonstrate the appropriate response, or if members of the species do so under highly specific conditions, then can the effect be called “pheromonal”? 2. The Aggression-Eliciting Pheromone of the Male Mouse It has been suggested that male mice produce a pheromone that elicits aggressive behavior from other males (Heyser et al., 1992; Mugford and Nowell, 1970,1971; Mugford et al., 1972). The laboratory demonstration of the aggression-eliciting pheromone has typically employed paired encounters where males— often trained fighters—engage in agonistic behavior towards a castrate stimulus male or female to which urine or preputial secretions from the target male animal are applied (e.g., Lee and Brake, 1971). The aggressioneliciting property of the stimulus is testosterone-dependent, since (1) male urine or preputial sebum from castrated males leads to fewer or briefer attacks when placed on the stimulus animals than those elicited by stimuli from non-castrate males (Heyser et al., 1992; Mugford et al., 1971), (2) a dose-response relation exists between the amount of testosterone injected into urine donor animals and the attack behavior elicited (Mugford et al., 1972), and (3) the efficacy of male urine is decreased by the administration of the anti-androgen, cyproterone acetate (Jones and Nowell, 1974; Nowell and Wouters, 1973). In general, female mice receive no or few attacks in this situation, and deodorizing males reduces aggressive responses directed towards them (Lee et al., 1971). Interestingly, female mice administered testosterone early in life also elicit agonistic responses, implying that this hormone somehow alters the female physiology such that the purported aggression-eliciting agent is produced (Lee and Griffo, 1973). Olfaction is implicated in mediating the response, although other factors seem to be involved as well. For example, Rowe and Edwards (1971) reported that male mice whose olfactory bulbs had been removed failed to attack castrates in a paired encounter, but when food deprived for 48 hours and paired with such mice in the presence of a food pellet, 70% of the bulbectomized mice fought for

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control of the food.* Nonetheless, bulbectomized males are much less prone to initiate agonistic encounters than normal mice (Ropartz, 1968). Strain differences in such behavior have also been noted (Kessler et al., 1975).† The fact that the putative aggression-eliciting substance is found in both the preputial glands and in the urine, as well as in the urine from female mice injected with testosterone early in life, suggests either that the same “pheromone” is present in multiple biological fluids or that there are several such pheromones. Assuming the former for the purpose of exposition, if the chemical signal is truly a “pheromone,” one would expect that responses to it would be relatively invariant, perhaps being expressed in proportion to the amount of substance being excreted by these various sources. However, a number of observations suggest that this is not the case. First, exposing a male mouse for one hour daily for 10 days to soiled woodshavings from the cage of a strange male mouse eliminates, for the most part, agonistic behaviors directed towards that male in subsequent encounters. Such exposure also mitigates, to a much lesser extent, such behaviors directed towards other mature male conspecifics (implying that familiarity with any conspecific male odor in this context decreases agonistic tendencies) (Kimelman and Lubow, 1974).† If such behaviors were being mediated by a pheromone, then one would have to postulate that each mouse has a different aggression-eliciting pheromone and that familiarity to one can, to some degree, influence the mouse’s behavior towards another. A seemingly more parsimonious explanation would be that the behavior is dependent upon the relative degree of olfactory strangeness or novelty, a concept with empirical support (Alberts and Galef, 1973; Mackintosh and Grant, 1966). Second, mice reared alone or isolated from other mice, when placed in subsequent encounters,

*One must be cautious in interpreting the effects of olfactory bulbectomy, since this operation has influences beyond simply altering the perception of environmental chemicals. Thus, it also effects vomeronasal organ input and, depending upon the species, can enhance, depress, or have no influence on tonic gonadotropin secretion (see Pieper and Newman, 1999, for a review). †Jemiolo et al. (1991) have chemically assessed the constituents of preputal gland secretion, and have found that two sesquiterpenic compounds, E, E,--farnesene and E--farnesene, elicit increased investigatory behavior relative to water or bladder urine in sexually naïve and experienced mice. †Conceivably the strangeness of the odor contributes to the elicitation of aggressive behaviors. Dixon (1982) found that increased aggression directed to mice injected with diazepam likely reflected changes in the odor of their urine.

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are more likely to initiate social/investigatory behaviors and are more prone to attack a conspecific male than mice reared in groups (Levine et al., 1965).* Both the duration and the nature or timing of the exposure can determine the magnitude of this phenomenon and in some cases may even result in its reversal (i.e., less aggression from isolated mice) (Cairns and Nakelski, 1971; King, 1957; King and Gurney, 1954). For example, mice who are housed alone for 24 days in the presence of the bedding of another mouse and then tested in agonistic encounters with either that mouse or a mouse whose odor is unfamiliar to them, exhibit more aggression towards the mouse whose odor is familiar to them than to the mouse whose odor is novel, demonstrating the lability and complexity of such behavior (Corridi et al., 1993; Telle, 1966). Third, male mice cohabitating with females are more aggressive towards strange males than male mice who are not living with females. Male mice who in the past had cohabited with a female for a short period of time exhibit intermediate levels of aggressive behavior (Goyens and Noirot, 1975). Presumably this reflects the testosterone titer of the aggressor, although psychological factors may also be involved. Fourth, commonly a strange mouse, or a mouse introduced into a group after having been removed from the group, is first investigated by the group members, and in most cases the attack behavior of the group depends upon the behavior of the intruder (Cairns and Nakelski, 1970). Often the degree of investigation directed towards the stranger depends upon group size. Thus, residents of small groups of rats (up to ~20 members) attack strangers and drive them away, whereas residents of large groups (80–100 members) do not, implying that when a local group of rats becomes large enough for individual members to be anonymous, it may no longer be closed (Carr et al., 1976; Telle, 1966). Fifth, in naturalistic settings (e.g., demes), aggression is minimal among group members, even though at least one male member of the group —the dominant male—undoubtedly has high testosterone titers and, hence, would be expected to produce the aggression-eliciting pheromone. This lack of aggression presumably depends on, in part, the development of a social dominance heirarchy and the scent-marking behaviors of the dominant individual(s) that familiarize the other deme members with their odor. Sixth, the existence of aggression-eliciting pheromones makes little sense from an evolutionary perspective. Brain et al. (1987) notes the following:

*Considerable species differences exist regarding the effects of isolation on agonistic behaviors. In guinea pigs, for example, longterm isolation results in more docility, not less (Sachser, 1986).

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Evans (1979) claimed that there is no logical way of arguing for the existence of a “pheromone” whose function is to release aggressive behaviour from other mice. This, to all intents and purposes, would involve a signal meaning “attack me please” and would result in a loss of fitness on the part of the signalling individual. The results of the present studies support this position and suggest that the odours really act as personal labels signalling “I am a threat to you”. For example, the odour of preputial sebum of dihydrotesterone-treated castrates probably identifies the donor as “an unfamiliar, mature, sexually active, territorial male” to non-habituated conspecifics who respond accordingly. In this way there is no loss of fitness. Novotny et al. (1985) have isolated two testosteronedependent volatiles, which they view as pheromones, from the urine of male mice [2-(sec-butyl)thiazoline and 2, 3dehydro-exo-brevicomin] that increase aggressive responses among males in standard behavioral bioassays. These agents do not work when added to water, but are synergistic when added to castrated male urine. Thus, outside of the mileau of a urine background, they seem ineffective. Even if one accepts them as key components of a secretion that signifies strangeness or elicits agonistic activity in conspecific male mice, can they be viewed as pheromones in light of the aforementioned issues? As noted by Alberts et al. (1973) in rats, familiarity is a key component in the initiation of aggressive responses: “the response of wild Norway rats to conspecifics is determined by a multitude of stimuli perceived via several sensory modalities. Response to a conspecific as such (amicable and sexual behavior) can occur in the absence of olfactory inputs. On the other hand, the initiation of aggression would appear to be dependent on olfactory stimuli arising from an unfamiliar individual. Both the duration and direction of aggressive behavior is further modified by the behavior [e.g., movement] of target animals.” A human analogy may be of value in describing the complexities of mammalian agonistic encounters. If the reader was to discover a large male stranger inside his or her living room in the morning upon awakening (assuming an all-night party had not been going on), it is likely that aggressive responses, whether verbal or physical, would be directed towards the intruder. However, the nature of such responses would likely depend upon the stranger’s size, age, and apparent intentions. If, for example, the intruder was holding a shotgun, the homeowner’s agonistic response would likely be more calculated. On the other hand, if the stranger was an old man in tattered rags and the weather outside was freezing, sympathy or the desire to help may overshadow any agonistic behavior on the part of

Mammalian Pheromones

the resident. In this situation, one does not postulate the presence of an aggression-promoting visuomone or audiomone, only a situation that requires a response or the mitigation of various response alternatives once an understanding of the situation has been established. 3.

Copulin—The Rhesus Monkey Vaginal Pheromone

In a series of highly publicized studies, Richard Michael and coworkers reported that vaginal secretions from female rhesus monkeys contain pheromonal substances that elicit copulatory behavior from males. Specifically, they found that males would perform a bar-press behavior to gain access to and to copulate with estrogen-treated females, presumably on the basis of olfactory cues (Michael and Keverne, 1968, 1970b). In other studies, often using the same subjects who had been trained in the aforementioned bar press paradigm, they applied ether or water extracts of vaginal secretions from estrogen-treated ovariectomized monkeys to the sexual skin of untreated ovariectomized females. During subsequent tests, application of the extracts resulted in an “immediate and marked stimulation of the sexual activity” of the male subjects (Keverne et al., 1971). These authors concluded “male sex-attractant pheromones, with powerful behavioral effects, are present in ether extracts of estrogen-stimulated vaginal secretions.” Chemical analysis of the estrous vaginal secretions resulted in the isolation of a series of volatile short-chain aliphatic acids—acetic, proprionic, isobutyric, n-butryic, and isovaleric— that these authors claimed to be the active pheromonal substance (Bonsall and Michael, 1971; Michael and Keverne, 1970 Michael et al., 1971). A mixture of these agents in specific proportions, which they termed Copulin, was prepared, found to be active, and patented in several countries for employment in human perfumes, suggesting that Michael et al. assumed the pheromone was not species specific, thereby violating one of the most common criteria for an agent being a pheromone. The involvement of aliphatic acids in primate sexual attraction is counterintuitive, however, since vaginal aliphatic acids primarily appear during the luteal phase of the cycle, rather than during the time of optimal fertility (Goldfoot et al., 1976; Michael et al., 1972). In humans, aliphatic acids are largely dependent upon bacterial fermentation of glycogen, which is highest during the luteal phase (Gregoire et al., 1973). In any event, Copulin’s effectiveness in altering human sexual behavior was subsequently found to be nil (Cowley and Brooksbank, 1991; Morris and Udry, 1978), which, ironically, would be expected if Copulin was truly a species-specific pheromone. Aside from the issue of species specificity, the validity or generalizability of these findings even among rhesus

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monkeys was called into question by Goldfoot et al. (1976). These authors carefully examined Michael et al.’s original data, noting that the male’s responsiveness varied considerably from male to male and in some cases tended to depend upon particular female, irrespective of an odor cue. They pointed out that in Michael et al.’s seminal work (Michael and Keverne, 1970b), a baseline consisting of as many as 60 pretests over 80 days was based on only two male subjects, and suggested that the application of the odorants after extinction of mounting could be explained on the basis of disinhibition, resulting in a resumption of mounting. Particularly damning to the vaginal pheromone concept was evidence, some from Michael et al.’s own studies, that removal of olfactory bulbs has no influence on male rhesus monkey mating behavior, implying that pheromones—at least ones whose effects are mediated via the olfactory pathways—are neither necessary nor sufficient for such behavior (e.g., Goldfoot et al., 1978; Michael and Keverne, 1968). Further problems for Michael et al.’s claims were Goldfoot et al.’s extensive behavioral and analytical studies (Goldfoot et al., 1976). In contrast to Michael et al.’s work, relatively large numbers of subjects were employed. For example, in the behavioral studies, a total of 19 adult male and 27 adult spayed female rhesus monkeys were used. Unlike in Michael et al.’s work, the donor females had not been recently paired with males in most of the test situations, and, thus, their vaginal secretions were not contaminated by male ejaculate. Despite careful quantitative assessment of a range of sexual behaviors (approach, genital inspection, contact, mounts, intromissions, ejaculations) under a variety of estrogen regimens and behavioral test conditions, no statistically significant differences between vaginal lavage and control treatments could be found, although a slight tendency for ejaculate-contaminated secretions to increase some behaviors was noted. Importantly, the amount and relative proportions of aliphatic acids found in the vaginal secretions differed markedly from those found by Michael and associates. For example, in contrast to Copulin, Goldfoot et al. did not detect any isovaleric acid in the secretions even after 29 days of estrogen treatment. Goldfoot et al. concluded that “comparison of our results to those from other laboratories [i.e., Michael et al.’s] suggests that the mechanism involved in positive effects may depend upon associative learning or upon extinction or disinhibition of sexual interest.” 4.

Dimethyl Disulfide—The Hamster Vaginal Attraction Pheromone

It has been reported that both sexually experienced and sexually inexperienced male hamsters are attracted to

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conspecific vaginal secretions of females. Singer et al. (1976), employing gas chromatography–mass spectrometry, identified dimethyl disulfide (DMDS) from volatile fractions of hamster vaginal secretions as the pheromone involved. In the bioassay, sexually experienced hamsters were employed, and the latency, duration, and number of animals “approaching, sniffing and digging” near a section of the cage under which either DMDS, the volatile fraction, or the vaginal secretion were located was compared. The number of animals exhibiting “positive responses” towards the whole vaginal secretion or the volatile fraction was 12 of 12. The number exhibiting such responses to DMDS varied with the DMDS concentrations chosen by the authors. At 128 and 22 ng, 8 of the 12 males responded; at 56 and 2 ng of material, 5 of the 12 males responded. In other words, at the best DMDS concentration concocted, only two thirds of the males responded to the “pheromone,” unlike the parent secretion or the volatile fraction derived from the secretion, where 100% of the animals responded. At the other concentrations, less than half of the animals responded to the agent. Of the responding subjects, the mean (  SEM) duration of “approaching, sniffing and digging” near the region of the cage under which the stimulus was located was 58(1) seconds for the whole vaginal secretion, 37(9) seconds for the volatile fraction, and 65(16), 23(10), 51(16), and 26(11) seconds for dimethyl disulfide at 128, 56, 22, and 2 ng concentrations, respectively. Responses to control stimuli (water) were nominal (i.e., 10 seconds or less, on average). It is interesting that the duration of behavior directed towards the dimethyl disulfide in the responding animals was greater, in some cases, than that directed towards the original secretion. Petrulis and Johnston (1995) reasoned that if dimethyl disulfide is truly a sex attractant pheromone, then (1) male hamsters should spend more time than female hamsters investigating it and (2) the attraction to the substance by males should be testosterone-related. In the first of two experiments, these authors found that males investigated female vaginal secretions more than did females, but this was not the case with dimethyl disulfide, where both sexes equally investigated the agent. In the second experiment, castrated males given testosterone investigated the vaginal secretions more than castrated males not given testosterone. However, neither castration nor testosterone repletion influenced attraction towards dimethyl disulfide or a control odor, leading these authors to conclude that “DMDS does not elicit sex differences in attraction and that in males the attraction to DMDS is not dependent on gonadal hormones. These results suggest that DMDS is not a sex attractant by itself nor is it a major component of an attractant mixture.”

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5.

The Erection-Eliciting Pheromone of the Rat

Some mature male rats exhibit penile erections and sexual behaviors in the presence of inaccessible estrous females (Sachs et al., 1994). This phenomenon appears not to require adult sexual experience on the part of the male and can be induced by airborne volatiles from estrous females, even ones who are anesthetized (Sachs, 1997). Such volatiles, however, appear to be effervescent, as bedding soiled by estrous females does not produce this effect (Sachs et al., 1994). This phenomenon is not dependent upon visual or auditory cues (Kondo et al., 1999; Sachs, 1997) and is eliminated by lesions of the olfactory, but not vomeronasal, nerve (Kondo et al., 1999). This is in spite of the fact that it is eliminated by lesions within the medial amygdala, a structure that serves as a major relay of the accessory olfactory system (Kondo et al., 1997). Sachs (1997) notes, “receptive female rats apparently broadcast a volatile pheromone that promotes erection. Pheromones are well known to attract mates and to act in concert with other stimuli to promote mating. However, this is the first mammalian evidence for a volatile pheromone acting alone to evoke a sexual fixed-action pattern and, in that sense, acting as an airborne aphrodisiac.” Since no tests of species specificity of this phenomenon have been made and no attempts have been made to identify the chemicals involved, its status as a pheromone—at least according to the primary criteria inherent to most definitions of pheromones—is unclear. It would be of interest, for example, to determine whether lactating female rats that exude a number of hormone-influenced odors also induce penile erection. It should be noted that this effect does not occur in a large majority of rats. Thus, in one study only 55% (11/20) of sexually inexperienced males displayed this effect; in another study within the same series, the same proportion (11/20) exhibited these erections, even though 5 of the 11 were sexually experienced (Sachs, 1997). One of the 11 rats was upwind from the odor source, suggesting the likelihood of a spontaneous erection. B.

Priming Pheromones

Since the 1960s, the effects of urine or body odors on conspecific reproductive endocrine responses have been attributed to priming pheromones. Most such agents have been described in mice and a few other rodent forms. Whether, or to what degree, such phenomena exist in a wide range of other species or whether, in fact, they even exist outside of the laboratory is a matter of controversy (see Bronson, 1979; Bronson and Coquelin, 1979; Labov, 1981). According to the classical mammalian pheromone paradigm, a pheromone exists in the urine of group-housed

Mammalian Pheromones

female mice isolated from conspecific males that produces, in other females, a lengthening of the diestrous component of their estrous cycles or, in some cases, pseudopregnancy. Another pheromone is purportedly present in the urine of noncastrate mature male mice, which, when presented to isolated females or females who have become acyclic as a result of being housed with other females in the absence of a male, intiates estrous cycling and induces shorter and more regular cycles. This or a similar pheromone is also said to accelerate puberty in young females, and a pheromone in the urine of females is reported to delay puberty in young females. The blockage of ovum implantation in a recently inseminated female by a strange, nonstud, male or his odor is also believed to depend upon a pheromone, although in this case the odor of the stud male is said to be remembered by the female in order for the strange male pheromone to be effective in inducing the blockage. In this section two so-called priming pheromones are examined—one associated with the blockage of pregnancy and the other with the acceleration of puberty. 1.

The Strange Male Pregnancy Blocking Pheromone (Bruce Effect)

In 1959, the endocrinologist Hilda Bruce reported that only 29% of a group of recently inseminated albino mice became pregnant when paired immediately after insemination with a nonstud male mouse of the wild-type strain, compared to 100% who were similarly paired with the stud male (Bruce, 1959). Housing with a nonstud albino male mouse decreased the pregnancy rate to 72%. The latter decrement was present regardless of whether the male albino mouse was intact or castrated (Bruce, 1960a), a finding now believed to be aberrant, given numerous subsequent reports—including ones from Bruce’s own laboratory— that castrates are ineffectual in blocking pregnancies (e.g., Bruce, 1965; Spironello-Vella and deCatanzaro, 2001). A number of studies have verified the “Bruce effect,” finding in some cases that same-strain (“strange”) male mice block pregnancies in 25–30% of the females, whereas different-strain (“alien”) males do so in up to 80% of the females (Parkes and Bruce, 1962). This general phenomenon has been described in numerous Mus strains (e.g., C3H, BALB/c, CBA), as well as in Peromyscus maniculatus, Microtus agrestis, Microtus ochrogaster, and Microtus pennsylvanicus (Bronson and Eleftheriou, 1963; Bronson et al., 1969; Bruce, 1959, 1960a; Clulow and Clarke, 1968; Clulow and Langford, 1964; Clulow et al., 1968; Stehn and Richmond, 1975; Terman, 1969; Watson et al., 1983). It apparently does not occur in some highly inbred Mus strains (Bruce, 1968; Kakihana et al., 1974) or in gerbils (Meriones unguiculatus)

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(Norris and Adams, 1979). The female appears to be most vulnerable to pregnancy block within 48 hours of coitus, with exposure during the first 12 hours being sufficient in the majority of cases (Bruce, 1961). The effectiveness of the pregnancy block is reportedly not augmented by increasing the number of males to which a female is exposed (Bronson et al., 1963; Bruce, 1963; Chipman and Fox, 1966a), although Chipman and Fox (1966b) reported six strange males blocked a greater percentage of pregnancies than a single male (85% vs. 42%), conceivably reflecting the intensity or complexity of the stimulus. The Bruce effect has been attributed to a pheromone, since (1) it occurs even when the strange male is separated from the female by a wire partition (Bruce, 1959), (2) it is eliminated by olfactory bulbectomy or damage to the female’s accessory olfactory system (Bellringer et al., 1980; Bruce and Parrott, 1960; Lloyd-Thomas and Keverne, 1982; Rajendren and Dominic, 1985; Reynolds and Keverne, 1979), and (3) urine or previously soiled bedding from a strange male is as effective, in many strains, as the strange or alien male himself in producing the effect (Dominic, 1964, 1965, 1966b). Memory of the stud male is believed to be important, since the effect is eliminated by infusion of phentoamine or other memoryblocking agents into the female’s accessory olfactory bulb after initial mating (Kaba and Keverne, 1988; Kaba et al., 1989). Nonetheless, at least limited physical contact with the male seems to be needed in some strains or test situations to block pregnancy (deCatanzaro et al., 1995b). While in one study exposure of recently mated Parks albino females to urine-soiled bedding from strange males in boxes was ineffectual (Bruce, 1960b), the pregnancy block could be produced by housing the mice in tall, poorly ventilated, glass jars containing bedding to which strange male odor urine had been added. Another study using the same type of albino mice found that once-daily renewal of the soiled bedding is markedly inferior to twice-daily renewal, suggesting that “. . . the operative substances are evanescent, highly volatile or highly labile, probably both” (Parkes et al., 1962). Three 15-minute exposures over a 4day test period have been found sufficient to produce the phenomenon in outbred Swiss strain females exposed to wild-type males (Chipman et al., 1966). The physiological basis for the Bruce effect is not entirely understood. It has been generally assumed to depend upon the leuteotrophic function of prolactin from the adenohypophysis,* since it is (1) blocked by injecting

*Crowding can influence postimplantation intrauterine mortality as well in a number of mammals, including housemice and deermice (Helmreich, 1960).

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recently inseminated females with the primary leuteogenic agent in the mouse, prolactin, or with progesterone during the strange male odor exposure period (Bruce and Parkes, 1960; Dominic, 1966b; Rajendren and Dominic, 1987),† (2) blocked by the implantation of a functional ectopic pituitary graft known to produce prolactin (Bronson et al., 1969; Dominic, 1966a, 1966b, 1967), (3) absent in postpartum pregnant females whose prolactin production is induced by suckling (Bruce and Parkes, 1961), and (4) blocked by the injection of reserpine (Dominic, 1966b, 1966c), an agent that depletes stores of catecholamines and serotonin in the brain and suppresses the inhibitory hypothalamic center that controls release of prolactin (Dominic, 1966b). Further evidence of a role of prolactin comes from the observation that exposure of recently inseminated females to strange male urine when prolactin surges induces the block, but not at other times (Rosser et al., 1989). Bromocriptine, a dopamine agonist, was just as effective as the exposure to strange male odor, suggesting that dopaminergic reduction of prolactin may be the basis of the effect. It has also been suggested that LH release may be the critical or at least an initial determinant of the pregnancy blockage (Chapman et al., 1970). Estrogen may be involved, since (1) the presence of males appears to enhance the synthesis and release of follicle-stimulating hormone (FSH) in gonadectomized female mice (Bronson and Desjardins, 1969), (2) the administration of estrogen, including minute amounts applied to the nose, eliminates successful implantation in nonlactating female mice (Bloch, 1971; deCatanzaro et al., 2001), and (3) antibodies to 17-estradiol prevent the Bruce effect in recently inseminated females (deCatanzaro et al., 1995a). Exogenous administration of androstenedione and dehydroepiandrosterone, agents that can be converted to estrogens, is capable of blocking a recently-inseminated female’s pregnancy; the major stress-related adrenal hormone corticosterone, which is not convertible into estrogens, fails to do so (de Catanzaro et al., 1991). Epinephrine is ineffectual, even though it does disrupt female mating behavior (deCatanzaro and Graham, 1992). Whatever the hormonal factors involved, it seems quite conceivable that they reflect responses to stress. As noted in several recent reviews (e.g., deCatanzaro and MacNiven, 1992; Marchlewska-Koj, 1997), blockage of implantation is not uncommon in rodents and can be induced by a variety of stressors, suggesting that the Bruce effect is one of a number of stressor-induced pregnancy

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blocks. In mice, the pregnancy of recently inseminated females can be blocked by (1) human handling (an affect eliminated by the injection of progesterone) (Chipman et al., 1966b; Runner, 1959; Weir and DeFries, 1963), (2) enforced swimming for 3 minutes and/or exposure to loud tones and open areas (Weir et al., 1963), (3) exposure to male rats (with or without tactile contact) (deCatanzaro, 1988), (4) exposure to predators (deCatanzaro, 1988), (5) exposure to male or female rat urine (deCatanzaro, 1988), and (6) the induction of nutritional or restraint stress (Euker and Riegle, 1973; McClure, 1959), the latter of which is reduced by the administration of estrogen antibodies during the preimplantation and early implantation stages (deCatanzaro et al., 1994). McClure et al. (1987) found that by starving and feeding mice in a series of alternating 2-day periods, the appearance of litters could be completely prevented. Moreover, the effectiveness of the pregnancy block is related to the aggressiveness and sexual behavior of the strange mouse, with intromissions being particularly important (deCatanzaro and Storey, 1989; Storey and Snow, 1990). In the deermouse, Peromyscus maniculatus, the nature of the postinsemination environment influences implantation (Eleftheriou et al., 1962), such that postinsemination housing in different-sized cages decreases pregnancy success by 30–60%, depending upon the difference in size of the new environment relative to that of the old. In rats, exposure to noxious sounds 4–6 days after mating reduces the number of pregnancies, as does chronic restraint (Euker et al., 1973; Zondek and Tamari, 1967). The adrenal gland is likely involved in some cases, since the Bruce effect is not present in some inbred mouse strains that exhibit attenuated adrenal responses (Marsden and Bronson, 1965; Snyder and Taggart, 1967) and, in some strains, is not present or is mitigated in adrenalectomized females (Snyder et al., 1967; however, see Sahu and Dominic, 1981). Exogenous adrenocorticotropic hormone (ACTH) is capable of inhibiting luteinization in adrenalectomized mice, but is more effective in mice with adrenal glands (Christian et al., 1965). Interestingly, endogenous estrogen rises in response to acute stress during early pregnancy as well as during nonpregnant states (MacNiven et al., 1992b; Shors et al., 1999), and injections of ACTH can produce increases in estrogen levels in some species (Arai et al., 1972; Strott et al., 1975), adding further credence to the notion that estrogen may be a primary factor in stress-induced blockage of implantation.*

†It

is noteworthy that progesterone is as effective as prolactin in this regard if presented early enough (Bruce et al., 1960; Dominic, 1966b; Rajendren et al., 1987).

*Estrogen also rises in response to chronic stress (MacNiven et al., 1992a)

Mammalian Pheromones

Evidence that stress-reducing manipulations can protect against the Bruce effect include the observations that (1) the presence of the stud along with a strange male mitigates the strange male’s blocking ability to some degree (Parkes et al., 1961; Terman, 1969), conceivably decreasing the strangeness of the situation or counteracting the strange male odor, (2) replacement of a strange male with an original stud who was present during the period of the previous pregnancy prevents the pregnancy block, whereas replacement with a stud who had limited contact with the female does not (Bloch, 1974),† (3) familiarization of the female with a male before she is mated with another male mitigates the familiarized male’s ability to block the pregnancy, regardless of the strain of the female, the strain of the stud male, or the strain of the familiarized male (Furudate and Nakano, 1981; Parkes et al., 1961),‡ and (4) the presence of other females or their urine—even urine from spayed or androgenized females—during the critical postcoital period can effectively prevent the pregnancy block in some strains, with the prophylactic effect being directly related to the number of females present (at least up to the largest number tested, i.e., 6) (Bruce, 1963; Dominic, 1965). Whether this reflects the calming effect of such odors, familiarity with such odors, prior conditioning induced in the suckling setting, or some type of chemical cross-adaptation to the strange male odor is not clear. In an atypically reactive housemouse strain, in which the threshold for pregnancy blockage is low, repeated handling of the rat pups in infancy decreases their susceptibility to the Bruce effect in later life (Bruce et al., 1968), presumably reflecting mitigation of later stress responses (Denenberg et al., 1977; King, 1959; Levine and Broadhurst, 1963). It is noteworthy that hyperprolactinemia is associated with suppression of stressor-induced elevations in plasma corticosterone levels (Drago et al., 1986; Endroczi and Nyakas, 1974) and that reserpine reverses the density-dependent adrenal hypertrophy and reproductive decrements observed in male housemice (Christian, 1956). It is of particular interest that most, if not all, drugs and hormones that have been shown to block the Bruce effect, with the

†One

study reports that duration of exposure required to make a stud male familiar to the female is 3–4.5 hours (Rosser and Keverne, 1985). ‡Some data do not support the familiarization hypothesis. For example, Lott and Hopwood (1972) reported that if the stud is removed from the female within 3 hours of mating, pregnancy block from a strange male is less likely to occur than if he remains with the female for 24 hours or longer, suggesting that exposure to the stud “sensitized” the female to subsequent pregnancy block.

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exception of those targeted on odor memory formation of the stud or strange male (e.g., phentolamine, anisomycin), have anxiolytic or antidepressant properties, including amitriptyline, chloropromazine, haloperidol, pimozide, progesterone, prolactin, propranolol, and reserpine (Bloch and Wyss, 1973; Dominic, 1966b, 1966c; Rajendren and Dutta, 1988; Sahu and Dominic, 1980; Saletu et al., 1975; Torner et al., 2001). Antidepressants such as fluoxetine, amitriptyline, desipramine, and buspirone have been demonstrated to enhance habitutation to novel stimuli in olfactory bulbectomized rats—rats that exhibit many characteristics of stress. This raises the possibility that such agents may depress the degree of novelty of a strange male or his odors on implantation (Mar et al., 2000). Of course, such actions need not be independent of the influences of these agents on the hypothalamic-pitutarygonadal axis. If one accepts the proposition that the Bruce effect is induced by stress, then it would seem that testosterone, or some other testicular-influenced agent, is involved in the production of the stress-inducing odor, since (1) urine from male castrates is typically ineffectual (Bruce, 1965; Spironello-Vella et al., 2001), (2) males are capable of blocking pregnancy only after the age of puberty (Bruce, 1965), (3) urine from males housed alone is less effective than urine from males housed near females (whose testosterone titer would be expected to be comparatively higher) (deCatanzaro et al., 1999), (4) the antiandrogen cyproterone acetate mitigates the pregnancy blocking ability of the urine (Bloch et al., 1973), and (5) male mice who have achieved sexual satiety are less effective than those who have not in producing the pregnancy blockage (Spironello and deCatanzaro, 1999), presumably reflecting decreased testosterone titer (Batty, 1978). The accessory sexual glands (e.g., the vesicular and coagulating glands) or the preputial glands are unlikely important, since pregnancy blocking efficacy remains in mice lacking these organs (Hoppe, 1975; Zacharias et al., 2000). Administration of epiandrosterone, androstenedione, androsterone, or testosterone to SJL female mice results in blocked pregnancies; administration of progesterone or dehydroepiandrosterone does not (Hoppe, 1975). Alternatively, it is possible that testosterone simply produces a strong salient smell that, without familiarization or adaptation on the part of the female, (1) is clearly discernible to the female, (2) allows for accurate differentiation between different males, and (3) elicits enhanced sensorineural activity. Urine from noncastrate male rodents is much more intense, even to humans, than that from castrates or females. Whatever the role of testosterone, it seems clear that the female rapidly learns the odor of the stud male and by further exposure accommodates herself to or adapts to his

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odor over time, making it either weak or familiar, and relatively nonstressful.* Presumably the degree of strangeness of the strange male is a function of the degree of qualitative difference between its odor and that of the stud—a difference likely learned and somehow discerned by, or dependent upon, the accessory olfactory system (Brennan and Keverne, 1997). If castrate males have relatively little smell, then they or their odors would probably not seem particularly strange or stressful to the female. Unfortunately, no control studies have been performed that have attempted to equate artificial odors on intensity or quality with noncastrate male urine odors, or to differentially odorize the stud and strange males to see if artificial odors can produce the Bruce effect. Inherently, the Bruce effect would seem not to be well described as being due to a pheromone, since the pheromone would have to vary from male mouse to male mouse as long as the mouse differs from the stud male in its identifying chemical composition or odor. Hence, there could be as many pheromones as there are male mice that can be identified as individuals, at least as individuals who have testosterone-related odors. Moreover, this phenomenon has a major learning component associated with it and seems to be a subclass of a group of phenomena that rely upon the fragile nature of the implantation process. 2. The Puberty-Accelerating Pheromone of Male Mice Prepubertal female mice housed or otherwise exposed to noncastrate males or their urine attain puberty sooner than females not so exposed, as measured, for example, by the time of vaginal opening (Andervont, 1944; Castro, 1967; Cowley and Wise, 1972; Vandenbergh, 1967,1969). This acceleration of puberty, which has been attributed to a pheromone, is stronger when the male is present, conceivably reflecting the addition of contact stimulation (Bronson and Maruniak, 1975; Drickamer, 1974, 1975). Application of male urine directly to the oral-nasal grooves of postweanling female mice accelerates the time of onset of first estrus by 4–6 days relative to contols exposed to tap water. Exposure for 3 days between the ages of 21 and 29 days is sufficient to advance puberty. Exposure to male urine tends to shorten the interval between the day of vaginal opening and the day of first estrus, implying that the

*A more common explanation of this phenomenon is that the inseminated female requires time to learn the characteristics of the stud male (Brennan et al., 1990; Bronson, 1976). Circumstantial support for the hypothesis that it reflects habituation comes from rats, where continuous exposure of a female to the same male produces fewer estrus cycles than successive exposures to different males (Cooper et al. 1972).

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substance accelerates sexual maturation rather than simply serving as a trigger for puberty (Colby and Vandenbergh, 1974). Urine from males whose preputial glands are removed is as effective as urine from intact males. Although conceivably strain differences are present, it is noteworth that (1) urine from dominant males produces greater acceleration than urine from subordinant males, (2) the male urine must be present for at least 2–3 hours per day, or the male must be present for 1 hour per day, to produce the acceleration, (3) urine from the same male presented each day produces the same degree of acceleration as urine from different males presented each day, (4) urine from the father or a full brother exerts the same degree of acceleration as urine from unrelated males, and (5) excreted or bladder urine from adrenalectomized males is as effective as urine from an intact male (for review see Drikamer, 1986b). However, the presence of an adrenalectomized male does not produce the same degree of acceleration as the presence of an intact male, possibly because adrenalectomized males pursue young females less and attempt fewer mounts (Drickamer, 1983). To date, the active “pheromone(s)” have not been identified, although they are said to be absent in food-deprived males and in males maintained under short photoperiods when testosterone would be expected to be low. Analogous to the situation with the Bruce effect, however, pubertal acceleration is not uniquely determined by male urine, and it is well established that a number of stimuli and stressors can accelerate the puberty of female mice and other rodents.* Thus, similar acceleration can be obtained by exposing prepubertal female mice to urine from (1) pregnant or lacting female mice (Drickamer, 1984) (2) estrous female mice (Drikamer, 1986a), and (3) male rats (Colby and Vandenbergh, 1974). The latter observation implies that if specific urinary agents are involved in this phenomenon, they may not be speciesspecific. Evidence that stressful situations can advance puberty in a number of mammals comes from several quarters. First, female rat pups that are handled and placed individually in separate containers for 3 minutes each day

*Not all stressful stimuli need to activate adrenal responses. For example, social isolation, long considered to be a “stressor,” can influence a number of behaviors (e.g., agonistic behavior, scent marking, emotionality) independent of clear changes in plasma corticosterone levels or adrenal gland weight (Spencer et al., 1973). Yoshimura (1980) found-prolonged isolation increased scent marking of male gerbils, a behavior known to be largely androgen dependent. The cholinergic antagonist scopoalmine, however, suppressed such marking behavior independent of any measurable changes in central acetylcholinesterase or choline acetyltransferase activity.

Mammalian Pheromones

from birth to 24 days of age display first estrus, on average, 10 days before unhandled controls (Morton et al., 1963). Second, handling female rats from birth to 30 days of age and rehousing them in small cages advances the time of vaginal opening. In contrast, housing handled female rats in groups of 10 in a large cage within an enriched environment delays the age of pubertal opening (Swanson et al., 1983). Third, applying intense visual, auditory, and/or electrical stimuli shortly before the time of normal physiological puberty accelerates the time of puberty in rats, although if such stimuli are applied much in advance of this time, puberty can be delayed (Árvay, 1967). Fourth, repeated exposure of young rats to cold stress brings about vaginal opening 3–4 days earlier than in the controls (Mandl and Zuckerman, 1952). Fifth, stress seems to accelerate the age of menarchy in humans. For example, girls from divorced families and families with greater interparental conflict tend to have an earlier menarche than girls from intact families (Wierson et al., 1993). These and other findings suggest that whatever factors influence male-induced pubertal acceleration, they are likely complex. Like many other situations, the rearing condition of female mice can influence the degree to which stimuli from males accelerate the time of puberty. For example, Mucignat-Caretta et al. (1995) reared females in three conditions: with both parents, with two females, or with two females and the presence of urine from adult males. Nine days after weaning, the females were exposed to either adult male urine or to prepubertal male urine. The adult male urine resulted in larger uteri and more cornified vaginal smears than the prepubertal male urine in the two groups reared with male odors (i.e., the one with both parents present and the one with two females and male odor). In the group reared with females only, no significant changes in uterus weight or vaginal smears were noted. The authors concluded that “the data support the notion that early experience of pheromonal cues may influence the response to pheromones in a later period, even if the preweaning exposure to males had no direct influences on early signs of puberty onset.”

VIII. NONLEARNED PHEROMONAL RESPONSES? Aside from the induction of stress-related changes in endocrine function, or possibly the influences of hormones or hormone-like agents from urine or other secretions on the reproductive processes of some species, are there examples where one or a few chemicals exert species-specific stereotypic or reflexive behaviors or endocrine responses that are not dependent upon, or significantly

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influenced by, prenatal or postnatal experience? If so, are they best described as being mediated by pheromones? While the examples described earlier in this chapter demonstrate the complexity of chemically mediated behaviors in mammals, some responses to isolated agents have been reported that, at first glance, would seem to be somewhat invariant and independent of obvious postnatal learning [e.g., the “aphrodisiac pheromone” protein in female hamster discharge (Singer et al., 1984, 1986, 1987)]. However, in most such cases the possibility of prenatal learning has not been addressed, and species specificity has not been established. Moreover, the isolated materials are often not as effective in inducing the behavior as the parent compound or may require urine or some other biological matrix to be effective (implying that other factors are involved, such as a combination of agents or symbiosis with releasing strata). Thus, evoking the pheromone concept in such cases would seem premature. There are, however, rare instances where a number of basic operational criteria found in a number of definitions of pheromones (e.g., species specificity, stereotypical response) have been tested and, for the most part, have been met. One of these exceptional cases is described below. Even in this case, however, there are questions as to whether the pheromone concept best describes the behavior, as postnatal learning may well intervene at some point and one might question whether the influences of intrauterine learning have been completely ruled out. Rabbit pups are said to be dependent upon a “shortrange pheromone” located on the doe’s belly to release and guide a steretotyped search behavior for locating the nipple—guidance that occurs even in preterm-delivered pups (Hudson and Distel, 1983). While at the time of the first suckling episode the nipple may be moistened with amniotic fluid, saliva is apparently not attractive (Hudson et al., 1983) and self-grooming does not seem to transfer an attractive agent to body areas distal to the nipple (Coureaud et al., 2001). Research suggests that two sources of attractive material may be present, one distributed over the nipple epidermis and one released within the nipple, perhaps via sebaceous structures (Moncomble et al., 2002). Apparently one or both of these stimuli forms a gradient that helps in guiding the pup towards the nipple (Coureaud et al., 2001). Such help is important, as a pup must find and attach to a nipple quickly to survive, since the mother is available for nursing only about 3 minutes each day (Coureaud et al., 2000).* Olfactory bulbectomy *Interestingly, during the nursing episode rabbit pups switch nipples periodically (average 2.6 times per minute), a behavior not dependent upon the amount of milk available from any one nipple (Hudson et al., 1983).

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eliminates the nipple search behavior and, hence, suckling (Distel and Hudson, 1985). In nonbreeding females, the emission of the substance is influenced by day length, peaking in the early summer, and is depressed by ovariectomy and restored by estrogen administration (Distel and Hudson, 1984). Its potency seems greatest during the immediate postpartum period, and it elicits the greatest interest on the part of pups just prior to the time of regularly scheduled nursing periods (Coureaud et al., 2001). Both progesterone and prolactin, probably in concert, seem to increase its emission in estrogen-primed does (Gonzalez-Mariscal et al., 1994), although oxytocin may also be involved (Fuchs et al., 1984). The nipple search response is not dependent upon an intact vomeronasal organ (Hudson and Distel, 1986). According to Hudson et al. (1983), the “nipple search releasing odour of rabbit does may be considered as a true pheromone as the behaviour elicited is reliable and highly stereotyped.” Nevertheless, it should be noted that female rabbit pups also display attraction towards abdominal odors of adult male rabbits, nonlacting female rabbits, and nonlactating nonpregnant female rabbits, although the odor of lactating females is more preferred (Coureaud and Schaal, 2000). Moreover, learning cannot be ruled out completely as a possible modifier of this response. Thus, if the rabbit mother is perfumed before nursing, the pups learn to respond to the novel odor with the characteristic nipple-search behavior in a single 3 to 4 minute nursing episode (Kindermann et al., 1991, 1994). In an insightful review stressing the importance of learning in the process of odor perception, Hudson (1999) notes that, despite the fact that rabbit pups delivered by caesarean section exhibit normal search and suckling behavior when placed by a lactating doe, “this does not exclude the possibility that the response is dependent on prenatal experience of chemical characteristics of the uterine environment. In fact, this might even be considered likely given the steep rise in pheromone emission in late pregnancy (Distel et al., 1984) and reports that rabbit pups are able to learn prenatally odor cues associated with their mother’s diet (Bilko et al., 1994; Hudson and Altbäcker, 1982; Semke et al., 1995; Coureaud, et al., 1997).” That being said, Schaal and associates have recently reported that a single rather specific compound found in doe milk elicits the aforementioned nipple search behavior —a compound whose meaning, they argue, is learned neither in utero nor post-natally (Schaal et al., 2003). In this study, active peaks were initially identified using a split stream gas chromatograph to establish biological reactions to various components of doe’s milk in a New Zealand–California cross-breed. These investigators performed a series of tests on this agent which they claim

Doty

meets the following criteria for a pheromone: (1) chemical simplicity; (2) unambiguous, morphologically invariant, and functionally obvious behavioral response of the receiver; (3) high selectivity of stimulus-response coupling; (4) species specificity of reception; (5) species specificity of emission; and (6) unconditioned stimulusresponse coupling. Among the studies performed on this compound, whose name has not yet been made available for proprietary reasons, were ones showing that (1) the agent, when placed on a glass rod, elicited stereotypic searching motions and attempts to orally grasp the source of the stimulus, (2) detectable impurities co-occuring with the commercially available agent do not elicit these behaviors, (3) a decrease in these responses parallels a decrease in the amount of this agent as the milk ages, and (4) replenishment of the aged milk with the substance reactivates the behavioral activity directed towards the milk. The compound’s effectiveness was, however, found to be concentration dependent, with optimal elicitation of the behavior occurring at concentrations 108 to 1010 g/mL. At concentrations above 10 4 g/mL, the “behavior efficiency vanished steeply.” None of a wide range of other compounds, tested across a range of concentrations, were found to produce this behavior, including (1) other volatiles found in rabbit milk, (2) volatiles not found in rabbit milk but found in other rabbit secretions, and (3) volatiles not found in rabbit milk but reportedly active in eliciting such behaviors in other neonatal mammals. When same-strain females were fed during gestation and lactation with two isocaloric diets composed of exclusive constituents bearing unique aromatic compounds, their pups responded to the same degree to the target substance, suggesting that its activity is not “dependent upon the individual bouquet of volatiles passed into the aminon or milk.” These authors state, “The high behavior efficiency of pure “agent x” in any of these contrasted chemoecological contexts proves the generality of its releasing properties within the breed under study, and mitigates the possibility that responses to “agent x” may be generated by experience in the individual-specific odor environment before and after birth.” This compound was effective in a number of different breeds of rabbits, implying that it is “an efficient releaser in newborns of O. cuniculatus regardless of their genotype.” The authors infer species specificity, since this agent did not elicit the searching and nipple grasping responses in newborns from another Lagomorph species, Lepus europaeus, or from several rodent species or cats (Rattus rattus, Mus musculus, Felis cattus). They further noted that freshly obtained colostrum and/or milk from rats, sheep, cattle, horses, and humans did not elicit the searchinggrasping response in the rabbit pups, and that upon

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chemical analysis bovine milk did not contain agent X. That the response is not learned postnatally was suggested from studies that found normal levels of attraction to this agent in (1) vaginally delivered pups immediately isolated from their mother or her secretions and (2) pups delivered by Cesarean section a day before gestational term. Lack of influences of intrauterine exposure to the agent was inferred from the following observations. First, when placed on glass rods, neither blood nor amniotic fluid produced the behavioral responses in 1 to 3-day-old pups. Second, “the dosage of X in the headspace developing over these substrates resulted in negative results.” The authors concluded, therefore, “that X may not directly contact the developing nasal chemoreceptors through either blood or amniotic pathways. Accordingly, the development of pup responsiveness to X apparently does not depend upon prenatal induction through stimulus exposure.” While one might argue that it unusual for a pheromone’s activity to be narrowly tuned to a range of concentrations, or that postnatal smelling of blood or amniotic fluid is not a conclusive test for the possibility that a preference was not learned in some way in utero, the authors of this study deliberately made a concerted effort to determine whether the agent they identified met a stringent set of operational criteria for a pheromone. Such an effort reflects their awareness of the need for an operational definition of this term. Only time will tell whether, in fact, others will agree that this specific agent is truly a pheromone.

IX.

CONCLUSIONS

The pheromone concept has attracted the imagination of scientists and laypersons alike and on the surface appears to provide a straightforward explanation for a number of mammalian behaviors. However, it is apparent from the material presented in this chapter that there is mixed agreement on what, in fact, constitutes a pheromone and how a pheromone is to be recognized. Many investigators assume that nearly every type of chemically mediated behavioral or endocrine response is mediated by a pheromone, even in the absence of any specific chemical stimulants and an agreed-upon set of criteria for distinguishing pheromones from nonpheromones. While a range of chemicals can significantly alter the behavioral and endocrine responses of mammals, the mechanisms underlying such influences are poorly understood and seeming complex. Moreover, by far the majority of phenomena attributed to pheromones can be evoked by other sensory stimuli, reflecting the redundancy and complexity of the communicative process. Since a primary function of the senses is to provide

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information about the environment, stimuli sampled by each sense are often fused into complex higher-order mental or cognitive constructs (Gibson, 1966; Marks, 1978). Thus, many apparent sensory redundancies are interactive and nonorthogonal, and caution is warranted in assuming that any one sense provides a totally unique contribution to the organism’s Umwelt (Doty, 1986). Are there cases where, in fact, employing the term pheromone aids in understanding or explaning chemically mediated mammalian behaviors or endocrine responses? It would seem to the present author that if one cannot practically define or test whether a “pheromonal” substance differs from a “nonpheromonal” substance, or if a large number of differing definitions are available for making such a distinction, the pheromone concept adds little to the scientific understanding of chemically mediated biological processes. If one accepts the common elements of most extant definitions of pheromones (e.g., species specificity, minimal influences of learning), one is hard-pressed to find verified examples of pheromones in mammals. Moreover, the general and uncritical use of the term is widespread and evokes a number of unwarranted, or at best untested, assumptions about the nature of the communicative process under consideration. Thus, even its name conjures up the idea that the social organization of animals is akin to the endocrine organization of an organism, with disparate parts being influenced by chemicals that circulate within the social milieu. For some nonvertebrates this may be true, given a relatively high degree of stereotypic behavior and evidence for comparatively simple stimuli that induce behavioral or endocrinological changes. However, for many vertebrates, particularly mammals, such a perspective would seem to be, with rare exception, an oversimplification of the underlying biological processes. While auditory and visual stimuli can alter hormone levels in birds and a number of mammals, including human beings, no scientist has found it necessary to evoke the terms “audiomones” or “visuomones” to describe such phenomena. Why, then, should the term pheromone be employed to describe chemically mediated behaviors? Even though there may be instances where hormones or hormone-like chemicals are detected by chemical receptors or are ingested or taken into the circulation of mammals via the lungs or via the highly vascularized nasal cavity, these instances appear to be the exception rather than the rule. Making the assumption that most chemically mediated social or endocrinological responses of mammals are due to pheromones would seem, therefore, to oversimplify complex phenomena, providing a term rather than an explanation for the observed responses. Thus, it would appear that the current less-than-judicial employment of the insect-derived pheromone concept in

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describing chemically mediated behaviors and endocrine responses of mammals is questionable. That being said, there is no doubt that biologically derived chemicals have profound influences on mammalian behaviors and endocrine responses, particularly among forms that live in burrows and are largely nocturnal or crepuscular. Moreover, one must not lose sight of the fact that chemical communication has some inherent advantages over communication using most other types of sensory stimuli. First, odorants can provide unique information about space occupancy or territory, being easily distributed in both space and time. “Time-coded” messages can be sent, such as the length of time since a given area has been visited or occupied or specific information about reproductive state. In the Norway rat, for example, estrous urine loses its attractiveness within 24 hours (Lydell and Doty, 1972). Second, chemical communication is useful in situations where the receiver and the sender are not present at the same point in time. Thus, chemical stimuli can remain in the environment for rather long periods of time even in the absent of the sender. If an animal emitted a continuous noise or visual signal in a manner analogous to leaving a long-lasting odor, an inordinate amount of energy would be expended and predators could easily locate the sender. This long-lasting property of odors allows a dominant male, for example, to make his odor nearly continuously present in the social environment. Third, chemical communication is efficient, making use of excretory and secretory products that often have other functions. Finally, scent marking makes it possible for the sender and receiver to communicate even outside of the range of hearing and sight, minimizing the physical harm, expenditure of energy, and exposure to predation that can result from direct physical encounters or inappropriately directed mating advances. ACKNOWLEDGMENTS This chapter was supported, in part, by the following grants from the National Institutes of Health: PO1 DC 00161, RO1 DC 04278, RO1 DC 02974, and RO1 AG 17496. I thank Lee Drickamer, Jack King, Igor Kratskin, Matthais Laska, Joel Maruniak, Michael Meredith, Vincent Sava, Benoist Schaal and John Stiller for their constructive comments on a previous version of this manuscript. REFERENCES A Dictionary of Science, 4th ed. Oxford: Oxford University Press, 1999. Alberts, J. R., and Brunjes, P. C. (1978). Ontogeny of thermal and olfactory determinants of huddling in the rat. J. Comp. Physiol. Psychol. 92:897–906.

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18 Psychophysical Evaluation of Olfaction in Nonhuman Mammals Lloyd Hastings University of Pennsylvania, Philadelphia, Pennsylvania, U.S.A.

1. INTRODUCTION

mechanisms underlying olfactory function. With the development of psychophysics, attention turned to investigating the relationship between changes in physical stimuli and the resulting psychological sensations in a quantitative manner. The initial objectives of psychophysical research were to determine the minimum detectable energy levels of stimulation (absolute threshold) and the minimum detectable difference between values on a continuum, e.g., difference thresholds. Numerous psychophysical techniques have since been developed to assess sensitivity, including ones that challenge the concept of a threshold as a fixed point, e.g., signal detection theory. Other techniques have been developed to investigate how people respond to odor stimuli that fall in the suprathreshold range, i.e., odors that are clearly perceptible. The latter includes procedures to examine (1) odor quality discrimination/generalization, (2) odor quality recognition/identification, (3) odor intensity, and (4) odor memory. In this chapter I describe various methods for assessing olfactory function in animals. The focus is on dogs, rats, and mice, given their widespread use in laboratory settings, although the basic procedures that are reviewed are applicable to other forms. These methods allow for determining the effects of numerous experimental manipulations on the ability to smell, including influences of drugs, brain lesions, and genetic manipulations. The reader is referred to Hübener and Laska (2001) for a recent review of nonhuman primate odor discrimination paradigms.

Experimental study of sense organs must be made on man because animals can not directly account to us the sensations which they experience. Claude Bernard, 1865 Results of studies employing animals in sensory research over the last 50 years have shown the above statement to be categorically wrong. In fairness, Bernard was partially right in that animals cannot directly convey their sensory experiences. However, he erred when he assumed that the only valid method for investigating sensory experience was by verbal report. His summary dismissal of animal testing can be better understood when it is realized that the field of animal experimental psychology, pioneered by Thorndike (1911) and Watson (1914), had not yet been developed. This chapter will show that, contrary to Bernard’s supposition, behavioral testing of animals can provide much important information about sensory systems, including olfaction, when the appropriate methodology is employed. Moreover, animal testing is the only way in which both behavioral testing and experimental physiological procedures, e.g., lesions, histological examination, genetic manipulation, etc., can be undertaken in the same organism. Early attempts to understand the olfactory system focused on developing classification systems based upon various “primary” odor groups. Since these attempts were usually subjective, there was little agreement and, consequently, little progress made in understanding the basic 385

386

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II.

HISTORICAL BACKGROUND

A.

Early Canine Studies

The olfactory powers of a number of animals have attracted considerable attention through the years, but the interest was usually casual in nature. Early observations were made concerning the ability of animals, like deer and kangaroos, to smell human scent from great distances, and of the ability of dogs and other animals to track game and to find escaped convicts. It was not until the late nineteenth and early twentieth centuries, however, that systematic application of the scientific method was employed to better understand such abilities. In 1910, Schmidt published one of the first books on canine tracking, Verbrecherspur und Polizeihund, which placed great emphasis on the necessity for the experimental controls and testing of hypotheses, in contrast to most contemporary approaches that were generally preoccupied with results and gave little detail on test conditions or procedures. Nearly two decades earlier, Passy (1892) conducted one of the first empirical studies of odor preferences in dogs (see Chapter 19 for a discussion of odor preference paradigms). Paper was dipped into alcoholic solutions of odorous substances and allowed to dry. When presented to a dog, a preference was scored if the dog smelled them attentively and tried to seize and eat the paper. If the dog turned its head away, a negative odor preference was scored. In this study, preference was operationally defined by the dog’s movement; motivational status of the subject was not considered. Thus, this early investigator was able to ascertain some quantitative information from animals about the perceived hedonics of olfactory cues. In 1907, Hamilton devised a operant box from which a young dog was rewarded by being allowed to escape if the correct of four pedals was pushed. Visual, olfactory, and other cues were associated with each pedal and randomized across trials. Unfortunately, the olfactory component of the study was attenuated when shock was employed to punish wrong responding, leading the subject to freeze thereafter when placed in the test situation. More extensive studies on the olfactory sensitivity or discrimination ability of dogs appeared in Germany around the same time, such as when Kalischer (1909) trained dogs to take food only when it was associated with a certain odor. He then proceeded to studies involving odor mixtures, where he mixed the original odor with as many as three or four additional odoriferous compounds before presenting the stimuli to the dog. Subsequently, Heitzenrohder (1913) recorded the respiratory movements of a dog using kymographical recordings, in an attempt to be more quantitative. He found, not surprisingly, that dogs responded forcefully to

the odors of other animals. His subjects also responded, however, to other odors such as camphor, carbon disulfide, and vanillin, which did not have any apparent biological significance. Using the same technique, Seffrin (1915) attempted to determine some “minima perceptible,” i.e., the absolute threshold, for some of the pure substances from Zwaardemaker’s classes, as well as a number of animal products such as urine. While the attempts to control the concentrations of the olfactory stimuli in these early studies can, at best, be considered crude, and the accuracy of the actual data collected as being suspect, these studies, along with the others still to be described in this chapter, nevertheless paved the way for the more rigorously controlled experiments of the last half of the twentieth century. In 1916, Henning conducted studies of discrimination behavior by training dogs to select a faintly perfumed handkerchief from a number of unadulterated ones. Conversely, he also trained dogs to ignore strong animal odors to which they normally responded vigorously. He concluded that methods based on motor responses revealed nothing about the olfactory acuity or hedonics of an odor, since absence of response did not necessarily indicate the inability to perceive the odor. This early observation presaged the necessity of well-developed instrumental and operant conditioning paradigms that clearly define the nature of the response to an odorant stimulus as a prerequisite for studying animal behavior and olfactory psychophysics. According to McCartney (1968), Neuhaus conducted the first well-controlled studies of canine olfaction in the early 1950s. Neuhaus developed not only an apparatus that attempted to regulate the olfactory cues presented to dogs, but also a protocol that incorporated a forced-choice paradigm. He determined the absolute thresholds for a variety of pure compounds as well as mixtures of compounds. He also performed one of the earliest animal studies to investigate the increase in stimulus concentration that was necessary to be judged different from a second one, i.e., a just noticeable difference (JND). In addition to his behavioral studies, Neuhaus performed histological examination of the canine olfactory region and counted the number of cells in different breeds of dogs. Finally, he included in his studies an investigation of the influences on olfactory thresholds of orally administered agents. About the same time of Neuhaus’s studies, researchers in the United States and England initiated studies in canine olfaction. Using a technique that insured the dog was responding only to olfactory cues and employing a large number of trials, Ashton et al. (1957) determined the absolute threshold for eight straight-chain fatty acids from formic to caprylic acid. When the threshold values obtained

Psychophysical Evaluation of Olfaction in Nonhuman Mammals

in this study were compared with those obtained by Neuhaus, they were found to differ by a factor of 107. These findings point out a monumental problem in olfactory research that does not presently exist for other sensory systems—namely, the marked difficulty in accurately measuring the stimulus reaching the nose of the subject. This is especially true in threshold studies, where stimulus concentrations may be well below the detection limits of available instruments. The similar inability to easily and accurately measure qualitative differences in olfactory stimuli also contributes to the uncertainty that accompanies many studies involving olfactory function and makes comparisons between studies difficult. B.

Ascension of Rodents in Olfactory Research

While most psychophysical studies of olfactory function during the first half of the twentieth century used dogs as subjects, the laboratory rat was making its ascent to prominence in psychological research. Liggett (1928) and Swann (1933) investigated olfactory function in rats using the buried food test—a test still widely used today. In the 1940s, Stone (1941) and Lashley and Sperry (1943) developed procedures for investigating olfactory discrimination behavior in rats using simple choice situation, which, unfortunately, allowed only crude control of relevant stimulus parameters. Among the first attempts to use operant conditioning techniques, as well as a functional olfactometer to control the presentation of stimuli, were studies of Pfaffmann et al. (1958) and Goff (1961). These investigators used flow-dilution olfactometers to accurately present the odorant stimuli and changes in the performance of various schedules of reinforcement to determine absolute thresholds. Once animal behavior could be reliably deciphered through the use of operant conditioning techniques and olfactory stimuli could be controlled (if not measured), the laboratory rat became a most useful subject in the investigation of olfactory function. Soon, more precise olfactometers were developed, as were more sophisticated operant conditioning protocols, and the investigation of olfactory function using animal behavioral assessment techniques began in earnest (Braun and Marcus, 1969; Davis, 1973; Pierson, 1974; Nigrosh et al., 1975). III.

METHODOLOGICAL ISSUES

A.

Stimulus Generation and Control

Until recently, research investigating the mechanisms underlying olfactory function has lagged behind similar research in vision and audition. This is due to a variety of reasons, but probably first and foremost is the fact that

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olfaction is not generally perceived as being as vital to humans as sight or hearing. In addition, another major impediment has been the inability to distinguish, a priori, between receptors that respond to different odorants. With the discovery of the large multigene family for olfactory receptors by Buck and Axel (1991) and greater knowledge of the molecular events in the transduction process, tremendous progress is now being made in delineating the basic mechanisms of olfactory function. Research on olfactory function that relies on behavioral measures, however, has not experienced a corresponding resurgence in growth. Those researchers interested primarily in the behavioral expression of olfaction, especially in animals, have always represented only a small minority in the field of olfactory research. This state of affairs is again due to a variety of factors, but with the development of sophisticated operant and instrumental conditioning paradigms, the presumed inability of animals to convey information about the sensory systems, as asserted by Bernard (1865), is not a primary reason. More likely the major impediment has been technical in nature, such as difficulties in accurately generating, controlling, and measuring olfactory stimuli. Not until the development of precision olfactometers, which allowed some control over the olfactory stimuli, was it possible to conduct meaningful studies on such psychophysical measures as absolute thresholds. Unlike the production of visual or auditory stimuli, generating and controlling olfactory stimuli are more arduous tasks. First, an olfactory stimulus must be volatile; if the compound is a liquid or solid, it must be converted to a vapor phase. To achieve the desired concentrations of a stimulus, a known quantity of the vapor phase is mixed with varying quantities of background air by the process of flow dilution. Also, in contrast to visual or auditory stimuli—which can be easily generated and turned on and off with great precision—olfactory stimuli linger until dispersed by diffusion or scavenged from the surrounding environs by a vacuum source. Furthermore, the presentation and removal of olfactory stimuli must be accomplished without producing extraneous cues, such as auditory cues or changes in temperature, flow, or pressure. Construction of an olfactometer that is capable of producing olfactory stimuli without such confounding artifacts requires considerable time and effort, as well as money (for review, see Prah et al., 1995). Although olfactometers have become more sophisticated and more precise, due mainly to the incorporation of computers and mass flow controllers in their design, one continuing deficiency in many applications is the failure to measure and verify the olfactory stimuli produced. Once again, unlike in auditory and visual research, there is no widely available instrument that can easily and routinely be

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used to accurately measure olfactory stimuli. Although great care may be taken in generating the stimuli, problems with leaks in the system, loss due to adsorption on the instruments walls, and/or contamination of the stock odorant can substantially alter the actual stimulus that reaches the subject. The failure to verify stimulus concentration, especially in threshold measurements, undoubtedly contributes to the large variability often found in published reports (Cain, 1977; Stevens et al., 1988). When stimuli are measured, gas chromotography is usually employed, but the technology is complicated and costly. A major challenge facing the field of olfactory research is finding new and more sensitive methods of measuring olfactory stimuli. Potential methodologies include “electronic noses” (see Chapter 14), but so far there has been very little use of these instruments in olfactory research. B.

Involvement of Other Systems in Olfaction

Besides the difficulties inherent in dealing with the generation and control of olfactory stimuli, care must also be taken to assure that the animal is responding only to olfactory cues, and not to cues from some other sensory system. While Cranial Nerve I (CN I) is the major neuronal system involved in olfactory function, there are other neuronal systems within the nasal cavity that may contribute to or at least subtly modulate the sense of smell. These include the trigeminal system (CN V), the vomeronasal organ (Jacobson’s organ), the septal organ of Masara, and the nervus terminalis. If understanding how the olfactory system functions is the primary goal, then the degree, if any, to which these other neuronal systems contribute to the sense of olfaction should be determined. Inherent in this statement is the implication that the function or purpose that these other systems perform or subserve is known; unfortunately, this is not entirely the case. One perplexing issue germane to this topic is the fact that when most of the olfactory epithelium in rodents is destroyed, olfactory sensitivity does not appear to be greatly affected (Hastings et al., 1991; Youngentob et al., 1997). Two theories exist to explain this phenomenon: (1) only a small percentage of the olfactory epithelium is actually needed to subserve this element of olfactory function, and/or (2) the animal is responding to cues generated by one of these other neuronal systems in the absence of a functional olfactory system. Some attempts have been made to establish the relative contributions of the different systems to olfactory function, but definitive information about the potential interaction of these other systems with olfaction is still lacking. Of the four, the trigeminal system (CN V) has been the most extensively studied. Its primary function is to detect and respond to airborne irritants (see Chapter 47).

Numerous studies have shown that (1) the threshold for activating the trigeminal system for most compounds is much higher than the olfactory threshold (Tucker, 1971), and (2) the trigeminal system can be eliminated and the olfactory system can still function in a normal manner (Silver et al., 1985). What has not been addressed, however, is whether the trigeminal system, when the olfactory system has been compromised or destroyed, can be used by animals to detect chemosensory stimuli to guide subsequent behavior. The possibility has been suggested that in humans, albeit in a minor way, the olfactory and trigeminal systems interact and that the sensitivity of one may change when the other is altered (Bouvet et al., 1987; Livermore et al., 1992). To what degree this is true in nonhuman mammals is not known. The vomeronasal organ (VNO), which shares many morphological and embryonic similarities with the olfactory system, is located in the anterior portion of the nasal cavity. The sensory fibers of the VNO terminate in the accessory olfactory bulb instead of the main olfactory bulb, unlike those of the olfactory neuroepithelium. The VNO appears to mediate sensory information important for reproductive physiology and behavior, although it does respond to some volatiles that are detected by CN I (Johnston, 1998). While olfactory function appears normal in the absence of the VNO (Brouette-Lahlou et al., 1994), and CN I and VNO neuronal pathways are relatively independent of one another, there is a remote possibility the VNO can mediate olfactory cues in the absence of a functional olfactory system. Such information is noteworthy, since damage to the VNO is often minimized during exposure to airborne toxic agents, compared to the olfactory epithelium (Gaafar et al., 1992; Youngentob et al., 1997). The third neuronal system in the nasal cavity, the septal organ (SO) of Masara, is a small patch of sensory epithelium located bilaterally on the septal wall in close proximity to the VNO. The structure of the neuroepithelium of the SO is very similar to that of the main olfactory epithelium, and its afferent fibers terminate in the caudal part of the olfactory bulb on glomeruli known as “septal glomeruli.” It was originally hypothesized that, due to its proximity to the entrance of the nasal cavity, it may have an alerting function (Rodolfo-Masera, 1943). That is, by continuously sampling the incoming olfactory stimuli during periods of rest (sleep), it performed an alerting function, which in turn might modify the function of the main olfactory system. An empirical test of this hypothesis (Giannetti et al., 1995) showed this not to be the case. No other role for the SO has been proposed, and its true functions remain unknown. The final neuronal system to innervate the olfactory epithelium is the nervus terminalis (NT), a plexiform, ganglionated nerve originating in the epithelium of the

Psychophysical Evaluation of Olfaction in Nonhuman Mammals

VNO (Schwanzel-Fukuda and Pfaf, 1995). Branches of the NT intermingle with afferent fibers of both the VNO and the trigeminal nerve as they course through the septal mucosa. The fibers eventually terminate in the accessory olfactory bulb as well as in the olfactory tubercle, the septal, the precommissural, and the preoptic areas of the brain. The function of the NT is poorly understood. It is possible that this nerve may be part of a luteinizing hormone–releasing hormone (LHRH) system involved in regulating the VNO “pump” (Wirsig-Wiechmann and Lepri, 1991). Another possible function includes mediation of some rather specific chemosensory responses (Kyle et al., 1987). Although the function of the olfactory nerve and the trigeminal system is apparent, and at least certain attributes of the VNO understood, the close proximity and intermingling of the fibers of the SO and NT make it very difficult to experimentally determine their actual function, as well as any role they may play in olfaction. If one is only interested in determining some psychophysical measure such as an absolute threshold in an intact, functioning animal, then there is little need to know the relative contributions that each of these subsytems may be providing to the process of olfaction. On the other hand, if one is interested in discerning some specific process of the olfactory system such as recovery of olfactory function after toxic insult or genetic manipulation of a gene for a specific odorant receptor, it then becomes very important to know the origin of the sensory information to which the animal is responding. Too often in the past, this information has not been known or even considered.

IV. PSYCHOPHYSICAL EVALUATION OF OLFACTION Stebbins (1970) defined animal psychophysics as an area of research in which the primary concern is the behavioral analysis of sensory function. The basic data consist of conditioned responses obtained from awake, intact animals in response to sensory stimulation. Function of the sensory system is then inferred from observation of the overt behavioral response. A change in the perception of a measureable parameter of an olfactory stimulus by the animal is reflected in a corresponding change in its conditioned behavior. As indicated earlier, psychophysical evaluation of olfaction in animals did not really begin until progress was made in stimulus control, i.e., olfactometry, and in behavioral analysis in animals, i.e., operant conditioning techniques. Earlier reviews of this topic include those by Passe and Walker (1985), Slotnick (1990), and Walker and Jennings (1991). In this section, protocols for assessing the perceived intensity and quality of olfactory stimuli in animals are described.

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A.

Assessment of Perceived Intensity

Determination of olfactory thresholds has long been considered the sine qua non parameter for best describing the overall function of the olfactory system. The absolute threshold is the measure of the minimal odorant concentration that can be detected from clean air, while the recognition threshold is the minimal amount that can not only be detected, but also identified. One variation of the absolute threshold measure introduced in recent years is the odor mixture threshold (Doty et al., 1999; Lu and Slotnick, 1998; Xu and Slotnick, 1999). In this procedure, the concentration of the test odorant is varied within the context of a second odorant, whose concentration remains constant and which also serves as the “blank.” The assumption, which may or may not be valid, is that since there is more “interference” in the system, the task should be more difficult to perform and, thus, more sensitive to any perturbation in the system. 1.

Establishing an Absolute Threshold via Operant Procedures

While the goal is the same, there are a variety of ways to determine the absolute threshold, including stimulus presentation procedures, such as the method of limits, staircases with reversals, and the method of constant stimuli (Figs. 1, 2). In all such procedures, the animal is taught to make some conditioned operant response, usually a bar press, nose poke, or lick, when an olfactory cue is detected and to withhold that response when only clean air is presented. This is termed a go/no-go paradigm. On rare occasions, a go/go differential response paradigm is used, e.g., go-right if odor is present/go-left if odor is absent. It is used less frequently, however, since more training trials are usually necessary to acquire the task. Studies that have used some type of operant conditioning paradigm to assess absolute detection thresholds are presented in Table 1. In most cases the absolute threshold measure was used to evaluate the functional status of the olfactory system after some form of insult, such as an anatomical lesion (olfactory bulb or CNS pathway), hormonal manipulation, or chemical damage of the olfactory neuroepithelium. Most studies have used either mice or rats, although the absolute threshold has been examined in dogs for a few specific compounds (Fig. 2). In the method of limits, olfactory stimuli consisting of a series of either ascending or descending concentrations of the odorant are presented to a subject. When used with animals, usually a descending series is used in order to maintain stimulus control. Threshold is reached when the subject’s ability to correctly detect the stimulus reaches

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values, usually in a randomized sequence, is presented and the threshold calculated from the generated response probabilities for the various stimuli. The method of constant stimuli has rarely been used in animal olfactory research, largely because of the large number of trials that are required and the propensity for adaptation. 2.

Figure 1 (top) Schematic diagram of the air dilution olfactometer used to assess sensitivity to ethyl acetate vapor. D1, D2, D3, and D4 are successive dilution stages. The airflows in channels A (odor) and B (clean air) were set at 0.1 L/min. The flow in channel C (carrier flow) was set at 1.9 L/min. (bottom) Schematic airflows in channels A and B were set at 0.05 and 1.95 L/min respectively. (From Bodyak and Slotnick, 1999.)

chance or some other prescribed criteria. In the staircase method, the same procedure is used as the method of limits, but the threshold region is traversed back and forth. The stimulus concentration continues to increase until a prescribed number of correct responses are made, and then the direction of stimulus change reverses until poor performance occurs. This procedure is repeated for a set number of reversals and then the reversal values are averaged. Thresholds obtained with this procedure are usually more reliable than those obtained with the standard method of limits, since more data are used in the determination. In the method of constant stimuli, a fixed set of stimulus

Establishing an Absolute Threshold Using Classical Conditioning or Related Conditioned Responses

While the majority of studies investigating olfactory function have used tasks based upon operant conditioning techniques, there are some that employ classical conditioning paradigms. These include studies based upon both conditioned avoidance (or approach) and conditioned suppression paradigms. In the conditioned avoidance paradigm, an odor is paired with negative reinforcement (positive reinforcement for conditioned approach) and the animal learns to perform some form of avoidance (or approach) response whenever it detects the odor. By successively lowering the concentration and noting when the conditioned behavior stops, i.e., when the animal can no longer detect the odor, a threshold measure is obtained. The conditioned suppression paradigm is very similar. The animal learns to suppress an ongoing operant response, e.g., licking, whenever it detects a specific odorant. As before, a threshold is determined by successively lowering the concentration and identifying the point at which the animal no longer suppresses its behavior. Both the method of limits and staircase stimulus presentation procedures can be used with either technique. Table 2 lists a number of studies that have made absolute threshold determinations using classical conditioning/conditioned response procedures. Included in this listing are three recent studies which have used an olfactory cue as the conditioned stimulus in a conditioned reflex paradigm (Hunt et al., 1997; Nsegbe et al., 1998; Richardson et al., 1999). While these studies did not actually measure olfactory detection thresholds, they could be easily modified to do so. As with the other examples, one only has to lower the concentration after the behavior has been established and look for alterations in the behavior. The benefits of using a conditioned reflex, like changes in heart rate, ventilation, or startle response, are that little training is required—compared to operant techniques—and that the techniques can be adapted to test very young animals. 3.

Assessment of Suprathreshold Stimuli

In humans, suprathreshold odor intensity is usually studied by either quantifying the growth in the magnitude of odor sensation as the odor concentration increases

Psychophysical Evaluation of Olfaction in Nonhuman Mammals

Figure 2 Apparatus for testing dogs in an odor-detection task. The test chamber is housed in a controlled environment room occupying part of a laboratory. For the purposes of illustration, many details have been simplified or omitted. (For example, a gas chromatograph and water reservoir bottles are normally housed on the roof of the chamber, and an air conditioning unit and purification stages lie on the roof of the room.) The olfactometer is shown in semi-schematic form. (From Moulton, 1977.)

(suprathreshold scaling) or by measuring the ability to detect small changes in odor concentration (differential threshold or JNDs) (Walker and Jennings, 1992). In human suprathreshold scaling studies, the increase in perceived intensity is quantified either by verbal report or by some cross-modal manipulation, e.g., adjusting the length of a line or arrow. No comparable paradigm is available for use with animals, so tests of differential sensitivity have been used with animals to investigate perception of suprathreshold stimulus intensity. When animals are asked to discriminate between two different odorants, it is assumed they can do so based on qualitative differences. However, since it is difficult to match the intensities of two qualitatively different odors, the animals could be responding to differences in intensity rather than quality. Knowing how sensitive animals are to differences in intensity is necessary to rule out any confounding effects in studies focusing on discrimination of qualitative differences. On a more practical note, generation of a suprathreshold stimulus is more reliable than a threshold stimulus, and instrumentation is available to measure these higher levels. This is usually not the case when dealing with olfactory threshold testing, where the required concentrations are usually below the detection limits of most instruments. Precise

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control of the olfactory stimulus, along with the ability to verify the actual concentrations presented to the subject, are required for the collection of reliable data. While the use of olfactory intensity-difference thresholds appears to have several advantages over the measurement of olfactory absolute thresholds, only a few such studies have been reported in the animal literature. Using this technique, Slotnick and Ptak (1974) compared olfactory intensity-difference thresholds in rats and humans. For rats, a go/no-go discrete trials, successive stimulus presentation was employed. Rats were exposed to either a reference concentration or to one of a series of concentrations, which varied according to a modified ascending method-of-limits procedure. The olfactory-difference threshold (Weber fraction) obtained was approximately 0.03 (compared to ~0.3 for humans). In two later studies (Slotnick and Schoonover, 1984; Slotnick et al., 1997), a Weber fraction approximately 10 times as great (or the same as humans in the previous study) was obtained. This difference was attributed to variations in the training protocol, reinforcing the principle that if behavioral measures are to be used to obtain sensory information in animals, such measures must be well defined. When used as a diagnostic test to evaluate whether olfactory function had been impaired by an experimental procedure, no effects were observed on the intensity-difference threshold in rats after lesions of the anterior amygdala (Slotnick, 1985). However, transection of the lateral olfactory tract (Slotnick and Schoonover, 1993) and application of intranasal zinc (Slotnick and Gutman, 1977) did have a greater effect on intensity-difference thresholds than on absolute detection thresholds. This suggests that the intensity-difference threshold test might be a more sensitive measure for assessing damage to the olfactory system than the more frequently measured absolute threshold. Table 3 lists studies that have measured olfactory intensity-difference thresholds in rats. B.

Assessment of Perceived Quality

Besides odor intensity, the other most frequently examined parameter is odor quality. It is the qualitative differences of olfactory stimuli that allow us to perceive, discriminate, and enjoy the myriad of smells in our environment. Probably the single most important issue in the field of olfactory research is the question of how the molecular properties of an odorant determine odor quality. Crucial to understanding this relationship was the discovery of the large multigene family of olfactory receptors (Buck and Axel, 1991). The question of whether a certain type of receptor responds to an individual odor or is broadly tuned to respond to many odors is being addressed through the tools of molecular biology, specifically the development of

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Table 1 Threshold Testing: Operant Conditioning Species Mice

Rats

Dogs

Experimental manipulation Technique Mutant Transgenic Lesion Technique Technique Baseline/compound Lesion Drug Drug Lesion Drug Lesion Lesion Drug Lesion Baseline/compound Lesion Lesion Drug Lesion Lesion Lesion Drug Drug Drug Lesion Baseline/compound Baseline/compound Baseline/compound Baseline/compound Baseline/compound Baseline/compound

Paradigm

Ref.

Go/no-go preference Go/no-go Go/no-go Go/no-go Go/no-go Go/no-go Go/no-go Go/no-go Go/no-go Go/no-go Go/no-go Go/no-go Go/no-go Go/no-go Go/no-go Go/no-go Go/no-go Go/no-go Go/no-go Go/no-go Go/no-go Go/no-go Go/no-go Go/no-go Go/no-go Go/no-go Go/no-go Go/no-go Go/no-go Operant/handler Operant/handler Operant/handler

Walker and O’Connell, 1986 Deiss and Baudoin, 1997 Youngentob and Margolis, 1999 Bodyak and Slotnick, 1999 Mihalick et al., 2000 Slotnick and Nigrosh, 1974 Marshall et al., 1981b Slotnick B. M. and Schoonover, 1984 Doty and Ferguson-Segall, 1987 Doty et al., 1988 Doty and Ferguson-Segall, 1989 Doty and Risser, 1989 Slotnick et al., 1987 Slotnick and Pazos, 1990 Doty et al., 1990 Hunt and Slotnick, 1991 Youngentob et al., 1991a Doty et al., 1991 Slotnick and Schoonover, 1992 Brosvic et al., 1996 Youngentob et al., 1997 Apfelbach et al., 1998 Lu and Slotnick, 1998 Doty et al., 1998 Dhong et al., 1999 Doty et al., 1999 Slotnick et al., 2000 Moulton and Marshall, 1976 Marshall et al., 1981a Marshall and Moulton, 1981 Arner et al., 1986 Kurz et al., 1994 Kurz et al., 1996

Table 2 Threshold Testing: Classical Conditioning/Conditioned Response Species Rats Rats Rats Rats,dog Rats Dogs Rat pups Mice Rats Rats Rats Rats Rat pups Rats Rats

Experimental manipulation Baseline/compound Baseline/compound Baseline/compound Baseline/compound Baseline/compound Baseline/compound Drug Chemical lesion Chemical lesion Technique Chemical lesion Technique/chemical lesion Conditioned reflex (change in heart rate) Conditioned reflex (ventilation) Conditioned odor potentiation of startle

Paradigm CA CA CA CS CS CS CA CA CA CA CS CA CR CR CR

CA
conditioned avoidance/approach; CS
conditioned suppression; CR
conditioned reflex.

Ref. Eayrs and Moulton, 1960 Moulton and Eayrs, 1960 Moulton, 1960 Davis, 1973 Pierson, 1974 Krestel et al., 1984 Welson et al., 1991 Kimura et al., 1991 Peele et al., 1991 Darling and Slotnick, 1994 Sun et al., 1996 Owens et al., 1996 Hunt et al., 1997 Nsegbe et al., 1998 Richardson et al., 1999

Psychophysical Evaluation of Olfaction in Nonhuman Mammals Table 3 Intensity-Difference Threshold Experimental manipulation

Paradigm

Technique/baseline Lesion Lesion Lesion Lesion Lesion Lesion

Go/no-go Go/no-go Go/no-go Go/no-go Go/no-go Go/no-go Go/no-go

Ref. Slotnick and Ptak, 1977 Slotnick and Gutman, 1977 Slotnick and Berman, 1980 Slotnick and Schoonover, 1984 Slotnick, 1985 Slotnick and Schoonover, 1993 Slotnick et al., 1997

genetically modified mice, e.g., transgenic and “knockout” mice, which have had genes added or deleted (Youngentob and Margolis, 1999). However, olfaction is a perceptual process, and even if there were specific receptors for each odorant, there would still be additional processing at the level of the olfactory bulb and other higher cortical centers. Only by examining an awake, responding organism is the fully integrated process of olfaction available for study. The issue of perceived quality can be examined in numerous ways. These differing approaches, which in themselves are not mutually exclusive, help define the nature of the questions addressed as well as the techniques employed to provide the answers. Thus, perceived quality can be examined in terms of discrimination behavior, as an issue of similarity/dissimilarity, and in terms of perceptual constancy. Discrimination involves the process of differentiating the elements of a group into two or more subgroups, based upon some attribute. This is a fundamental property of any sensory system; exactly how the olfactory system accomplishes this and the nature of the basic mechanisms or processes are not known. On a more practical level, assessing the ability of an organism to discriminate among odorants is frequently used as an index of the functional status of the system. While the discrimination process seeks to separate elements into different groups, usually in a binary (e.g., same, different) fashion, studies on stimulus generalization seek to investigate in greater detail the relationships that exist among the various elements. The testing situation is thus structured so that the degree of similarity (or dissimilarity) among stimuli is reflected in a performance gradient. It is hoped that by examining these relationships, a clear understanding of how the system processes olfactory stimuli can be gained. Tests of similarity / dissimilarity can also be used in a diagnostic mode, e.g., intensity-difference thresholds. The final issue, perceptual constancy, examines how the system maintains its functional integrity over time. The primary olfactory receptor cells, which also are the first order afferents, are replaced over time without any adverse effects on the func-

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tional capacity of the system. This is also an important issue in recovery of function after the system has experienced extensive damage. 1.

Olfactory Discrimination Behavior

As with the absolute threshold task, the predominant paradigm employed in most studies of olfactory discrimination behavior has been the go/no-go, successive trials paradigm, which incorporates a learned operant response such as a nose poke or bar press. Since most problems presented in an olfactory discrimination task are binary in nature, e.g., differentiate between odor A and clean air or between odor A and odor B, the go/no-go task is the most efficient way to answer the question. While the same testing paradigm was used in almost all the studies listed in Table 4, the rationale behind the testing did differ. Sometimes discrimination testing was used to assess the functional status of the system after local insult to the olfactory epithelium. In other studies, discrimination testing was used to assess the effects of lesions to the olfactory bulb or other CNS pathways in an effort to understand the neuronal circuitry and structures mediating olfaction. Other times, when it was apparent that the organism could perform simple olfactory discrimination behavior, more complex tasks were used or parameters such as varying delays in responding, changes in intertrial intervals, and reversal learning procedures were incorporated in the testing design to examine the role learning and memory played in the processing of olfactory cues. An attempt was made to identify the predominant question being asked in the studies listed in Table 4. However, the distinctions made between the various study rationales are not absolute, and learning is obviously inherent in all tasks. 2.

Stimulus Generalization

Studies on discrimination behavior have been used in a diagnostic manner to investigate which neuronal structures or pathways are necessary for olfaction and, to a lesser degree, how the system actually processes qualitative differences/similarities. In the discrimination process, the general goal is to elucidate the differences between/among stimuli in order to differentiate them. In studies on stimulus generalization (Table 5), the goal is to determine how closely different stimuli are perceived as being similar in quality. By focusing on the degree of perceptual similarity among a group of stimuli, it is hoped that a continuum of similarity can be found among either some physiochemical property of the odorants, a commonality among members of the olfactory receptor gene family, or some other variable that will aid in under-

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Table 4 Discrimination Tasks Experimental manipulation Technique Baseline/ learning-seta Lesionc Lesionb Lesionb Metabolic activityb Odor masking in mixturesb Lesionc Lesionc Baselinec Chemical lesiona Lesionb

Paradigm

Ref.

Go/no-go

Nigrosh et al., 1975

Go/no-go Go/no-go Go/no-go Go/no-go Go/no-go

Slotnick and Katz, 1974 Slotnick and Kaneko, 1981 Slotnick, 1985 Slotnick et al., 1987 Slotnick et al., 1989

Go/no-go Go/no-go Go/no-go Go/no-go Buried food Go/no-go

Laing et al., 1989 Lu an Slotnick, 1990 Slotnick and Risser, 1990 Slotnick et al., 1991 Hastings et al., 1991 Slotnick and Schoonover, 1992

Baseline/match to samplec Lesionb Chemical Lesiona Odor memoryc

Go/no-go Go/no-go Go/no-go Runway

Lesionc Lesionb

Go/no-go Go/no-go

Chemical lesiona Lesionc Lesionb Transgenic Lesion

Go/no-go Go/no-go Go/no-go Buried food Buried food

Chemical lesion Diet Drug Chemical lesion

Go/no-go Go/no-go Go/go Go/no-go

Lu et al., 1993 Lu and Slotnick, 1994 Evans et al., 1995 Lovelace and Slotnick, 1995 Thanos and Slotnick, 1997 McBride and Slotnick, 1997 Setzer and Slotnick, 1998 Zhang et al., 1998 Lu and Slotnick, 1998 Smith et al., 1998 Berger-Sweeney et al., 1998 Xu and Slotnick, 1999 Greiner et al., 1999 Winters et al., 2000 Slotnick et al., 2000

a

To assess functional status of neuroepithelium. To assess effect of manipulation on olfactory function (nonneuroepithelium effects). c To assess learning/memory function. b

was, in essence, a go/no-go paradigm, the response metric —fixed-ratio responding—allowed for a gradation of responding, as opposed to the usual single nose poke or bar press response. While some of the steric properties of the odorant molecules could be shown to correlate with the gradation in responding, there were too few data to substantiate any hypothesis. Almost 20 years elapsed before another example of a study that examined stimulus generalization in rats appeared in the literature (Duncan et al., 1992). It was similar in design to that of Braun and Marcus (1969), but instead of using fixed-ratio responding as the operant, a touch response was employed in the go/no-go paradigm. Odorants were assigned as S and S and the rats trained to a certain level of proficiency. On test days, a percentage of the trials was replaced with unreinforced probe trials, in which new test odorants were presented. Performance on the probe trials was graded; the more the test odorant was like the S, the better the performance. One caveat offered by the authors applies to most generalization studies of similar design: “generalization responses must be compared to levels of S responding; a low level of response does not mean that the test stimulus is similar to S.” A procedure that has considerable potential as a tool for investigating the degree of similarity/dissimilarity among different odorants in animals is an animal analog of the confusion matrix task used to evaluate human olfactory function (Wright, 1987) as developed by Youngentob and coworkers (Kent et al., 1995; Youngentob et al., 1990, 1991b, 1995) (Fig. 3). In this procedure, rats are initially trained to sample a test odorant at a central location and then to traverse a specially marked alley to obtain reinforcement. Similar training is given for all test odorants. During this training period, the rat is not allowed access to any alley except the one associated with the test odorant. In the testing phase, all Table 5 Stimulus Generalization Studies Paradigm

standing how the system actually processes qualitative differences of odorants. The initial publication of a technique to study stimulus generalization among odorants in rats occurred over 30 years ago (Braun and Marcus, 1969). Braun and Marcus taught rats to respond on a fixed-rate schedule in the presence of one odorant and to refrain from responding in the presence of a second odorant. On a nonrewarded probe trial, a third, novel odor was presented and the degree to which the rat responded was recorded as a measure of the similarity to the original S odorant. Although this task

Fixed ratio responding during S,S with probe trial Go/no-go: masking by similar/ dissimilar odorants Confusion matrix Confusion matrix Go/no-go with probe trials Approach/avodiance Confusion maxtrix Confusion maxtrix Buried odor mixtures

Ref. Braun and Marcus, 1969 Laing et al., 1989 Youngentob et al., 1990 Youngentob et al., 1991b Duncan et al., 1992 Heth et al., 1992 Youngentob et al., 1995 Kent et al., 1995 Linster and Smith, 1999

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395

reinforcement. By manipulating the composition of the odorant cue in the scented cup they were able to demonstrate that rats can generalize between a component and a binary mixture that contains that component. While this task requires much less baseline training than some of the aforementioned tasks, its overall utility is limited by the lack of rigorous control over stimulus generation and presentation. Even with this limitation, the modified buried food task can still provide useful information concerning odor identification and processing, 3.

Figure 3 Five tunnel differential response chamber for the animal odorant confusion matrix: (A) top view; (B) side view. (From Youngentob et al., 1990).

response alleys become available and the test odorant is varied from trial to trial. By examining the pattern of errors made during a session, the similarity/dissimilarity of all odor combinations can be investigated. The major drawbacks of this procedure are the long and complicated training and testing periods required to perform the task, as well as the need for a highly sophisticated testing apparatus. Not all procedures for examining stimulus generalization are based upon operant conditioning paradigms conducted in operant chambers. Heth et al. (1992) used positioning of the nest and food store and the preferred location of the tested animal (mole rats) as indicators for preference/aversion to various pairs of enantiomeric compounds. Linster and Smith (1999) employed one of the oldest tests available for assessing olfactory function—the buried food test—to investigate generalization between binary odor mixtures in rats. Instead of simply allowing the rats to dig in sand to find food reinforcement, they trained the rats to choose between a scented cup with reinforcement and an unscented cup without

Perceptual Constancy

As noted earlier, olfactory receptor neurons (ORNs) are replaced throughout life on a continuous basis. Whenever the neuroepithelium is damaged after exposure to toxic compounds or diseases, this process is accelerated. The fact that perceptual quality remains more or less constant over long periods suggests that during normal replacement, new ORNs either reestablish the same bulbar connections as their predecessors or are reprogrammed once they successfully connect to the bulb. Furthermore, under normal conditions, only a small percentage of the total number of neurons is being replaced, leaving the bulk of the system intact to function in a normal manner. The situation is quite different when the olfactory neuroepithelium is extensively damaged by toxic agents, disease, or experimental nerve transaction. Under these conditions, almost all normal connections are lost and the system must reconstitute itself, de novo. While a number of studies have examined the functional status of the olfactory system after extensive insult, usually by employing threshold or discrimination tasks, only a few have attempted to establish whether perceptual quality remains constant during and/or after insult. Anecdotal reports suggests that perceptual qualities can be severely disrupted after pervasive damage to the olfactory system. To address the question of perceptual constancy, there must be a way to assess perceptual quality with enough sensitivity to detect even small perturbations to the system. Few studies have attempted to address this challenging issue. Yee and Costanzo (1995) manually trained hamsters to perform a go/no-go task that involved responding initially to an odorized container while ignoring a nonodorized container, followed by analogous discrimination training between two odorants. After successfully learning to discriminate the two odorants, the olfactory nerve fibers were severed. Return of olfactory-mediated behavior was first seen 19 days after surgery, with complete recovery evident by day 40. While these results confirmed that replacement ORNs regained full functionality, it left unanswered whether the system

396

displayed perceptual constancy during the recovery process. This was because testing after surgery used reinforced trials. Thus, the complete recovery of olfactory function could have been due to recovery of the system and performance of the originally task, or the animals could have learned to discriminate between the two odors based upon the associated reinforcement contingencies, even if the odors were being perceived as two new, distinct odors. To address this issue, Yee and Costanzo (1998) repeated the study, but did not test animals until 40 days after surgery, to allow time for the new neurons to fully mature. Sham-operated animals were able to perform the original discrimination task after 40 days of rest, but the nerve-transected animals could not, when tested without reinforcement. When reinforcement was reinstated, rats could perform the discrimination. This suggested that the nerve transection and subsequent regrowth had changed the odor perceptual qualities of the stimuli and that the animals had to relearn the task based upon the reinforcement contingencies of the new stimuli. The only other study to assess perceptual constancy of odorant quality during recovery of function after insult to ORNs was conducted by Youngentob and Schwob (1997). Rats were trained on a five-odorant identification confusion matrix task and then their nasal neuroepithelium was almost completely destroyed by exposure to the olfactotoxin methyl bromide. Like the preceding study, rats were not tested until the olfactory neuroepithelium had been reconstituted (2 months for methyl bromide). Under these conditions, control rats retained the identification task at a 75% level, in contrast to methyl bromide–treated rats, who performed at a 30% level. It also took the lesioned rats almost three times as long as the control rats to once again achieve criterion performance. The authors interpreted this poorer performance by the lesioned rats as an indication that odorant quality perception had been altered. This is in contrast to the findings of their earlier study, where they reported no change in absolute thresholds for similarly lesioned rats (Youngentob et al., 1997). This would suggest that the absolute threshold test might not be the most sensitive test to use to evaluate the functional status of the olfactory system after insult. However, additional studies are necessary to establish whether this is true. V. BRIEF SCREENING TESTS FOR ASSESSING OLFACTION IN GENETICALLY MODIFIED MICE Development of methodologies in molecular biology that allow direct manipulation of genes has opened an entirely

Hastings

new era in olfactory research. Much progress has been made in delineating not only the large multigene family of olfactory receptors, but also the physical location of these receptors within the nasal cavity. The next crucial step is to link specific receptors and / or families of receptors with the detection of individual odorants. Some progress has been made using cell cultures (Zhao et al., 1998) or electrophysiological measures such as the electro-olfactogram (Buiakove et al., 1996) and patch-clamping (Ma et al., 1999). While these studies provide very important information, they cannot address the issues of perception that occur in an intact, responding organism. The development of genetically modified mice in which the ORN genes have been manipulated, or mice that mimic a disease known to be associated with olfactory dysfunction, e.g., Alzheimer’s disease (Doty, 1991), along with suitable techniques for assessing olfactory function offers promise in this regards. An approach is outlined below, which incorporates many of the techniques already discussed, that can be used to assess olfactory function in genetically modified mice. A.

Adults

Although mice can be evaluated using the lengthy psychophysical paradigms described earlier, there is a need for rapid tests that can be used to assess large numbers of mice. Three screening tests—the buried food test, a conditioned odor aversion task, and a odor habituation task— have been found to be useful for this purpose. In the simplest form of the buried food test, food reinforcement is buried in bedding or sand and the mouse must locate it and dig it up within a specified time frame (Liggett, 1928; Hastings et al,. 1991). The test can be made more sophisticated by requiring the mouse to discriminate between two odors or odors of different concentrations (BergerSweeney et al., 1998; Linster and Smith, 1999; Smith et al., 1998). In conditioned taste aversion, an aversion to a flavored solution is induced by following ingestion with toxicosis (Garcia et al., 1966). However, development of an aversion to an odor alone is much more difficult (Hankins et al., 1973). Holder and Garcia (1987) have shown, though, that if a taste is paired with an odorant, a strong aversion to the odorant alone can be obtained. Only one trial conditioning is needed, and the learned association is quite persistent. Tests involving both detection thresholds and discrimination tasks can be developed using conditioned odor aversion (Darling and Slotnick, 1994; Kimura et al., 1991). In an odor habituation paradigm, one odor is presented repeatedly in short trials until investigatory interests in it wanes. A novel odor is then presented, producing a dramatic increase in sniffing, thereby demon-

Psychophysical Evaluation of Olfaction in Nonhuman Mammals

strating detection. If differences are found in the olfactory function of genetically modified mice using these screening tasks, more sophisticated psychophysical tasks can then be used to evaluate subtle changes in olfactory function. B.

ACKNOWLEDGMENTS Supported, in part, by Grants RO1 DC 02974 (R. L. Doty), RO1 DC 04083 (I. Kratskin) and R43 DC 04024 (L. Hastings). The comments of Dr. R. L. Doty during the preparation of this manuscript were especially helpful.

Neonates

Since the senses of hearing and sight are underdeveloped at birth, mouse pups must rely almost entirely on their sense of smell for bonding and nutrition. In fact, any manipulation that results in anosmia at birth would most likely be lethal to the pup. Nonetheless, some genetic alterations may influence olfactory function in neonates. The use of neonates could also be cost effective, since they do not have to be housed until they reach maturity. One task that has been used to evaluate olfaction in mouse pups involves a preference test for soiled “home-cage” bedding or the dam (Montella and Reddy, 1991; Wong et al., 2000). A novel task that is used extensively with neonatal rats and could be easily adapted to mice is associative conditioning using odorants. Pups are exposed to an odor paired with tactile stimulation (stroking the skin with a paint brush), with preference measured as time spent near the odor versus a nonodorized space (Weldon et al. 1991). Another approach to allow greater quantification of responses is to establish conditioned reflexes, e.g., heart rate, to odorant cues (Hunt et al. 1997; Nsegbe et al., 1998; Richardson et al., 1999). It should be noted, however, that, given the limited response repertoire of neonates, it can be difficult to detect subtle deficits with these tasks. VI.

397

SUMMARY AND CONCLUSION

Contrary to Bernard’s supposition that humans are the only animals for whom the study of sensation and perception is possible, nonhuman animals have been used to study the sense of olfaction with much success. Techniques have been developed that allowed behavioral responses of animals to be interpreted and quantified as well as the precise generation and presentation of olfactory stimuli. Currently, animals are routinely used to study the olfactory process, including the coding of odor intensity and quality. The effects of a wide variety of experimental manipulations, including lesions, drugs, and various learning paradigms, on the ability to perceive odors have been employed. Future application of these techniques to the study of olfactory development and the genetic basis of olfactory function in mice are now being investigated.

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401 Youngentob, S. L., and Schwob, J. E. (1997). Changes in odorant quality perception following methyl bromide induced lesions of the olfactory epithelium. Chem. Senses 22: 830–831. Youngentob, S. L., Markert, L. M., Mozell, M. M., and Hornung, D. E. (1990). A method for establishing a five odorant identification confusion matrix task in rats. Physiol. Behav. 47:1053–1059. Youngentob, S. L., Hornung, D. E., and Mozell, M. M. (1991a). Determination of carbon dioxide detection thresholds in trained rats. Physiol. Behav. 49:21–26. Youngentob, S. L., Markert, L. M., Hill, T. W., Matyas, E. P., and Mozell, M. M. (1991b) Odorant identification in rats: an update [comment]. Physiol. Behav. 49:1293–1296. Youngentob, S. L. Kent, P. F., Sheehe, P. R., Schwob, J. E., and Tzoumaka, E. (1995). Mucosal inherent activity patterns in the rat: evidence from voltage-sensitive dyes. J. Neurophysiol. 73:387–398. Youngentob, S. L., Schwob, J. E., Sheehe, P. R., and Youngentob, L. M. (1997). Odorant threshold following methyl bromideinduced lesions of the olfactory epithelium. Physiol. Behav. 62:1241–1252. Zhang, Y., Burk, J. A., Glode, B. M., and Mair, R. G. (1998). Effects of thalamic and olfactory cortical lesions on continuous olfactory delayed nonmatching-to-sample and olfactory discrimination in rats (Rattus norvegicus). Behav. Neurosci. 112:39:53. Zhao, H., Ivic, L., Otaki, J. M., Hashimoto, M., Mikoshiba, K., and Firestein, S. (1998). Functional expression of a mammalian odorant receptor. Science 279:237–242.

19 Methods for Determining Odor Preferences in Nonhuman Mammals Richard L. Doty University of Pennsylvania, Philadelphia, Pennsylvania, U.S.A.

also McIndoo, 1926). Quantitative assessment of odor preferences and aversions in nonhuman mammals, however, appeared a number of decades later and, with rare exception (e.g., Swann, 1933), largely followed the development of formal taste preference testing in the 1930s (e.g., the two-bottle preference test) (see Richter, 1936, 1939; Richter and Campbell, 1940). Nonetheless, anecdotal reports regarding mammalian odor preferences appeared at earlier dates (e.g., Binet and Passy, 1895, 1896; Hamilton, 1907), and attempts to understand the tracking abilities and preferences of dogs have a long history (e.g., Romanes, 1887; von Uexküll and Sarris, 1931) (see Chapter 18). In 1949, Beach and Gilmore employed boxes containing sawdust over which the urine was sprinkled to quantitatively demonstrate that male dogs spend more time sniffing urine odor from an estrous than from an anestrous female and that sexual experience or competence may be associated with this behavior. Le Magnen (1952), using a Y-maze, showed that adult male rats (Rattus norvegicus) prefer the odor of receptive females to nonreceptive ones, whereas prepubertal or castrated rats do not (unless they have been injected with testosterone). Four years later, Godfrey (1958) found, using a similar maze, that estrous female bank voles (Clethrionomys) prefer homospecific male odors to heterospecific male odors and that hybrids were discriminated against. Subsequent workers like Carr et al. (1965, 1970) devised even simpler preference paradigms that could be placed within the subject’s home cage, demonstrating, among other things, the

. . . the more we study animals, the more that we perceive that smell is the chief agent which provokes attractions and aversions between animals themselves and between animals and men. —Gilbert Coleridge, 1920

I.

INTRODUCTION

Operationally, one stimulus is assumed to be preferred to another if an animal behaviorally increases the probability of being stimulated by that stimulus over the probability of being stimulated by the other. In general, behavioral inferences about preferences or aversions are established on a relative basis, although marked preferences or aversions are easily identified. Increased or decreased stimulus access on the part of an animal can be achieved in a number of ways, such as by spending more or less time in the physical proximity to a stimulus or by increasing or decreasing sniffing behavior in its presence. Despite the fact that preference paradigms have been used to infer discrimination, it should be emphasized that a lack of preference does not necessarily mean a lack of discrimination. Numerous invertebrate odor preference studies appeared in the late nineteenth and early twentieth centuries, such as those examining the behavior of paramecia (Jennings, 1906) and the pomice fly (Barrows, 1907; see 403

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important role of sexual experience in establishing heterosexual odor preferences. In this chapter, basic methodologies are described for determining odor preferences and aversions in mammals, with an emphasis on rodents. Five types of paradigms are conceptualized and critiqued: approach, forced approachavoidance, bar-press stimulus presentation, odor trail, and sniff-rate analysis. Although these paradigms are not necessarily mutually exclusive, they provide a useful taxonomy for the classification and comparison of most odor preference test situations. Tests designed to measure detection sensitivity, discrimination, and learning, including conditioned aversion paradigms, are not reviewed in this chapter, and the reader is referred elsewhere for information on such procedures (e.g., Slotnick, 1990; Stevens, 1975) (see also Chapters 18, 20, 40). Results of specific studies are discussed only if they add to the description of a particular procedure.

II.

APPROACH PARADIGM

The most popular odor preference test paradigm is one in which two or more odorants are placed at different locations within a home cage or test chamber or room. In some cases, anesthetized conspecifics to whom an odor has been added are employed. The duration and/or frequency of investigation of the odorant or odorized stimulus object usually serves as the dependent measure, although other measures have been employed as well (e.g., the amount of food or water consumed near the stimulus) (see Barnett and Spencer, 1953; Drickamer, 1972; Millman, 1968). Some investigators have required subjects to dig through sand, sawdust, or wood-chip bedding towards an odor source, using, for example, the time required to locate the stimulus as the dependent measure (e.g., Heth et al., 1992; Howard et al., 1969; Swann, 1933). Studies employing discrete one-trial choices between portions of a test apparatus containing experimental odors (e.g., Y-or T-maze arms) commonly assess the proportion of subjects choosing the appropriate section (e.g., Godfrey, 1958; Huck and Banks, 1984; Leone and Moltz, 1971; Thiessen et al., 1971). When duration of investigation is the dependent measure, such times are either recorded on stopwatches or via photoelectric cells or mechanical tripping devices connected to electrical recording systems or computers (Additional examples of early studies to employ this paradigm include Baran and Glickman, 1970; Beach and Gilmore, 1949; Beauchamp, 1973; Carr et al., 1965, 1970; Doty, 1971, 1972; Doty and Anisko, 1973; Doty and Dunbar, 1974; Doty et al., 1971; Godfrey, 1958; LeMagnen, 1952; Lydell and Doty, 1972; Mainardi et al., 1965; Marr and Gardner, 1965; Moore, 1965; O’Connell and Meredith,

1984; Pfaff and Pfaffman, 1969; Smith, 1965; Stern, 1970; and Thiessen et al., 1971.*) The approach paradigm has a number of advantages. First, it is easy to set up and can be placed within the subject’s home cage or home range in some instances, making it amenable to both laboratory and field situations. Second, several odorants can be tested simultaneously. Third, given its reliance upon simple exploratory or foraging behaviors, there is no need for elaborate training or shaping procedures (of either the subject or the technician) to produce the desired behavior. Unfortunately, this paradigm also has a number of potential disadvantages. First, depending upon the nature of the test situation, the possibility of odor diffusion and mixing cannot be ruled out, particularly where exhaust systems are absent or poorly planned, adding noise to the measurements. Second, this paradigm assumes that a positively preferred odor elicits marked approach behavior in the subject. This may not always be the case, particularly if the animal can stand and simply sniff an odor stream without approaching its source. Third, position biases or preferences, particularly in males, are common in such test situations (see Barnett and Spencer, 1953; Doty, 1971). Such preferences potentially limit repeated testing of the same subjects, require careful counterbalancing of the positions of the stimulus presentations, and may necessitate the use of a relatively large number of subjects to decrease error variance. Fourth, test situations which use the amount of food or water consumed as their dependent measure may confound olfactory and gustatory factors (e.g., some of the “odor” stimuli may become ingested and tasted), limiting the degree of generalization of their results to other situations. Such confounding may vary with the animal’s deprivation level. For example, the odor of a novel flower may elicit more investigation than that of food pellets in a satiated rat, whereas such a flower odor associated with food may be avoided by a hungry rat in favor of food pellets not so odorized or food pellets associated with a different odor. Imagine the odor of roses in your breakfast cereal at 7:00 a.m.! Fifth, this procedure does not allow accurate measurement of negative odor preferences or aversions. For example, both a neutral and a highly aversive odor may receive an initial exploratory sniffing bout by an animal and then receive no more attention during a test

*

These examples reflect studies from the first few decades of the widespread employment of this methodology and should not be viewed as inclusive or even representative of the hundreds of studies that have since used this paradigm.

Determining Odor Preferences in Nonhuman Mammals

session. A literal interpretation of the data would suggest, erroneously, that both odorants are equally attractive. A sixth limitation of this paradigm is related to the potential interaction between a subject’s activity level and the preference measure. During periods of high circadian running activity, for example, a preference observed during other periods may wash out, depending upon the type of test apparatus (Doty, 1971). III. FORCED APPROACH-AVOIDANCE PARADIGM Under the assumption that an animal is motivated to approach attractive odorants and avoid aversive ones, a simple test employing a relatively small two-chamber apparatus can be employed. Air or some other presumed neutral stimulus is typically present or contingent upon the subject’s presence in one chamber, whereas the experimental odorant is present or contingent upon the subject’s presence in the other chamber.* Attraction of the subject to the odorant is operationally defined as his or her spending more than 50% of a test period on the odor side, and avoidance is defined as spending less than 50% of the test period on that side. A preference measure between two odorants can be inferred indirectly by comparing the relative time spent with each odor when they are individually paired against a control alternative in separate tests. Experimental test situations that can be categorized as forced approachavoidance include those of Brown and Willner (1983), Carter (1972), Doty (1973), Rottman and Snowdown (1972), and Cornwell-Jones (1981). The major advantage of the forced approach-avoidance paradigm is that both aversive and attractive qualities of odorants can be determined. However, this procedure has many shortcomings. First, developing preference measures for a number of odorants becomes quite time consuming. Only one odorant can be tested at a time, and large numbers of subjects must be used to decrease error variance resulting from large individual differences in the preference measure. Second, since the animal must be on one side of the apparatus or the other, his or her position is constantly being recorded during the test session, even though it is unlikely that he is attending to the environmental

*Some investigators employ two odors, rather than an odor and a blank, in this paradigm (e.g., Brown and Willner, 1983). This is usually done so that all pairs of a set of odorants can be tested against one another. Although this does allow for the development of a preference hierarchy, it does not allow for a delineation of the degree to which a given finding is due to attractive or aversive qualities of the odorants involved.

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odors throughout the entire period. Third, it is conceivable that a preference measure inferred from this paradigm is not the same as a preference measure obtained from paradigms in which more direct comparisons of two odor stimuli are possible (Doty, 1973). Although few data are available that directly test this point, results from a study using a bar-press stimulus presentation paradigm suggest this possibility. Tapp and Long (1971) found that a hierarchy of odor preferences obtained by giving a rat a choice between a lever producing a puff of air and one producing a puff of odorized air correlated only 0.02 with a hierarchy obtained from a paired-comparison two-lever situation. Research is needed to determine if similar preferences are obtained from the approach and forced approach-avoidance paradigms. Fourth, this paradigm requires the testing of subjects in a test chamber independent of the home cage, where competing cues are not present. Finally, a number of the shortcomings of the approach paradigm are also present in the forced approach-avoidance situation. For example, circadian activity and position preferences within the test apparatus conceivably influence the dependent measure.

IV. BAR-PRESS STIMULUS PRESENTATION PARADIGM A number of studies from a single laboratory have used a test paradigm in which a puff of odorized or nonodorized air is made contingent upon a bar press in a two-lever modified Skinner box (e.g., Long and Stein, 1969; Long and Tapp, 1967, 1970; Tapp and Long, 1968, 1971). Odors are subsequently exhausted from the test apparatus by means of a flow-through exhaust system. This test situation differs from the approach paradigm primarily in its brief presentation of the odorant (usually 1-second puff/bar press) and in its use of an extremely objective and measurable dependent variable. This paradigm has a number of positive features. First, various parameters of the stimulus can be independently manipulated with maximal precision and minimal difficulty (e.g., duration of odor puff, concentration of odorant, schedule of reinforcement, magnitude of energy expenditure required to produce an odor puff). Second, the use of bar presses as a dependent variable allows the reinforcing properties of various odorants to be determined using standard operant conditioning equipment. Third, response rates elicited in food deprived rats for odors of powdered food (see, e.g., Long and Tapp, 1967) are quite high in comparison to response rates elicited in nondeprived ones, suggesting the sensitivity of this measure to biological need states of the organism. Fourth, since the subject must

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perform a discrete behavior in order to smell an odorant, bypassing of the recording system is not possible, as can occur in the approach paradigm. Fifth, a number of odorants can be presented simultaneously in this test situation by simply adding additional levers. Sixth, a measure of odor aversion could be determined, at least theoretically, by requiring a subject to push a lever to turn off an odorant. Major drawbacks of this paradigm include: (1) its reliance upon complex mechanical and electrical components; (2) potential difficulties involved in adequately cleaning odorants from the valves and tubes after their use; (3) potential influences of position preferences and activity levels upon the subjects’ responses; and (4) the necessity of shaping animals to press the bars in some test situations.

V.

an increase in running speed is an even more difficult task unless one can assume that running speed is related to the amount of odorant reaching the olfactory mucosa, an untested and somewhat counterintuitive notion in the context of a run down a short T-maze runway. Common sense would suggest that an odor (or lack of odor) that does not signify “frustration” or “impending illness” would be preferred to an odor that does. If we assume that running speed has no significant influence on olfactory function, then the finding that rats actually slow down in the presence of “nonreward” rather than “reward” odors would make speed inversely related to preference. Given, however, the fact that such measures are contaminated by freezing or other emotion-related responses, it is difficult to see how this specific paradigm would be generally useful in establishing odor preferences.

ODOR TRAIL PARADIGM

Many mammals are capable of detecting subtle differences in odor trails laid down by conspecifics and preferentially follow one trail over another (e.g., Eibl-Eibesfeldt, 1970; Ewer, 1968). According to our earlier definition of a preference, the trail that is followed is in effect preferred to the nonfollowed one. This paradigm differs from the ones mentioned earlier in terms of the unique presentation of the stimuli and the types of odorants typically tested. Douglas (1966) systematically examined various cues used by rats in producing “spontaneous alternation” in the T-maze and found that the rat’s tendency to avoid its own recently laid odor trail was a major determinant of the alternation. Klein and Brown (1969) found that rats made either anosmic or both anosmic and blind did not alternate at rates significantly above chance levels, whereas control and osmic blinded rats did alternate at such levels, supporting Douglas’s findings. These studies imply that the rats preferred earlier laid trails or clean flooring to recently laid ones in this test situation. Studies using straight alley runways indicate rats deposit a distinctive odor trail when their expectation of food reward has been recently thwarted or when they have encountered a cue related to impending illness (e.g., Batsell et al., 1990; Ludvigson and Sytsma, 1967; Morrison and Ludvigson, 1970; Seago et al., 1970). The deposited odor trails can be used as discriminative stimuli in learning situations and produce decrements in the running speed of animals passing over them. Although the tracking of one odor trail rather than another clearly meets our general definition of preference, the real preference decision was made at a choice point and the time spent on the trail can be indicative of a number of factors, including the length or integrity of the trail. Hence, calculations of preferences based upon time on trails is problematic. Attempting to define a preference in terms of

VI.

SNIFF-RATE ANALYSIS PARADIGM

Teichner (1966) and Teichner et al. (1967) developed a sophisticated electronic system to monitor and record the number, duration, and intensity of sniffs by unrestrained rats in an odor-controlled environment. The “repellency” and “attractiveness” of various concentrations of a number of different odorants were determined by comparing the amount of sniffing during a control period to that during a period when an odorant was present. Decreased sniffing was the measure of aversiveness and increased sniffing the measure of attractiveness. It was found that the less restriction to movement imposed upon their subjects, the more they sniffed. The sniff-rate analysis paradigm is one of the more interesting and underutilized preference test situations reported in the literature and, when used prudently, should provide important information about relative preferences to odorants. However, one must be careful in interpreting what changes in sniffing rates signify. Welker (1964), for example, reports that “mildly novel” auditory, olfactory, tactile or visual stimuli are capable of arousing a characteristic sniffing pattern in the rat and that removal of the olfactory bulbs has no marked effect on the average rate of sniffing or upon the integration of various behaviors related to sniffing. Furthermore, strong stimulation of any of the aforementioned modalities tended to inhibit sniffing in a number of subjects. Keeping these potential issues in mind, the sniff-rate analysis paradigm still appears to have many merits and potential applications in the development of odor preference hierarchies. VII.

CONCLUSIONS

There are many reasons for wanting to know an animal’s preference for one odorant over another. Aside from better

Determining Odor Preferences in Nonhuman Mammals

understanding animal behavior and establishing factors responsible for mediating odor communication among animals, knowledge gained from preference testing is critical in applied settings. For example, developing lures as well as repellants is critical to modern wildlife-management programs, and preference tests are essential in such development. Animal preference paradigms are key components of research within the pet food industry and, in some situations, even have applications in the development of model systems for human food preferences. Although the present review is not a comprehensive assessment of all olfactory preference studies reported in the literature, it does provide a framework that includes most odor preference test situations devised to date. ACKNOWLEDGMENTS The preparation of this chapter, an update of an earlier one, Doty (1975), was supported, in part, by Grants PO1 DC 00161, RO1 DC 04278, RO1 DC 02974, and RO1 AG 27496 from the National Institutes of Health, Bethesda, MD (R. L. Doty, principal investigator). REFERENCES Baran, D., and Glickman, S. E. (1970). “Territorial marking” in the Mongolian gerbil: A study of sensory control and function. J. Comp. Physiol. Psychol. 71:237–245. Barnett, S. A., and Spencer, M. M. (1953). Responses of wild rats to offensive smells and tastes. Br. J. Animal Behav. I:32–37. Barrows, W. M. (1907). The reactions of the pomice fly, Drosophila ampelophila Loew, to odorous substances. J. Exp. Zool. 4:515–537. Batsell, W. R. J., Ludvisgon, H. W., and Kunko, P. M. (1990). Odor from rats tasting a signal of illness. J. Exp. Psychol. Anim. Behav. Proc. 16:193–199. Beach, F. A., and Gilmore, R. W. (1949). Response of male dogs to urine from females in heat. J. Mammal. 30:391–392. Beauchamp, G. K. (1973). Attraction of male guinea pigs to conspecific urine. Physiol. Behav. 10:589–594. Binet, A., and Passey, J. (1895/1896). Contribution à l’étude de l’olfaciton chez le chien. C. R. 24ms Session, Assoc. Frac. Avance. Sci. ii, pp. 659–661. Bronson, F. H., and Caroom, D. (197.1). Preputial gland of the male mouse: Attractant function. J. Reprod. Fertil. 25:279–282. Brown, R. E., and Willner, J. A. (1983). Establishing an “affective scale” for odor preferences of infant rats. Behav. Neur. Biol. 38:251–260. Carr, W. J., Loeb, L. S., and Dissinger, M. E.(1965). Responses of rats to sex odors. J. Comp. Physiol. Psychol. 55:415–417. Carr, W. J., Krames, L., and Costanzo, D. J. (1970). Previous sexual experience and olfactory preference for novel versus

407 original sex partners in rats. J. Comp. Physiol. Psychol. 71:216–222. Carter, C. S. (1972). Effects of olfactory experience on the behavior of the guinea-pig (Cavia porcellus). Animal Behav. 20:54–60. Coleridge, G. (1920). Animal attractions and repulsions. Contemp. Rev. 117:539–545. Cornwell-Jones, C. A. (1981). Conspecific odor preferences of male albino rats are reversed by intracerebral 6-hydroxydopamine. Brain Res. 213:379–385. Doty, R. L. (1971). Homospecific and heterospecific odor preferences in sexually-naive Peromyscus maniculatus bairdi and Peromyscus leucopus noveboracensis. Unpublished doctoral dissertation, Michigan State University. Doty, R. L. (1972). Odor preferences of female Peromyscus maniculatus bairdi for male mouse odors of P. m. bairdi and P. leucopus noveboracensis as a function of estrous state. J. Comp. Physiol. Psychol. 81: 191–197. Doty, R. L. (1973). Reactions of deer mice (Peromyscus maniculatus) and white-footed mice (Peromyscus leucopus) to homospecific and heterospecific urine odors. J. Comp. Physiol. Psychol. 84:296–303. Doty, R. L., and Anisko, J. J. (1973). Procaine hydrochloride olfactory block eliminates mating behavior in the male golden hamster. Physiol. Behav. 10:395–397. Doty, R. L., and Dunbar, I. A. (1974). Attraction of Beagles to conspecific urine, vaginal, and anal sac secretion odors. Physiol. Behav. 35:729–731. Doty, R. L., Carter, C. S., and Clemens, L. G. (1971). Olfactory control of sexual behavior in the male and early-androgenized female hamster. Horm. Behav. 2:325–335. Douglas, R. J. (1966). Cues for spontaneous alternation. J. Comp. Physiol. Psychol. 62:171–183. Drinkamer, L. C. (1972). Experience and selection behavior in the food habits of Peromyscus: use of olfaction. Behaviour 41:269–287. Eibl-Eibesfeldt, I. (1970). Ethology: The Biology of Behavior. Holt, Rinehart and Winston, New York. Ewer, R. F. (1968). Ethology of Mammals. Plenum, New York. Gleason, K. K., and Reynierse, J. H. (1969). The behavioral significance of pheromones in vertebrates. Psychol. Bull. 71:58–73. Godfrey, J. (1958). The origin of sexual isolation between bank voles. Proc. R. Phys. Soc. Edinburgh 27:47–55. Goff, W. R. (1961). Measurement of absolute olfactory sensitivity in rats. Am. J. Psychol. 74:384–393. Hamilton, G. van T. (1907). An experimental study of an unusual type of reaction in a dog. J. Comp. Neurol. 17:329–341. Heath, G., Nevo, E., Ikan, R., Weinstein, V., Ravid, U., and Duncan, H. (1992). Differential olfactory perception of enantiomeric compounds by blind subterranean mole rats (Spalax ehrenbergi). Experientia 48:897–902. Howard, W. E., Palmateer, S. D., and Marsh, R. E. (1969). A body capacitor-olfactometer for squirrels and rats. J. Mammal. 50:771–776. Huck, U. W., and Banks, E. M. (1984). Social olfaction in male brown lemmings (Lemmus sibiricus
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408 collared lemings (Dicrostonyx groenlandicus): I. Discrimination of species, sex, and estrous condition. J. Comp. Psychol. 98:54–59. Jennings, H. S. (1906). Behaviour of the Lower Organisms. Columbia University Press, New York. Klein, D., and Brown, T. S. (1969). Exploratory behavior and spontaneous alternation in blind and anosmic rats. J. Comp. Physiol. Psychol. 68:107–111. LeMagnen, J. (1952). Les phenomenenes olfacto-sexuels chez le rat blanc. Arch. Sci. Physiol. 6:295–332. Leone, M., and Moltz, H. (1971). Maternal pheromone: discrimination by preweaning albino rats. Physiol. Behav. 7: 265–267. Long, C. J., and Stein, G. W. (1969). An analysis of the reinforcing properties of food odor. Can. J. Psychol. 23:212–218. Long, C. J., and Tapp, J. T. (1967). Reinforcing properties of odors for the albino rat. Psychonom. Sci. 7:17–18. Long, C. J., and Tapp, J. T. (1968). An apparatus for the assessment of the reinforcing properties of odors in small animals. J. Exp. Anal. Behav. 11:49–51. Long, C. J., and Tapp, J. T. (1970). Significance of olfactory tracts in mediating response to odors in the rat. J. Comp. Physiol. Psychol. 72:435–443. Ludvigson, H. W., and Sytsma, D. (1967). The sweet smell of success: apparent double alternation in the rat. Psychonom. Sci. 9:283–284. Lydell, K., and Doty, R. L. (1972). Male rat odor preferences for female urine as a function of sexual experience, urine age, and urine source. Horm. Behav. 3:205–212. Ludvigson, H. W., and Sytsma, D. (1967). The sweet smell of success: apparent double alternation in the rat. Psychonom. Sci. 9:283–284. Mainardi, D., Marsan, M., and Pasquali, A. (1965). Causation of sexual preferences of the house mouse. The behavior of mice reared by parents whose odor was artificially altered. Atti Soc. Ital. Sci. Naturali e del Museo Civico di Storia Naturae di Milano 104:325–338. Marr, J. N., and Gardner, L. E. (1965). Early olfactory experience and later social behavior in the rat: preference, sexual responsiveness, and care of young. J. Genet. Psychol. 107: 167–174. McIndoo, N. E. (1926). An insect olfactometer (to measure attraction to non-flowering plants). J. Econ. Entomol. 19:545–571. Millman, B. (1968). Odor preferences and aversions in the rat. Unpublished master’s thesis, University of Calgary, Canada. Moore, R. E. (1965). Olfactory discrimination as an isolating mechanism between Peromyscus maniculatus and Peromyscus polionotus. Am. Midland Naturalist 73: 85–100. Morrison, R. R., and Ludvigson, H. W. (1970). Discrimination by rats of conspecific odors of reward and nonreward. Science 167:904–905. Moulton, D. G. and Eayrs, J. T. (1960). Studies in olfactory acuity. II. Relative detectability of n-aliphatic alcohols by the rat. Q. J. Exp. Psychol. 12:99–109. O’Connell, R. J., and Meredith, M. (1984). Effects of volatile and nonvolatile chemical signals on male sex behaviors mediated

Doty by the main and accessory olfactory systems. Behav. Neurosci. 98:1083–1093. Pfaff, D, and Pfaffmann, C. (1969). Behavioral and electrophysiological response of male rats to female rat urine odors. In Olfaction and Taste: Proceedings of the Third International Symposium, C. Pfaffman (Ed.). Rockefeller University Press, New York, pp. 258–267. Ralls, K. (1971). Mammalian scent marking. Science 171: 443–449. Richter, C. P. (1936). Increased salt appetite in adrenalectomized rats. Am. J. Physiol. 115:115–161. Richter, C. P. (1939). Salt taste thresholds of normal and adrenalectomized rats. Endocrinology 24:367–371. Richter, C. P., and Campbell, K. H. (1940). Taste thresholds and taste preferences of rats for five common sugars. J. Nutr. 20: 31–46. Romanes, G. J. (1887). Experiments on the sense of smell in dogs. Nature 36:273–274. Rottman, S. J., and Snowdon, C. T. (1972). Demonstration and analysis of an alarm pheromone in mice. J. Comp. Physiol. Psychol. 81:483–490. Seago, J. D., Ludvigson, H. W., and Remley, N. R. (1970). Effects of anosmia on apparent double alternation in the rat. J. Comp. Physiol. Psychol. 71:435–442. Slotnick, B. M. (1990). Olfactory perception. In Comparative Perception. Vol. 1, Basic Mechanisms, M. A. Berkley, W. C. Stebbins (Eds.). John Wiley & Sons, New York, pp. 155–214. Smith, M. H. (1965). Behavioral discrimination shown by allopatric and sympatric males of Peromyscus eremicus and Peromyscus californicus between females of the same two species. Evolution 19:430–435. Stern, J. J. (1970). Responses of male rats to sex odors. Physiol. Behav. 5:519–524. Stevens, D. A. (1975). Laboratory methods for obtaining olfactory discrimination in rodents. In Methods in Olfactory Research, D. G. Moulton, A. Turk and J. W. Johnston, Jr. (Eds.). Academic Press, London, pp. 375–394. Swann, H. G. (1933). The function of the brain in olfaction. I. Olfactory discrimination and an apparatus for its test. J. Comp. Physiol. 15:229–241. Tapp, J. T., and Long, C. J. (1968). A comparison of the reinforcing properties of stimulus onset for several sense modalities. Can. J. Psychol. 22:449–455. Tapp, J. T., and Long, C. J. (1971). Olfactory preferences in rats. Unpublished manuscript, Vanderbilt University. Teichner, W. H. (1966). A method for studying olfaction in the unrestrained rat. J. Psychol. 63:291–297. Teichner, W. H., Price, L. M., and Nalwalk, T. (1967). Suprathreshold olfactory responses of the rat measured by sniffing. J. Psychol. 66:63–65. Thiessen, D. D., Lindzey, G., Blum, S. L., and Wallace, P. (1971). Social interaction and scent marking in the Mongolian gerbil (Meriones unguiculatus). Anim. Behav. 19:505–513. Uexküll, J. von, and Sarris, E. G. (1931). Das Duftfeld des Hundes (Hund und Eckstein). Z. Hundeforsch. 1 (3/4):55–68. Welker, W. I. (1964). Analysis of sniffing of the albino rat. Behaviour 22:223–244.

20 Olfactory Memory Aras Petrulis Georgia State University, Atlanta, Georgia, U.S.A.

Howard Eichenbaum Boston University, Boston, Massachusetts, USA

I.

olfactory nucleus, piriform cortex, olfactory tubercle, cortical amygdala, and the entorhinal cortex (ENT). The piriform cortex (PIR), the largest area and the one most extensively innervated by the OB, is heavily interconnected with other olfactory structures and provides the majority of olfactory input to the orbitofrontal cortex (OFC) both directly and indirectly via the mediodorsal thalamus (MDthal). PIR also has extensive bidirectional connections with ENT, which, in turn, provides the majority of cortical connections with the hippocampus. Not surprisingly, these interconnected components are all involved in olfactory memory, but each area appears to have different and unique functional properties in both animals and humans (Eichenbaum, 1997; Savic et al., 2000).

INTRODUCTION

A wealth of data exists on olfactory memory and its neural substrates in experimental animals and, increasingly, in humans. This review is an attempt to comprehensively survey this research within the domain of studies on learning and memory mediated by the main olfactory system in adult mammals. Even within these limitations, there is a voluminous literature from many different behavioral paradigms. To organize this review, we have subdivided the literature to highlight important differences in mnemonic and cognitive processes inherent in various memory tasks. We deal first with olfactory discrimination learning, a process in which odors are learned by association with positive and negative reinforcers. Next, we consider several paradigms that assess the recognition of odors. Third, we examine the existing literature on more complex forms of olfactory memory that involve the formation of associations between odors and other stimuli (including other odors). Lastly, we include evidence showing how olfactory learning is important for the social lives of animals. Our aim is to understand how information flows within the main olfactory system and its projection targets. The anatomy of this system is well known and has been reviewed extensively (e.g., Haberly, 1985; Shipley and Ennis, 1996) (see also Chapters 1–9). Briefly, olfactory receptor neurons project to the apical dendrites of mitral/tufted cells in the olfactory bulbs (OB). These projection neurons make reciprocal synapses with inhibitory granule cells, via their basal dendrites, and project mainly through the lateral olfactory tract (LOT) to the anterior

II.

DISCRIMINATION LEARNING

A.

Appetitive Conditioning of Odor Cues

1.

Behavior

Some of the earliest attempts to train animals to discriminate between odors have involved appetitive conditioning; that is, rewarding animals with water or food for performing some action (nose-poke, bar-press) after detecting particular odors but not after detecting other odorants (Slotnick, 1990) (see Chapter 18). This basic technique has persisted and has undergone refinements over time that have introduced better temporal and spatial control over odor stimulation (Nigrosh et al., 1975; Slotnick and Katz, 1974; Slotnick and Nigrosh, 1974). The use of computer-controlled olfactometers, in particular, led 409

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to substantial progress in understanding the behavioral and neural processes underlying olfactory memory and perception in nonhumans (Slotnick, 1990). Moreover, increased interest in studying olfactory discrimination and memory followed the realization that olfactory cues are highly salient stimuli for laboratory rodents and can be used to probe the cognitive and neural architecture of stimulus representation, learning, and memory (Davis and Eichenbaum, 1991; Slotnick, 1990). Rodents learn to discriminate odors more rapidly than auditory or visual cues (Nigrosh et al., 1975; Slotnick, 1984), even when extremely similar odors such as those of individual hetero- and conspecifics are used (Bowers and Alexander, 1967; Gheusi et al., 1997; Schellinck et al., 1991; Yamazaki et al., 1990). Furthermore, rats rapidly develop a learning set for odor discriminations, reflecting their acquisition of abstract rules or procedures, such as “win-stay, loose-shift” (Jennings and Keefer, 1969; Nigrosh et al., 1975; Slotnick, 1984; Slotnick and Katz, 1974). Although this interpretation has been questioned (Reid and Morris, 1992, 1993), subsequent research revealed that learning set formation is robust (Slotnick, 1994). Rats also develop reversal learning sets for odor discrimination (Nigrosh et al., 1975; O’Grady and Jennings, 1974). The strength and nature of odor discrimination learning has been explored using measures of positive savings in relearning discriminations and of negative savings in learning the reversal of a previously acquired discriminations. These assessments show that animals can remember many different pairs of odors for at least several weeks, indicating that olfactory memory is both quite large and highly resistant to interference and degradation [rats (Slotnick et al. 1991; Staubli et al., 1987a), squirrel monkeys (Laska and Hudson, 1993; Laska et al., 1996)]. Rats encode both the positively and the negatively reinforced odors within a discrimination, rather than simply ignoring one of the cues, and they appear to encode, like humans (see Chapter 10), multicomponent odors as unitary, “gestalt” stimuli and not as a collection of independent components. Also, rats quickly learn olfactory discriminations with intertrial intervals ranging from several seconds to 30 minutes, demonstrating that they form odor-reward associations even with long delays between each odor presentations (Lovelace and Slotnick, 1995). Olfactory discrimination has also been explored by training rats to differentiate electrical stimulation of distinct regions of the olfactory bulbs or lateral olfactory tract. Rats retain discriminations between “electrical odors” over long periods, form learning sets for them, and can discriminate stimulation of extremely close regions of the bulb (Mouly and Holley, 1986; Mouly et al., 1985; Roman et al., 1987).

Petrulis and Eichenbaum

2.

Lesion Studies

Before the mid-1970s, some studies failed to find deficits in olfactory discrimination tasks following substantial lesions to cortical and subcortical components or projections of the main olfactory system (Brown, 1963; Kimble and Zack, 1967; Lashley and Sperry, 1943; Schuckman et al., 1969; Swann, 1934, 1935), although other reports indicated deficits following lesions of temporal lobe in monkeys (Brown et al., 1963; Santibanez and Hamuy, 1957). More recent lesion studies, outlined below, have shed light on the differential roles of distinct components of this system. a. Olfactory Bulb and Cortex. The PIR is widely considered the olfactory system structure most likely to be critical for olfactory memory (Ambros-Ingerson et al., 1990; Haberly and Bower, 1989; Hasselmo et al., 1990; see also Davis and Eichenbaum 1991). PIR damage severely impairs discrimination of odor mixtures that share components and prevents development of an odor learning set (Staubli et al., 1987b). However, pretraining and use of simple odorants can alleviate these deficits (Staubli et al., 1987b; Zhang et al., 1998). Transection of the lateral olfactory tract sparing OB and anterior PIR impairs odor discrimination learning with brief, but not long, intertrial intervals (Slotnick, 1985; Slotnick and Berman, 1980; Slotnick and Risser, 1990; Slotnick and Schoonover, 1992; Thanos and Slotnick, 1997). In addition, olfactory information needed for discrimination performance can also reach the PIR via fibers running within the anterior commissure as well as the LOT (Bennett, 1968; Slotnick and Schoonover, 1992). This pattern of results suggests that the posterior PIR and ENT may be necessary for longer-term odor memories and that short-term memories can be maintained in anterior PIR. Furthermore, PIR may be necessary for discriminations between highly similar odorants, but is not critical for easy discriminations, which may be supported by other anterior olfactory structures such as the AON (Hamrick et al., 1993; Slotnick and Schoonover, 1992). In addition, posttraining inactivation of the OB impairs retention of discriminations of “electric odors,” suggesting a role for the OB in memory consolidation (Mouly et al., 1993). b. Hippocampus. Early studies reported no deficit in olfactory discrimination following hippocampal lesions (Allen, 1940; Swann, 1934; Kimble and Zack, 1967). In addition, several early studies reported that damage to septal nuclei and its connections to the hippocampus via the fornix, as well as damage to the hippocampus proper, can facilitate acquisition of odor discriminations (Carlson and

Olfactory Memory

Vallante, 1974; Schmajuk and Isaacson, 1984; Vom Saal et al., 1975). By contrast, lesions of the lateral entorhinal cortex (ENT) severely impair the acquisition of simultaneous olfactory discriminations acquired with long intertrial intervals and impaired retention of a discrimination learned one hour before (Staubli et al., 1984). However, ENT lesions do not affect preoperatively acquired odor discriminations, suggesting this area is not the critical site of memory storage (Staubli et al., 1986). Spared, impaired, or facilitated performance on olfactory discrimination following hippocampal system damage depends on whether odors are presented in a way that facilitates or hinders learning relationships between them. Rats with damage to the hippocampal system, via fornix transection (FNX) or ENT lesions, acquire odor discriminations and learning set for the task as quickly as normal animals if the odors are presented successively in a go/no-go task (hold nose in odor port for rewarded odors, withdraw for unrewarded odors) (Eichenbaum et al., 1986, 1988; Otto et al., 1991b). By contrast, animals with FNX and ENT damage perform poorly when two odors are presented simultaneously and the animal is rewarded for nose-poking into the source of the positive odor (Eichenbaum et al., 1988; Otto and Garruto, 1997), although they can learn some discriminations by treating the odors as a compound stimulus (Eichenbaum et al., 1989). It has been argued that this task requires not only that animals remember individual odor cues and their valences but also encourages the animal to learn the relationship between simultaneously presented odors. Shortterm maintenance of memory for individual odors does not depend on processing by either the hippocampus or ENT (Eichenbaum et al., 1994). However, maintenance of odor memories for longer periods does seem to require the posterior PIR and/or the ENT (Thanos and Slotnick, 1997). c. Orbitofrontal Cortex and Mediodorsal Thalamic System. Lesions of the OFC and the MDthal result in a pattern of deficits different from that observed following hippocampal damage. Like hippocampal or ENT lesions, damage to the OFC and MDthal does not impair basic olfactory perception, in that detection ability and odor thresholds are similar to sham animals. In contrast, rats with either OFC or MDthal lesions display severe deficits in go/no-go successive odor discrimination (Eichenbaum et al., 1980). Furthermore the impairment could be attributed to increased perseveration of responses, a hallmark of prefrontal damage (Fuster, 1989; Kolb, 1984). In this case the deficit is selective to the olfactory domain, as OFC rats are not impaired in spatial alternation (Eichenbaum et al., 1983a).

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The deficits following MDthal ablations are less severe than those after OFC damage, although animals with MDthal damage are more impaired in discriminations of qualitatively similar and novel odors (Eichenbaum et al., 1980; but see Lu and Slotnick, 1990). In addition, using simultaneous presentations of odors in an olfactory maze, Staubli et al. (1987b) demonstrated that rats with MDthal lesions were severely impaired in initial learning, but partially recovered with extensive overtraining. Also, MDthal lesioned rats are severely impaired in odor reversal learning (Slotnick and Kaneko, 1981) and do not form learning sets during olfactory discrimination training (Lu and Slotnick, 1990). However, MDthal is not critical for retention of preoperatively learned odor discriminations (Slotnick and Risser, 1990). This combination of results suggests that the MDthal projections to OFC allow animals to rapidly organize odor-reward associations (McBride and Slotnick, 1997). d. Amygdala. Generally, lesions or disconnection of the amygdala do not impair olfactory discrimination learning in rats or rhesus monkeys (Eichenbaum et al., 1986; Schuckman et al., 1969; Slotnick, 1985). It is likely that the amygdala is not critically involved in memory formation that requires incremental learning involving small rewards. Instead, it may be primarily engaged when learning about odors with strong affective associations (see below). 3.

Cellular Correlates

a. Olfactory Bulb and Cortex. Some of the earliest evidence for physiological correlates of olfactory learning came from EEG recordings in the OB and PIR of rabbits. Stable changes in the amplitude and location of OB EEG bursts were observed following presentation of conditioned odors, suggesting the EEG primarily reflects the reinforcement history of odorants rather than their sensory qualities (Di Prisco and Freeman, 1985; Freeman, 1991; Freeman and Schneider, 1982; Grajski and Freeman, 1989). Also, large-scale changes in the synaptic dynamics between OB and PIR have been observed during sniffing to conditioned odors, but not to unreinforced odors (Bressler, 1988). These findings suggest the OB plays a dynamic role in the formation of odor memories. Other studies have examined changes in PIR synaptic physiology following odor discrimination learning. Roman et al. (1987) demonstrated that “electrical odor” discrimination learning leads to long-term potentiation (LTP) in PIR. Furthermore, population responses of PIR neurons to LOT stimulation increased with the number of successful discrimination trials but not if the animals were presented with familiar but unconditioned odors (Roman et al.,

412

1993a). Increased excitability of PIR neurons, as well as synaptic facilitation between PIR neurons, was also observed following odor discrimination training (Saar et al., 1998, 1999). Given the importance of the piriform cortex in olfactory perception and memory, it is surprising that only a handful of studies have evaluated PIR neuronal firing patterns during odor discrimination learning. McCollum et al. (1991) reported that most PIR neurons rapidly habituate to discriminated odors. Moreover, very few cells responded to more than two odors presented to the rats, indicating a sparse coding in the PIR (see also Schoenbaum and Eichenbaum, 1995a). Most interestingly, like the OB (Kay and Laurent, 1999), PIR neurons respond to a variety of discrimination task parameters that are not obviously olfactory in nature, including trial initiation cues and reward (Schoenbaum and Eichenbaum, 1995a). Some PIR neurons also fire associated with the acquired reward-valence of odors, with predictive relationships between odor cues when one odor occurred reliably prior to another, and with the expectation of reward. Thus, PIR neurons encode associations between nonolfactory and olfactory cues that underlie odor discrimination performance, suggesting PIR might be a locus of longterm odor memories. b. Orbitofrontal Cortex. OFC neurons fire in association with many events throughout all periods of an odor discrimination trial. Many OFC neurons are responsive to particular odors, but these responses are usually not specific to a single odor (Tanabe et al., 1975a), and most OFC cells fire more associated with the valence of particular odors and the consumption of water reward that with odor quality (Alvarez et al., 1999; Schoenbaum and Eichenbaum, 1995a). Like PIR, some OFC neurons also encode predictive relationships between odors and the expectation or anticipation of reward. However, ensemble activity in the OFC is predominantly correlated with “essential” trial information, such as odor identity and valence, and not with incidental information such as predictive odor-odor relationships (Schoenbaum and Eichenbaum, 1995b). During initial acquisition, OFC neuronal responses to odor identity and valence increase in selectivity with improved performance and continue to change, even in animals that have learned the task well (Alvarez et al., 1999). c. Hippocampus. During successive and simultaneous odor discrimination, learning neurons in the CA1 region encode the full array of task-relevant parameters (Eichenbaum et al., 1987; Wiener et al., 1989). In particular, many cells respond preferentially to presentation of

Petrulis and Eichenbaum

rewarded odors, irrespective of odor identity. In addition, hippocampal neurons responded to highly specific combinations of odor and location and in association with particular odor pairs or sequences, supporting the idea that the hippocampus is primarily involved in processing the relationship between various stimuli (Eichenbaum et al., 1994). During the acquisition of odor discriminations, the hippocampal theta rhythm synchronizes with sniffing (Macrides et al., 1982). Also, hippocampal neurons tend to fire in high-frequency bursts in phase with the ongoing theta rhythm, suggesting a link between conditions optimal for synaptic plasticity and periods of odor stimulus evaluation and learning (Otto et al., 1991a). For example, discrimination of “electrical odors” results in the enhanced excitability of ENT inputs to the dentate gyrus (DG) (Chaillan et al., 1996). Enhancement of the DG field potential emerges early in training and the degree of potentiation is correlated with discrimination performance (Chaillan et al., 1999). These findings suggest that plasticity at the first steps of hippocampal processing occurs when the animal is learning the significance of stimuli. d. Interactions Between Systems. Using expression of the c-fos gene as a marker for neuronal activation, Hess et al. (1995a,b, 1997) have shown that different areas of the brain are activated during distinct stages of olfactory discrimination learning. During exploration of the olfactory maze prior to training and during the learning of the operant task (nose-poking), all divisions of the hippocampus, as well as PIR, the granule cell layers of the OB, and the anterior medial amygdala rats were activated. The basolateral amygdala was also activated during discrimination learning, suggesting that this area is engaged, even if its role is not necessary to odor discrimination (Eichenbaum et al., 1986; Slotnick, 1985). During initial learning, CA3 is more activated than CA1, and the reverse was observed later, indicating a shift of processing within the hippocampus. Finally, with overtraining, PIR and the DG activity became correlated, suggesting a facilitation of information transfer between cortex and the hippocampus (Hess et al., 1995b).

4.

Biochemical Substrates

a. Glutamatergic System. Antagonists of glutamatergic N-methyl-D-aspartate (NMDA) receptor impair acquisition of discriminations between low-intensity odors presented at long intertrial intervals, suggesting that NMDA receptors are involved in persistent synaptic changes (Griesbach et al., 1998; Staubli et al., 1989). Also, drugs that increase fast (AMPA-mediated) glutamatergic conductance facilitate olfactory discrimination learning

Olfactory Memory

(Larson et al., 1995; Staubli et al., 1994b; see also Morris and Davis, 1994). Manipulation of glutamate receptors after learning are ineffective, suggesting a selective role in encoding odor memories (Staubli et al., 1994a, 1996; see also Miserendino et al., 1990; Morris et al., 1986). In addition, reduction of synaptic plasticity via inhibition of cellular protease also impairs acquisition of olfactory discriminations (Staubli et al., 1985). b. Cholinergic System. Acetylcholine (ACh) has been shown to modulate PIR synaptic physiology by increasing the excitability of pyramidal cells and suppressing intrinsic, but not afferent, connections of pyramidal cells (Hasselmo and Bower, 1993). Formal models suggest that increased ACh release puts PIR into an acquisition mode, whereas decreased ACh tone facilitates retrieval of patterns stored within pyramidal cell ensembles (Hasselmo and Bower, 1993). This modulation may be critical for allowing new odor representations to be incorporated into the network without massive interference between the new and old representations (Hasselmo, 1995, 1999). In support of this model, injections of ACh receptor antagonists preferentially impair a rat’s ability to discriminate odor pairs when one odor in the pair had a different reward history (De Rosa and Hasselmo, 2000). Conversely, increasing ACh activity facilitates discriminations between compound odors and their elements (Doty et al., 1999). The major source of ACh to the olfactory system is from the horizontal limb of the diagonal band (HLDB) (Linster and Hasselmo, 2000, Zaborszky et al., 1986). HLDB stimulation replicates the effects of application of ACh agonists on the dynamics of piriform cortex physiology, indicating that increased activity of HLDB is critical for putting the PIR into an acquisition mode (Linster et al., 1999). Furthermore, rats with HLBD lesions are impaired in acquiring an odor discrimination with long but not short intertrial intervals, and these animals rapidly forget odor discriminations (Roman et al., 1993b). c. Monoaminergic System. Noradrenergic (NE) release in the olfactory system has profound modulatory effects on both the OB and PIR. NE decreases intrinsic activity within the OB and PIR without greatly affecting afferent input (Hasselmo et al., 1997; Jiang et al., 1996). This may facilitate processing of weak odor stimuli by the OB (Jiang et al., 1996) and may prime the PIR for acquisition of olfactory information (Hasselmo et al., 1997). Perfusion of the OB with ß-adrenergic antagonists prevents the normal change in EEG patterns following exposure to conditioned odors during olfactory discrimination training (Gray et al., 1986). In addition, intrabulbar injections of NE antagonist can impair long-term retention of odors when

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administered during acquisition (Mouly et al., 1990). Injections of NE directly into the OB during discrimination training stimulates the EEG changes normally seen following exposure to novel odors and delayed EEG habituation to repeated presentations of unreinforced odors (Gray et al., 1986). Moreover, NE is released in the OB of mice performing an odor discrimination task (Brennan et al., 1998). These findings support the idea that NE is involved in odor learning (Gray et al., 1986) and may be important for odor memory consolidation (Sara et al., 1999). B.

Aversive Conditioning of Odor Cues

Learning about aversive situations must, at some level, require different neural circuits than those that underlie appetitive learning. These differences may be expected in pathways for reinforcement and behavioral output, as well as in the autonomic nervous system. At the same time, one might expect similarities of responses in the olfactory system itself. The early literature does suggest similarities in the neural systems underlying olfactory discrimination learning using aversive and appetitive reinforcers. For example, in studies of conditioned paw-lifting to odors following odor-shock pairing (Allen, 1937), lesions of the piriform cortex, amygdala, fornix, hippocampus, and neocortex did not impair odor conditioning, although both piriform and prefrontal cortex damage resulted in a lack of discrimination between odors (Allen, 1938, 1940, 1941). Rats with similar lesions could also acquire shock-motivated olfactory discriminations (Brown and Ghiselli, 1938). Moreover, only damage to various hypothalamic structures and the piriform cortex impaired retention of a shock-motivated odor discrimination in rats, whereas lesions to various neocortical structures, mediodorsal thalamus, hippocampus, amygdala, septum, and cerebellum had little effect (Thompson, 1980a,b,c). These findings, indicating that only damage to primary olfactory structures consistently impairs simple olfactory discriminations using aversive stimuli, are consistent with the results on olfactory learning using appetitive reinforcers. (Long and Tapp, 1970; Swann, 1935; Thanos and Slotnick, 1997). More recent studies have demonstrated that odors can serve as conditioned stimuli in Pavlovian fear-conditioning paradigms (Otto et al., 1997; Richardson et al., 1999) and that the neural substrates underlying performance on this task are somewhat different than those underlying performance on appetitive learning tasks. Otto et al. (1997) found that rats retain conditioned freezing to an odor for more than 2 weeks after repeated pairing with foot shock. Similarly, odor-shock pairings result in an increased startle response to a loud tone in the presence of the conditioned odor but not other odors (Richardson et al., 1999).

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As observed in visual or auditory fear-conditioning tasks (LeDoux, 1995), lesions of the basolateral amygdala (BLA) impairs freezing both to the conditioned olfactory cues and to shock, implicating the amygdala as a critical node in the expression of fear responses (Cousens and Otto, 1998). Rats with lesions of the anterior perirhinal cortex (PRC), a structure with substantial and reciprocal connections to the BLA and olfactory areas, also are impaired in olfactory fear conditioning (Herzog and Otto, 1997, 1998). By contrast, lesions of the BLA or the PRC do not impair odor discrimination using water reward (Eichenbaum et al., 1986; Otto et al., 1991b; Slotnick, 1985), suggesting differences in the neural substrates underlying appetitive and aversive olfactory memory. In a more direct comparison, rats with BLA lesions normally prefer an odor previously paired with a sucrose reward but do not avoid odors previously paired with footshock (Cahill and McGaugh, 1990), indicating a selective deficit in aversive odor learning. However, BLA lesions do not impair odor aversion induced by pairing with quinine, a mildly aversive stimulus, indicating that the BLA is involved in olfactory memory only when learning involves highly arousing stimuli. Consequently, the observed specificity of the amygdala in aversive olfactory conditioning may simply reflect the fact that few appetitive stimuli produce high levels of arousal, whereas aversive stimuli often do. Nevertheless, the differential involvement of PRC in aversive versus appetitive olfactory learning suggests that real differences may exist in how positive and negative valence is attributed to odor cues. Conditioned affective responses to odors may also involve a part of the caudate-putamen, because damage to the ventrolateral aspect of this area impairs acquisition of conditioned suppression of water licking when exposed to an odor previously paired with foot shock (Viaud and White, 1989). Processing of this conditioned emotional response involves dopaminergic activity in the ventrolateral neostriatum as posttraining injections of amphetamine or dopamine D2 receptor agonist into this area facilitate retention of the conditional response (Viaud and White, 1989; White and Viaud, 1991).

III. RECOGNITION MEMORY A critical feature of adaptive behavior in animals and humans is the ability to remember previously encountered stimuli over relatively long time intervals and to judge the relative familiarity or novelty of current percepts. The ability to recognize prior occurrence of individual stimuli has been well characterized at the behavioral and neural levels (Eichenbaum et al., 2000).

Petrulis and Eichenbaum

A.

Delayed Match and Nonmatch to Sample Tasks

1.

Behavior

Several simple recognition memory tests have been used to develop animal models of amnesia. Of these, the most successful and most widely adopted is the delayed nonmatching to sample test (DNMTS) (Eichenbaum et al., 2000; Gaffan, 1974; Mishkin, 1978). As first applied to monkeys, this test consists of a sample phase during which an animal is rewarded for displacing a novel complex object, then a variable delay period, then a test phase where the animal is rewarded for selecting a novel (nonmatching) object over the familiar one (Eichenbaum et al., 2000). Other studies have used a continuous recognition variant in which a series of stimuli are presented, and the animal must respond differentially to each stimulus depending on whether it is a match or nonmatch with the previous stimulus. Rats are able to rapidly acquire and perform odorguided DNMTS and match to sample (DMTS) tasks at levels of accuracy observed in monkeys on visually guided versions of the task, and performance is sensitive to similar parametric manipulations (Lu et al., 1993; Otto and Eichenbaum, 1992a). For example, both species show more forgetting if the delay between the sample and choice phases is increased (Koger and Mair, 1994; Otto and Eichenbaum, 1992a). Also, in both species performance decreases substantially when the same stimuli appear more frequently (Koger and Mair, 1994; Otto and Eichenbaum, 1992a). In both species, performance is sensitive to aging (Zyzak et al., 1995). 2.

Lesion Studies

a. Hippocampus. Selective lesions of the PRC and ENT of rats and monkeys dramatically impair object-cued DNMTS performance, whereas damage to the hippocampus or to FNX produces either no deficit or less severe and transient deficits (Mumby and Pinel, 1994; Mumby et al., 1992; Murray and Mishkin, 1998; Zola-Morgan et al., 1989). Similarly, hippocampal removal or FNX transection does not impair olfactory-guided DNMTS performance with memory delays of less than 15 minutes (Mair et al., 1998; Otto and Eichenbaum, 1992a; Sutherland et al., 1989) but may impair performance over longer delays (30–60 min) (Dudchenko et al., 2000). In contrast, rats with damage to the PRC and ENT are impaired at delays of 30 and 60 seconds (Otto and Eichenbaum, 1992a). These animals acquire the task normally and perform well at short delays (3 sec), indicating the PRC and ENT are selectively involved with maintaining the memory of the odor for extended periods.

Olfactory Memory

b. Orbitofrontal Cortex. Lesions of the OFC result in a pattern of deficits on DNMTS different from those following hippocampal system lesions. Unlike hippocampal or PRC-ENT lesions, OFC lesions severely impair DNMTS acquisition (Koger and Mair, 1994; Otto and Eichenbaum, 1992a). However, once the task is learned, OFC lesions do not affect memory, except in conditions of high stimulus interference and longer delays. MDthal lesions have minimal effects, indicating that the task-relevant information used by the OFC is not transmitted through its connections with the MDthal (Zhang et al., 1998).

c. Olfactory Cortex. Lesions of PIR impair retention and reacquisition on DNMTS irrespective of the memory delay and level of interference (Zhang et al., 1998). This suggests that short-term recognition of odors requires PIR processing. In addition, both systemic and intraolfactory bulb injections of ACh receptor antagonists impair DMTS performance at 30-second but not 4-second memory delays (Ravel et al., 1992, 1994). These results, along with other evidence (see below), indicate an important role for ACh in memory early in the central processing of olfactory cues. 3. Cellular Correlates Several studies have reported task-related neuronal activity in the hippocampus, PIR, ENT, and OFC of rats performing DNMTS tasks. Although neurons in all of these areas respond to virtually all aspects of task performance, differences exist in the proportion of cells encoding various aspects of the task, and some of these differ between versions of the task. For example, in a DNMTS task in which animals nose-poked into an odor port for reward, CA1 neurons in the hippocampus responded indiscriminately to all match/nonmatch decisions rather than to the odor identity on any given match/nonmatch episode (Otto and Eichenbaum, 1992b). In contrast, when rats performed the formally identical task in a rich spatial environment where digging in odorized sand for food was the operant response, hippocampal cells encoded not only the abstract match/match rule and positional information, but also odor identity (Wood et al., 1999). Wiebe and Staubli (1999) reported that CA1/CA3 hippocampal neurons also fire differentially to odors during the sample choice phases of the DNMTS task. Interestingly, neurons in different subfields of the hippocampus had distinctive firing patterns depending on the particular phase of their version of the task. Most strikingly, CA1 cells showed the greatest odor-selective activity during the sample phase, whereas dentate gyrus cells

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showed the preponderance of odor-selective firing during the choice phase. In addition, the odor-specificity of match/nonmatch neural responses decreased from the DG to CA3 to CA1, suggesting an increased level of abstraction through the trisynaptic circuit. Finally, although increased or decreased neuronal activity was observed across delays in hippocampal neurons, none of these responses was specific to any particular odor, suggesting that representations of specific odors are not maintained in the hippocampus. Neurons in the parahippocampal region (PRC and ENT) and OFC differ from hippocampal neurons in their responses associated with DNMTS events (Ramus and Eichenbaum, 2000; Young et al., 1997). In contrast to the hippocampal neurons, many cells in PRC-ENT and OFC are odor-selective during odor sampling and maintain this odor-selectivity during the delay period. In addition, unlike hippocampal neurons, PRC-ENT and OFC neurons fire in association with match/nonmatch status of specific odors. Differences are also apparent between PRC-ENT and OFC neurons. More OFC cells fire associated with the reward and to match/nonmatch features of the task than PRC-ENT cells. By contrast, more PRC-ENT cells show stimulusselective activity firing over delay periods (Ramus and Eichenbaum, 2000; Young et al., 1997). In general, OFC cellular responses appear to correlate with important task parameters, whereas PRC-ENT neurons maintain the representations of particular odor stimuli over delay periods.

B.

Juvenile Recognition

1.

Behavior

Recognition memory can also be demonstrated using ethologically relevant tasks that do not require extrinsic rewards. Thor and Holloway (1982) described a test of social memory that takes advantage of a rat’s inherent curiosity in novel conspecifics. In the initial study, adult male rats investigated a juvenile for several minutes whereupon, after a variable delay, the adult was presented with either the same juvenile or a novel juvenile. After delay intervals of 30 minutes or less, male rats investigated the familiar juvenile less than they had on the first trial, whereas males presented with a novel juvenile maintained a high rate of investigation. This phenomenon does not appear to be due to differences in behavior of novel and familiar juveniles, as juveniles are not able to remember the adult animal over delays exceeding 10 minutes (Thor and Holloway, 1982) and almost all social encounters are initiated by the adults (Gheusi et al., 1994). The ability to recognize a familiar juvenile over long delays is facilitated by repeated exposure to the familiar juvenile (Sekiguchi et

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al., 1991b) and can be impaired by repeated exposure to a novel juvenile during the delay period (Thor and Holloway, 1982), suggesting the memory is subject to interference. Female rats and mice are able to remember juveniles over longer delays (up to 2 hours) and, unlike male rats, do not appear to require the vasopressinergic system for modulation of this memory (Bluthe and Dantzer, 1990; Bluthe et al., 1993). Several lines of evidence indicate that juvenile recognition is mediated by olfactory cues. First, adults decrease their investigation of juveniles if their odors were presented on the initial trial (Sawyer et al., 1984). Second, adult investigation of juveniles is primarily centered on the ano-genital area (Gheusi et al., 1994), and removal of the preputial glands, a prominent source of chemical signals in rats, impairs recognition of juveniles (Popik et al., 1991a). Third, removal of the olfactory bulbs eliminates the reduction in investigation of familiar juveniles (Dantzer et al., 1990). Although juvenile recognition has been widely attributed to functions of the vomeronasal organ (VNO), removal of the VNO results in only transient impairments (Bluthe and Dantzer, 1993), whereas relatively selective peripheral olfactory lesions impair recognition (Popik et al., 1991a), indicating that the main olfactory system may be more important. 2. Biochemical Substrates a. Neuropeptides. The juvenile recognition paradigm has been widely adopted by behavioral pharmacologists as a fast and inexpensive assay for the mnemonic effects of various drugs and endogenous neurochemicals. Most studies have focused on the role of the neuropeptide arginine-vasopressin (AVP). Several lines of evidence support the idea that increased vasopressin release in the rodent septo-hippocampal system, after investigation of a juvenile, improves memory. Stimulating release of AVP or giving injections of AVP into the septum result in recognition of the familiar juvenile over long delays (Dantzer et al., 1988; Engelmann and Landgraf, 1994, 1995; Engelmann et al., 1994; LeMoal et al., 1987; Popik et al., 1991b; Sekiguchi et al., 1991a), whereas blockade of AVP action in the septum/hippocampus impairs recognition over short delay intervals (Dantzer et al., 1987; Landgraf et al., 1995; van Wimersma Greidanus and Maigret, 1996). It is unclear what processes are being affected by AVP that ultimately lead to improved memory. For example, many of the manipulations of the AVP system that modulate juvenile recognition also modulate fear and anxiety-like behaviors in nonsocial tests as well as aggressive behavior (Everts and Koolhaas, 1999; Koolhaas et al., 1990, 1998; Landgraf et al., 1995; Liebsch et al., 1996). However, evi-

Petrulis and Eichenbaum

dence exists that septo-hippocampal AVP may be more involved in juvenile recognition than in other hippocampal-dependent memory tasks (Engelmann et al., 1992, 1996). In addition, AVP may have effects on early stages of olfactory processing; injections of AVP directly into the olfactory bulbs (OB) improve memory, and this effect appears to be due to AVP modulation of NE activity, as depletion of bulbar NE eliminates this facilitatory effect (Dluzen et al., 1998, 2000). Other peptides, such as oxytocin (OXY) and cholecystokinin (CCK), also modulate juvenile recognition. For example, OXY release facilitates recognition, although this effect is more important for female than male rats, and appears to have effects on different neural structures than does AVP (Engelmann et al., 1998; Popik and van Ree, 1991; van Wimersma Greidanus and Maigret, 1996). CCK can either facilitate juvenile recognition by stimulating CCK A receptors in the periphery (via the vagus nerve) or impair memory for juveniles via CCK B receptors in the CNS (Lemaire et al., 1992, 1994a,b), suggesting multiple mechanisms for peptide action on mnemonic processes. b. Neurotransmitters. Modulation of more traditional neurotransmitter systems also can affect recognition of juveniles. Acute depletion of norepinephrine (NE) in the central nervous system (CNS) impairs juvenile recognition at 30-minute delays, whereas increasing NE release facilitates memory in the face of retroactive interference (Griffin and Taylor, 1995). However, increased NE release postinvestigation does not extend recognition after a long delay, suggesting that NE does not directly aid consolidation but may, instead, reduce interference from intervening stimuli (Griffin and Taylor, 1995). While the site of NE action on juvenile recognition is not well characterized, the amnestic effect of CNS-wide NE depletion cannot be attributed to effects on the OB, as selective depletion of NE in the OB does not impair recognition (Dluzen et al., 1998). Recognition can also be facilitated by dopaminergic activity in the nucleus accumbens (Ploeger et al., 1991). Similarly, increasing cholinergic (ACh) activity following investigation of juveniles or ovariectomized females facilitated memory over long delays, whereas blockade of muscarinic ACh receptors impaired memory over short delays if given directly after the encounter (Perio et al., 1989; Soffie and Lamberty, 1988; Winslow and Camacho, 1995). c. Genetic Manipulations. Unlike rats, grouphoused mice, but not isolated mice, are able to recognize juveniles after delays of 1–7 days. This long-term memory appears to involve protein synthesis as injections with

Olfactory Memory

protein synthesis inhibitors impair recognition over long delays (Kogan et al., 2000). One particular protein, cAMPresponsive element-binding protein (CREB), previously implicated in the cellular cascade surrounding hippocampal-dependent, nonsocial memory (Silva et al., 1998), appears to be involved in juvenile recognition as mutant mice with a specific knock-out of the gene that produces CREB are unable to show long-term memory for individual juveniles (Kogan et al., 2000). 3.

Lesions

Although it is clear that the septum is involved in juvenile recognition, the role of the hippocampus has been more difficult to evaluate. Indirect manipulations that damage the hippocampus such as ischemia and perforant path transections impair recognition of familiar juveniles at 30-minute delays (Andersen and Sams-Dodd, 1997; Lemaire et al., 1994a). Moreover, transection of FNX, but not lesions of the BLA, resulted in overall reduced investigation of juveniles and an apparent deficit in recognition, although damage to other limbic system pathways makes interpretation of hippocampal involvement problematic (Maaswinkel et al., 1996). Selective lesions of the hippocampus in mice impair recognition after a 30-minute delay, but not if recognition was tested immediately after the first exposure, implicating the hippocampus in this type of long-term recognition memory (Kogan et al., 2000). C.

Habituation/Discrimination

1.

Behavior

Habituation paradigms have also been used to investigate the kinds of information available in social odors and how these individualized odors are generated in several species (e.g., Halpin, 1986; Johnston et al., 1993, 1994; Schellinck et al., 1995). Although there are several variants on the task (Gregg and Thiessen, 1981; Johnston, 1993; Sundberg et al., 1982), in each animals are repeatedly presented with a particular odor from one individual and decrements in sniffing directed at the odor occur over repeated presentations. On a subsequent test trial, the same odor from a novel individual is presented either alone or opposed to the now-familiar odor, and increased or preferred sniffing of the novel odor is taken as a reflection of recognition of the familiar one. Hamsters and guinea pigs demonstrate recognition of social odors over delays of several seconds to several weeks (Beauchamp and Wellington, 1984; Johnston, 1993). This procedure taps into a general olfactory recognition process because animals are able to discriminate between individual odors of other species

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(Johnston and Robinson, 1993; Schellinck et al., 1995) and between artificial odors, although the magnitude of olfactory investigation is reduced (Hunter and Murray, 1989). 2.

Lesions

The physiological underpinnings of social odor recognition have not been well characterized. However, initial evidence indicates that social odor recognition shares neural substrates with other forms of recognition, such as those critical to DNMTS. Recognition of individual odors in female hamsters depends on the integrity of the olfactory, rather than vomeronasal, system (Petrulis et al., 1999a), and requires processing by the posterior parts of the main olfactory bulb projection zone (Petrulis et al., 1999b). Hamsters with lesions of OFC or medial amygdala (Petrulis and Johnston, 1999) showed no deficits in either habituation or discrimination (Petrulis et al., 1998), whereas lesions of the parahippocampal region selectively impaired recognition of a novel individual’s odor with little effect on recognition of the familiar odor (Petrulis et al., 2000). FNX transaction (Petrulis et al., 2000) and selective lesions of the hippocampus (Petrulis and Eichenbaurn, 2000) do not eliminate individual odor recognition in hamsters. Similar results have been observed in rats: lesions of the hippocampus or septum do not impair recognition of odors, whereas animals with parahippocampal region damage appear to have deficits in recognizing novel urine odors (Hunter and Murray, 1989; A. Petrulis and A. Armenakis, unpublished observations). Surprisingly, lesions of ENT in rats actually facilitate the memory for familiar, artificial odors, in that lesions allow rats to recognize familiar odors over longer delays than normal rats (Wirth et al., 1998). This result is difficult to reconcile with either the impairments on the DNMTS task using artificial odors after parahippocampal lesions (Otto and Eichenbaum, 1992a) or the deficits in recognition of social odors. 3.

Biochemical Substrates

A limited set of pharmacological manipulations suggests that the neurochemical substrates underlying habituation and discrimination of odors are similar to those involved in other tests of olfactory-guided recognition memory. For example, administration of scopolamine to rats, prior to testing, impairs habituation to artificial odors as well as preventing increased investigation of novel odors (Hunter and Murray, 1989). Scopolamine is ineffective in blocking recognition if given after repeated exposures to the familiar odor but before presentation of the novel odor, suggesting that the cholinergic system is necessary for encoding the odor rather than involved in retention or retrieval of the memory. Similarly, scopolamine injections in male mice

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impaired habituation to repeated presentations of ovariectomized females, whereas blockade of acetylcholinesterase facilitated habituation (Winslow and Camacho, 1995). These manipulations of the ACh system may selectively affect the OB or PIR-ENT, as lesions of the horizontal diagonal band, the major source of ACh in olfactory areas, eliminate habituation to artificial odors (Paolini and McKenzie, 1993), whereas lesions of the medial septum, the main source of ACh to the hippocampus, have little effect (Hunter and Murray, 1989). As in other olfactory memory paradigms, the NE system may be involved in social odor recognition. Depletion of NE in OB of rats did not greatly impair habituation to social odors, but after the first presentation of a novel social odor, animals failed to show reinvestigation of subsequent, novel urine odors (Guan et al., 1993). The lack of interest was not observed if animals were used as the familiar and novel stimuli, suggesting that, lacking a functioning olfactory system, recognition of individual animals may be achieved using nonolfactory cues.

cues, suggesting that spatial cues are sufficient. However, providing either visual or olfactory cues significantly increased correct food site identification. Olfactory cues were more salient than visual markers when these two cues were dissociated, suggesting that olfaction is the dominant modality for local detection of objects. Moreover, rats can use both environmental and self-generated odor cues to orient themselves in space if no illumination is available (Lavenex and Schenk, 1996, 1998). Rats are also able to associate odors sampled at a fixed location with rewards delivered at places differentiated by visual, tactile and positional properties (Youngentob et al., 1990, 1991). OFC may be one area where these cross-modal representations are formed. Using a task in which rats were trained to detect a unique odor at each of four locations, OFC neurons responded not only to specific odors or places, but also demonstrated odor-specific firing during arrival at the location where the odor was presented previously (Lipton et al., 1999). B.

Odor-Tactile Associations

4. Cellular Correlates The few studies that have investigated physiological correlates of olfactory habituation have recorded from anesthetized animals and often use extremely long odor exposures (Buonviso et al., 1998; Buonviso and Chaput, 2000), and so have limited direct comparisons with behavioral recognition. Nevertheless, recent studies by Wilson demonstrate that neurons in PIR show decrements in response to repeated presentations of the same odor and that this decrement occurs even though the OB is highly active (Wilson, 1998a). Intracellular recordings indicate that some of this reduction is due to reduced efficacy of synaptic potentials generated by OB afferent input to PIR (Wilson, 1998b). The habituation of PIR neurons appears specific to particular odors as presentation of highly similar or overlapping mixtures of odorants leads to cellular dishabituation (Wilson, 2000). This pattern of results strongly suggests that even highly similar odors are processed and stored as separate representations in piriform cortex. IV.

STIMULUS-STIMULUS ASSOCIATIONS

A.

Odor-Place Associations

Several paradigms have been used to assess how animals associate odors with places in the environment. Lavenex and Schenk (1995, 1998) trained rats where to find food hidden at various open field locations flagged either by odors or by visual cues or left unmarked. Latency to find the food was unaffected by eliminating olfactory or visual

Tomie and Whishaw developed a task in which rats were required to pull strings to obtain attached food rewards (Tomie and Whishaw, 1990). By using strings of different thickness and texture and painted with different odorants, they showed that rats could be trained to use particular configurations of string texture and odor to access food rewards (Tomie and Whishaw, 1990). Acquisition of these configural discriminations, but not simple discriminations, was impaired in animals with large hippocampal lesions (Whishaw and Tomie, 1991), although eventually other brain systems could support the association. In particular, large lesions of OFC, but not MDthal (Tomie and Whishaw, 1996), permanently impaired acquisition and retention of this task (Whishaw et al., 1992), suggesting an alternative pathway.

C.

Odor-Odor Associations

1.

Formal Learning Tasks

Several odor-odor association paradigms have been developed to investigate the role of the hippocampus in “declarative” memory in animals (Eichenbaum, 1997; Eichenbaum and Cohen, 2000). This kind of memory can be studied in nonlinguistic species by characterizing it as memory for relationships between memories that can be expressed “flexibly,” that is, in situations different than repetition of the learning event (Cohen and Eichenbaum, 1993; Eichenbaum, 1997).

Olfactory Memory

In humans, paired-associate learning depends on hippocampal function (Cohen and Eichenbaum, 1993). This task involves presenting a list of two-word (object, etc.) pairs, such as “hamster-dance, army-table,” and then, after some delay, presenting them with one word from each pair and requiring recall or recognition of the second, associated word. Using an olfactory variant of this task, Bunsey and Eichenbaum (1993) demonstrated that rats can learn to discriminate assigned pairings of odors from “mispairs” of the same odors and that lesions of the PRC-ENT block this capacity. In contrast, rats with selective lesions of the hippocampus are able to form odor-odor associations (Bunsey and Eichenbaum, 1996; Li et al., 1999). However, these associations are highly inflexible and are bound to the circumstances in which they are learned. Hippocampal rats are unable to infer a transitive relationship between overlapping odor pairs (e.g., A is associated with B and B is associated with C, therefore A is associated with C). And they do not demonstrate symmetry in their responses to odor pairs (A is associated with B, therefore B is associated with A). Taken together, these data suggest that the PRC-ENT, but not the hippocampus proper, is critical to form stimulus-stimulus associations and that the hippocampus is needed for a representation that allows flexible expression of these associations. Disconnection of the hippocampus, either by fornix transection or PRC-ENT lesions, also results in severe impairment in flexible memory expression in another odor-guided transitive inference task (Davis, 1992). In this study, rats were trained on a series of overlapping two-odor discriminations that could be organized to form a hierarchy (AB, BC, CD, DE, where “”
is selected over). If animals form the hierarchical representation, they should be able to judge between any two odors (especially BD), even though both of these odors had been equally rewarded during training. Whereas normal rats demonstrate this transitive inference, rats with hippocampal system damage do not, even though they learned the premise pairs and could discriminate the odor that was always rewarded from the one that was never rewarded (AE) (Dusek and Eichenbaum, 1997). Lesions of the hippocampal system also impair acquisition of an olfactory version of transverse patterning, a protocol that involves a circular organization of odors (AB, BC, CA) (Dusek and Eichenbaum, 1998). 2. Social Transmission of Food Preferences a. Behavior. One of the most critical decisions that animals have to make is which foods to eat and which foods to avoid. Although animals have ingestive mechanisms that reduce the risks of consuming toxic substances (Palmerino et al., 1980), social living animals can also

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learn the palatability of different substances through social interactions with conspecifics. The social transmission of diet preference has been studied experimentally in both domestic and wild rats and has resulted in fascinating insights into the information dynamics of animal social groups (Galef and Allen, 1995; Galef and White, 1997), adaptive foraging (Galef, 1993), and how odor-odor associations are formed naturally in animals (Galef and Wigmore, 1983). Three studies, published nearly simultaneously, reported that naïve rats (observers) show a substantial preference for the food that the demonstrator had eaten after an interaction with the demonstrator (Galef and Wigmore, 1983; Posadas-Andrews and Roper, 1983; Strupp and Levitsky, 1984). Exposure to the diet alone does not lead to an increased preference (Galef et al., 1985), and the critical cues appear to be olfactory: (1) preferences are not formed by anosmic animals or by animals separated by a Plexiglas screen (Galef and Wigmore, 1983); (2) rats can show a preference after investigating the snout of anesthetized demonstrators (Galef and Stein, 1985); and (3) diet preferences can be induced by pairing constituents of rat breath (e.g., carbon disulfide) with a particular diet odor (Galef et al., 1988). The memory can last for 4 weeks, depending on the number of interactions with demonstrators (Galef, 1989; Galef and Whiskin, 1998; Galef and Wigmore, 1983), does not suffer from interference between successive demonstrators (Galef, 1983), is insensitive to the level of food deprivation, social familiarity, age, sex, and health status of observers and/or demonstrators (Galef et al., 1984, 1991; Galef and Smith, 1994; Galef and Whiskin, 1998), and can reverse toxicosisinduced diet aversions (Galef, 1985, 1986b) and prevent formation of new food aversions (Galef, 1986a, 1987). Socially acquired diet preferences can be formed even to foods that are relatively unpalatable, protein-deficient, and require long handling times (Galef, 1986b; Galef and Whiskin, 1995). b. Lesions. This natural learning of associations between odors has proved to be an attractive paradigm for the study of the neural mechanisms underlying relational memory. As with other forms of relational memory (Eichenbaum, 1997), rats with lesions of the hippocampal formation or the parahippocampal region, but not MDthal, were able to learn the association but were not able to retain this information over 1–2 days (Alvarez et al., 2001; Bunsey and Eichenbaum, 1995; Winocur, 1990). One recent study did not replicate this observation but had to use four times the normal odor concentrations to obtain learning by normal rats, suggesting that a different kind of learning may have guided the observed preferences (Burton et al., 2000). Rats with hippocampal

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damage also showed amnesia for odor preferences learned a few days prior to surgery, indicating that the hippocampus is involved in consolidation of this association (Winocur, 1990). c. Biochemical Substrates. New molecular techniques have provided further support for the critical role of the hippocampus in this form of olfactory memory. Selective deletion of a subunit of the glutamatergic NMDA receptor, a key component of synaptic plasticity (Morris and Davis, 1994), in the CA1 region of the mouse hippocampus produced substantial deficits in long-term retention of socially-induced diet preference (Rampon et al., 2000). Manipulations of other biochemical systems involved in synaptic plasticity and learning have also lead to deficits in retention of diet preferences. Mutant mice lacking several forms of CREB, a substance previously implicated in synaptic plasticity and other forms of memory (Silva et al., 1998), demonstrated impairments in retaining the diet preference over 24 hours, but memory was normal on an immediate test (Kogan et al., 1997). In addition, several experiments have demonstrated a role for AVP in both recall and retention of socially acquired diet preferences. Injections of AVP facilitated recall when rats were tested at delays that result in no diet preference but showed impaired recall if tested on delays that animals will normally show preferences over (Strupp et al., 1990). Thus, AVP modulates memory retrieval depending on how well animals remember the stimulus associations and may play a role in consolidation (Popik and Van Ree, 1993). D.

Odor-Taste and Odor-Toxicosis Association

1. Behavior Strictly speaking, any form of discrimination learning in which odors are paired with water and food reinforcers includes, in part, odor-taste associations. However, since water and food reinforcers contain other types of stimuli, such as texture and physiological reactions to ingestion, and not just gustatory stimuli, the precise association formed during odor discrimination learning is not clear. Consequently, in this section we will be primarily concerned with the formation of associations between odors and well-defined taste cues. The vast majority of information about taste-odor associations comes from studies of taste-potentiated odor aversions (TPOA) and from variations on this paradigm. In this task, rats are allowed to drink a solution adulterated with both an odorant and a taste stimulus or with an odor presented in direct proximity to the taste solution. After a variable delay, rats are administered LiCl to induce gastric

distress. Thereafter, rats avoid drinking water in the presence of the taste or the odor alone. If presented with only an odor and then poisoned after delays of 15 minutes or longer, rats do not show aversion to that odor, whereas a rat presented with only a tastant will show robust avoidance of that taste even at delays of several hours (Durlach and Rescorla, 1980; Palmerino et al., 1980). Rats can show odor-toxicosis learning but only if the interval between consumption of the compound stimulus and toxicosis is sufficiently short (Durlach and Rescorla, 1980; Pain and Booth, 1968; Palmerino et al., 1980). Consequently, the potentiation of odor aversion by taste refers to the ability of taste in compound with odor to extend the duration of the sensory trace until it can be associated with toxicosis. The literature surrounding the psychological nature of this association has been contentious and filled with contradictory reports (e.g., Bouton and Whiting, 1982; Droungas and LoLordo, 1991; Durlach and Rescorla, 1980; Holder et al., 1987; Miller et al., 1986a; Palmerino et al., 1980). However, it now seems clear that taste does not have privileged access to learning about illness as previously postulated, but that odor, taste, or texture can support learning over long delays (Martin and Lawrence, 1979). In particular, strong odors in drinking solution that, by themselves, have no taste, potentiate aversion to tastes that are ineffective when conditioned alone (Darling and Slotnick, 1994; Slotnick et al., 1997). In spite of the controversy, much is known about neural mechanisms underlying odor-taste associations by using TPOA and related tasks. 2.

Amygdala System

Several lines of evidence point to the basolateral amygdala (BLA) as a critical convergence site between olfactory and gustatory information in TPOA. The BLA is one of the first regions that receive both olfactory and gustatory cues (Alheid et al., 1995). Temporary inactivation of the amygdala (Bermudez-Rattoni et al., 1983; Ferry et al., 1995), selective lesions of the BLA (Bermudez-Rattoni et al., 1986; Ferry et al., 1995; Hatfield et al., 1992), and catecholaminergic depletions of the amygdala (FernandezRuiz et al., 1993) all impair TPOA acquisition. Also, rats with injections of NMDA antagonists into the BLA are impaired in TPOA acquisition but are able to express previously learned TPOA (Hatfield and Gallagher, 1995). In all cases, rats demonstrated aversion to the taste stimulus alone, indicating that these manipulations interfere with the association between odor and taste, rather than impairing associations with toxicosis. Conversely, stimulation of BLA after ingestion facilitates odor-toxicosis learning, allowing for longer delays between odor and poisoning, suggesting that the BLA is involved in

Olfactory Memory

prolonging the olfactory memory until it can be paired with gastric distress (Ferry and DiScala, 1997). Lastly, in a task in which rats need to discriminate between one odor paired with an attractive taste stimulus and another paired with an aversive taste, neurons in the BLA anticipate the appearance of the positive or negative taste following odor sampling, and this selectivity emerges prior to the animal’s discriminative behavior (Schoenbaum et al., 1998). In addition, when BLA neurons fire during odor sampling, they appear to be primarily encoding the valence of the odor stimuli and not odor identity per se, as these neurons quickly reverse their selectivity to an odor when its reward value changes (Schoenbaum et al., 1999). Collectively, these results strongly suggest that the BLA is important for making odor-taste and odor-toxicosis associations and, by extension, is a region where odor valence, based on taste or visceral information, is generated. The mechanism of this association is not yet known, but it is likely that it involves activation of NMDA receptors, a type of glutamate receptor previously implicated in other forms of associative memory (Morris and Davis, 1994). 3.

Orbitofrontal Cortex System

The OFC and insular cortex compose another prominent region of convergence between odor and taste information (Rolls, 1997). Early studies suggested that the OFC-insular cortex is involved in odor-taste association (Tanabe et al., 1975a,b) and, more generally, in learning contingencies between odor and reward (Eichenbaum et al., 1980, 1983b). Using both conditioned taste aversion (CTA) and TPOA paradigms, Lasiter et al. (1985) defined the anterior insular cortex as being the most likely site of odor-taste association as lesions within this area selectively impaired TPOA. More recently, evidence for the integration of odor and taste in the OFC-insular cortex has been provided by the observation that removal of one OB and the contralateral ventrolateral OFC impairs the ability of rats to distinguish an olfactory-taste compound from its component features (Schul et al., 1996). Physiological evidence also suggests an intimate relationship between odors and taste in the OFC-insular cortex of rhesus monkeys and rats. During performance of discrimination tasks that demand the formation of associations between odors and attractive or aversive tastes, different populations of OFC neurons coded odor identity and odor valence (taste) in both rats and monkeys (Critchley and Rolls, 1996; Rolls et al., 1996b; Schoenbaum et al., 1998, 1999). OFC neurons showed selectivity in their firing during odor presentation only after the animal had learned the task, and few cells reversed this selectivity after the reward contingencies for

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the odors were changed (Rolls et al., 1996a; Schoenbaum et al., 1999). Surprisingly, correlated firing between cells in the OFC of rats increased after reversal, suggesting that the original odor-taste associations are still maintained within the network of OFC neurons (Schoenbaum et al., 2000). This pattern of results, when compared to the firing characteristics of BLA neurons, has been interpreted as showing that OFC is responsible for accessing information about the significance of an odor from BLA and then linking this representation with the appropriate behavioral output (Schoenbaum et al., 1999). 4.

Hippocampal System

The hippocampal system also plays a role in odor-taste association learning. Lesions of the hippocampus prevent the acquisition of both CTA and TPOA; this impairment is not attributable to deficient neophobia observed after these lesions (Miller et al., 1986b). Blockade of ACh receptors prior to aversion training enhanced the strength of TPOA, whereas increasing ACh activity in the hippocampus impaired the acquisition of TPOA and odor-toxicosis associations (Bermudez-Rattoni et al., 1987). Although hippocampal damage impairs TPOA, lesions of the ENT, which is the source of cortical input to the hippocampus, facilitate odor-toxicosis learning in that ENT-lesioned animals showed aversions even after long delays between odor and poisoning (Ferry et al., 1996). This facilitatory effect requires interactions of ENT with the BLA because inactivation of BLA in rats with ENT lesions prevents them from displaying odor-toxicosis learning with long delays between odor and poisoning (Ferry et al., 1999). 5.

Olfactory Bulb and Cortex

Lastly, using a task in which odors are paired in solution with either attractive or aversive tastants, Kay and Laurent (1999) demonstrated that mitral cell activity in the OB reflects the reward (taste) contingencies of odors. While modulation of OB activity by nonolfactory factors, such as hunger, satiety, or malaise, have been observed previously (Chaput and Holley, 1976; Pager et al., 1972; Pager and Royet, 1976), this report showed that the strongest predictor of mitral cell firing was not odor identity but odor valence and task-relevant behaviors, such as licking for water reward. For example, apparent odor-selective neurons changed their firing patterns when the taste with which it is paired was changed and some mitral cells even fired in anticipation of reinforcement (Kay and Laurent, 1999), much like BLA and OFC neurons (Schoenbaum et al., 1998, 1999). These results suggest that even the earliest stages of olfactory processing can be strongly influenced by prior experience and that odor memory may be

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highly distributed between primary olfactory areas such as the OB and PIR. This interpretation is in agreement with current theoretical and empirical work in other neural systems showing that sensory and motor memory can be encoded by the same neurons that initially process this information (Fuster, 1995).

Familiarity can either increase (Corridi et al., 1993) or decrease agonistic behavior (Daly, 1977; Halpin, 1976; Kimelman and Lubow, 1974). Conversely, aggressive interactions can alter attraction and/or preference for odors of familiar animals. For example, following agonistic interactions, male guinea pigs spend more time near the odors of animals that they had defeated than near the odors of the male that had dominated them (Martin and Beauchamp, 1982; see also Nyby et al., 1970). Similarly, rats that win agonistic encounters increase scent marking and olfactory investigation of odors from defeated males, more so than to unfamiliar animals (Brown, 1992). Moreover, the odors of familiar dominant rats potentiate freezing responses of defeated animals during presentation of shock, indicating that social odors can be readily conditioned by the aversive experience of defeat (Williams and Scott, 1989; Williams et al., 1990). Although little is known about neural mechanisms underlying learning about odors within agonistic circumstances, the medial amygdala (MeA) may play a critical role in this process. For example, MeA lesions in subordinate rats attenuated their defeat-induced behaviors, such as the reduction of olfactory investigation of dominant animals (Bolhuis et al., 1984; Luiten et al., 1985).

cues as animals preferred anesthetized novel animals or their odors over those of a prior mate (Carr et al., 1970, 1979, 1980; Huck et al., 1984; Johnston and Rasmussen, 1984). Furthermore, destruction of the olfactory epithelium, but not vomeronasal organ removal, eliminates preferences of male hamsters for novel females (Johnston and Rasmussen, 1984). Although the neural circuitry underlying this form of olfactory recognition remains to be identified, it may involve increased dopamine release in the nucleus accumbens (Fiorino et al., 1997) as well as processing by PRC-ENT, but does not require an intact hippocampus (Petrulis and Eichenbaum, 2000). Olfactory-based, long-term recognition of mates has been observed in pair-bonding species such as prairie voles (Newman and Halpin, 1988) and Djungarian hamsters (Vasilieva and Sokolov, 1994), as well as solitary species such as collared lemmings (Huck and Banks, 1979). Much work in prairie voles has shown that pair-bonding develops either by long-term cohabitation or, more usually, following mating and depends on increased activity of the vasopressin and oxytocin systems in the brain (Carter et al., 1995; Young et al., 1998). Recently, a role for dopamine in pair formation has been described following the observation that dopamine antagonists given before or immediately after mating impair pair-bonding (Wang et al., 1999). This effect of dopamine (DA) appears to be related to consolidation and not sensorimotor processing, as antagonist given 24 hours after mating did not impair mate recognition. Dopamine may be acting through the nucleus accumbens (NAcc) as injections of dopamine D-2 antagonists into this region impaired pair-bonding whereas D-2 agonists facilitated pair formation (Gingrich et al., 2000). Evidence from both lesion and c-fos activation experiments have implicated other neural structures, such as the cortico-medial amygdala and BLA, in the pair-bonding process (Demas et al., 1997; Kirkpatrick et al., 1994a; Wang et al., 1997). Although formation of pair-bonds also requires a functioning vomeronasal/olfactory system (Kirkpatrick et al., 1994b; Williams et al., 1992), there is little direct evidence to link changes in oxytocin, vasopressin, or other neurochemical systems to the olfactory memory underlying long-term attachment.

B.

C.

V.

SOCIAL BEHAVIOR AND ODOR LEARNING

Recognition of individual identity can alter interactions between animals in many contexts and is mediated, to a great extent, by odors in several macrosmatic taxa (Brown and Macdonald, 1985) (see Section III. C. and Chapters 15 and 17). A.

Aggressive Behavior

Mate Recognition and Pair-Bonding

Olfactory memory is also important for mate recognition in both polygamous and monogamous pair-bonding species. In species that do not form long-term social attachments, such as rats and hamsters, both males and females increase preference for novel mates shortly following copulation with another conspecific. This “Coolidge effect” appears to be dependent on olfactory

Sexual Behavior

Sexual behavior in animals is often characterized as a stereotyped and rigid process that is impermeable to learning. However, the available evidence suggests that adaptive expression of courtship and copulatory behavior may require the formation of associations between the odors of conspecifics and copulatory stimuli. For example, male mice produce ultrasonic vocalizations (USV) as part of

Olfactory Memory

their precopulatory behavior and when they encounter urinary cues produced by females (Nyby and Whitney, 1980). If males are not allowed to interact with females, the number of USVs in response to female urine declines over time; this decrease is reversed by brief exposures to females (Dizinno et al., 1978; Nyby and Whitney, 1980). The maintenance of male USV depends on the formation of associations between odor cues emitted by the female and some aspect of their interaction with them. Males interacting with artificially perfumed females will later produce USVs in response to the perfume when presented alone (Kerchner et al., 1986; Nyby et al., 1978). Similarly, the hormonal response of male rats to sexually receptive females can be conditioned to neutral odors by pairing these odors with female presentation (Graham and Desjardins, 1980). Moreover, pairing neutral odors with access to receptive females also results in increased number of ejaculations with females scented with this odor (Kippin et al., 1998). The nucleus accumbens, a region long implicated in reward learning (Robbins and Everitt, 1996), may be important for the association of odor with the rewarding stimuli of sociosexual interaction. In anesthetized rats, the presentation of odors previously paired with copulation elicited greater neuronal activity in this area than unpaired odors or odors paired with unreceptive females (West et al., 1992). D.

Maternal Behavior

Olfactory learning is also important during maternal behavior (Fleming and Korsmit, 1997; Lee et al., 1999). One particularly good example of this is the formation of a selective bond between a recently parturient sheep and their offspring. After birth, the ewe recognizes her mobile offspring and selectively feeds it while repulsing other lambs. The formation of the bond is rapid and limited to a short time after birth, such that an alien lamb can be fostered successfully during this period and the ewe’s own lamb will be rejected if the ewe has not interacted with it during this time (Poindron and Le Neindre, 1980). Altering the olfactory cues originating from the lamb or removing main olfactory input in the ewe impairs bond formation, implicating the olfactory system in this imprinting phenomenon (Levy et al., 1994; Poindron and Le Neindre, 1980). The critical stimulus that precipitates maternal behavior and bonding is the stimulation of the cervix and vaginal tract during delivery, as nonpregnant ewes can be induced to show selective bonding with lambs by artificial vaginocervical stimulation (VGS) (Kendrick et al., 1991; Keverne et al., 1983). The effect of VGS on olfactory imprinting appears to be due, in part, to its influence on both intrinsic and neuromodulatory systems of the main

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olfactory bulbs (OB). For example, birth or VGS leads to a release of centrifugal pathway neurotransmitters such as NE and ACh, as well as stimulating release of intrinsic neurotransmitters, such as GABA and glutamate (Kendrick et al., 1988a,b). The behavioral plasticity that underlies recognition of offspring is correlated with changes in the responsiveness of OB mitral cells to lamb odors. Mitral cells normally do not respond to lamb odors prior to parturition or after the close of the sensitive period for bonding, but they do show increased activity to lamb odors during the time when bonding would normally occur (Kendrick et al., 1992). More specifically, parturition results in changes in the dendrodendritic reciprocal synapses between mitral cells and the inhibitory granule cells in the OB. Levels of both glutamate (from mitral cells) and GABA (from granule cells) are increased by exposure to lamb odors, and proportionally more GABA is released in response to glutamate postpartum than before birth, suggesting that these synapses are altered by VGS associated with the birthing process (Kendrick et al., 1992). Nitric oxide (NO), a retrograde messenger molecule implicated in synaptic plasticity, is also produced in the OB during birth and appears to be required for olfactory memory formation (Kendrick et al., 1997). Inhibiting NO release in the OB during bonding blocks the formation of a selective bond but does not impair recall. NO is formed in the OB by glutamate acting via the NMDA receptor and appears responsible for the increased glutamate release from the mitral cells during exposure to lamb odor since blocking NO impairs this increase (Kendrick et al., 1997). The release of extrinsic NE and ACh into the OB associated with the period of selective bonding also appears to be a critical event for the acquisition of the olfactory memory. Lesioning the source of NE innervation of the OB prevents ewes from forming selective bonds with her own lambs (Pissonnier et al., 1985). Similarly, blockade of -adrenergic receptors in the OB during imprinting significantly impaired formation of the olfactory memory (Levy et al., 1990). Manipulating the ACh system also has profound consequences for memory formation. Injections of scopolamine around parturition as well as immediately after bond formation impaired the ability of ewes to bond with their own lamb, but did not impair recall of a previously learned odor memory (Ferreira et al., 1999; Levy et al., 1997a). However, it is likely that NE and ACh release in the OB is also involved in recalling or processing lamb odors, as ACh and NE are also released in response to lamb odor after bonding has already occurred (Kendrick et al., 1992). The precise role of ACh and NE in OB dynamics during imprinting remains obscure but may function to increase signal effi-

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cacy and/or to facilitate the storage of nonoverlapping memory representations (Jiang et al., 1996; Linster and Hasselmo, 1997). Although much is known about the pharmacology of olfactory memory formation in ewes, comparatively less is known about what brain systems, other than the OB, are involved in selective bonding. Based on immediate early gene expression, several olfactory structures are activated following parturition and exposure to lambs including PIR, ENT, OFC, and the dentate gyrus (DaCosta et al., 1997). Clearly more research is needed to identify the critical site(s) of plasticity responsible for the olfactory imprinting phenomenon in sheep.

VI.

HUMAN OLFACTORY MEMORY

Can the extensive body of knowledge on olfactory memory in experimental animals provide insight into the neural basis of olfactory memory in humans? Before considering this question, one must confront several problems. First, the complex and unavoidable relationship between linguistic and olfactory processes may result in different patterns of activation between animals and humans, making meaningful comparisons difficult (see Serby and Chobor, 1992). Second, the sensory world of humans, like many other Catarrhine primates, is dominated primarily by visual and auditory cues, and, not surprisingly, we devote much less brain space to olfaction than do most other mammals, including rodents. This raises the possibility that the mechanisms and areas that we study in animals are selective adaptations to macrosmatic life and, thus, may differ in complexity and function from odor processing in microsmatic humans. Lastly, observations on human olfactory memory are contradictory on even the most basic of issues and therefore lack the consensus needed for reasonable comparisons with animal research (for reviews, see Herz and Engen, 1996; Richardson and Zucco, 1989; White, 1998). Nonetheless, there are parallels between the neural mechanisms underlying human and animal olfactory memory. Neuropsychological evaluations of patients have shown that damage to olfactory structures results in impairments on tests of odor discrimination and memory. For example, damage to right OFC impairs odor-quality discrimination, common odor identification, and odor recognition memory (Jones-Gotman and Zatorre, 1988, 1993; Potter and Butters, 1980). In most cases OFC lesions do not greatly affect odor detection thresholds or discrimination of nonolfactory stimuli. Patients with Korsakoff’s syndrome have severe damage to the MDthal and perform quite poorly on odor discrimination tasks (Jones et al.,

1978; Mair et al., 1980; Potter and Butters, 1980). However, their deficit appears less selective than those observed in OFC patients, as several reports indicate altered olfactory thresholds, attentional deficits, and problems with nonolfactory tasks in Korsakoff’s patients (Jones et al., 1978; Mair et al., 1986; Potter and Butters, 1980). Deficits in odor identification and recognition memory are also observed after unilateral temporal lobe damage, with the greatest deficit seen after right side damage (Jones-Gotman and Zatorre, 1988; Rausch and Serafetinides, 1975; Rausch et al., 1977). Severe deficits in odor recognition were observed in patient H. M., who had bilateral resection of the medial temporal lobe. H. M. performed normally on odor detection or in odor intensity discrimination. However, he was completely unable to make same-different judgments using odor quality, match previously sampled odors to current odors, or identify common objects by smell (Eichenbaum et al., 1983b). At present, it is not clear what structures in the temporal lobe are critical for odor discrimination, but recent evidence points to damage in the piriform cortex located along the anterior medial temporal lobe (Jones-Gotman et al., 1997). Results from functional imaging of human brain activity during olfactory stimulation have been largely congruent with the neuropsychological literature by showing that the OFC, ENT, insular and piriform cortex are all activated in response to odor stimulation (Kettenmann et al., 1997; Levy et al., 1997b; Savic et al., 2000; Sobel et al., 1998a, 2000) (see Chapter 12). Odor exposure also activates structures more remotely connected to the olfactory system, such as the cerebellum and the cingulate cortex, with at least some of this activation related to the control of sniffing behavior (Levy et al., 1997b; Savic et al., 2000; Sobel et al., 1998b). Several recent studies have explicitly assessed the brain areas involved in olfactory memory using functional imaging. Royet et al. (1999) observed increased activation of the right OFC, as well as in other frontal and cingulate regions, during judgments of odor familiarity (Royet et al., 1999). Savic et al. (2000) observed that discrimination of odor quality specifically activates the hippocampal formation, as well as the caudate, but these areas are not activated during recognition of an odor learned one hour previously. In contrast, during recognition large areas of association cortex, including OFC and PIR, showed increased activity. This widespread activation of temporal and parietal cortex may reflect the reactivation of representations of nonolfactory stimuli that invariably surrounded the initial encoding of odor stimuli. Finally, the amygdala is activated primarily to unpleasant and presumably highly arousing odors (Zald and Pardo, 1997). Taken together, the observations from neuropsychological

Olfactory Memory

and brain imaging studies on humans is largely consistent with the descriptions of the neuroanatomical substrates that underlie olfactory memory in animals. Across species, widespread areas of the old “rhinencephalon” participate in odor processing in characteristic patterns associated with particular types of olfactory memory.

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Petrulis and Eichenbaum, 2000; Petrulis et al., 2000). Neither of these structures appears to be the final site of memory storage, a view consistent with the literature on the role of the hippocampal system in nonolfactory memory (Cohen and Eichenbaum, 1993). 2.

VII.

CONCLUSIONS

Although a clear and comprehensive synthesis of this field is not yet possible, several themes emerge from this review. First, olfactory memory is not a unitary phenomenon. Instead, it a highly distributed process that involves the activation of a distinct set of pathways when particular cognitive demands are placed on the animal. In correspondence with this view, damage to different parts of olfactory system and its projections selectively disrupts a particular kind of odor memory, or shifts the strategies used in learning the task (Eichenbaum and Cohen, 2000). Second, although it has been known for some time that structures within the olfactory system are highly interconnected, recent evidence has provided insights into the functional consequences of reentrant connections within the olfactory system. Lastly, modeling and empirical studies of ACh and NE effects on the olfactory system are beginning to provide an understanding of neuromodulatory systems that regulate the dynamics of memory formation. In particular, research on the neurochemistry underlying juvenile odor recognition has provided hitherto unsuspected insights into the complex nature of consolidation of olfactory information. We consider each of these topics in more detail below. A.

Multiple Memory Systems

1.

Hippocampal System

The observations from both lesion and electrophysiological studies indicate that different aspects of olfactory memory require or engage distinct brain pathways depending on the required cognitive operations. The hippocampus is involved in situations that require animals to rapidly learn the relationships between odor stimuli, or between odors and other cues, and then express this knowledge in situations different from those of acquisition (Eichenbaum, 1997; Eichenbaum et al., 1994, 1999). In contrast, the hippocampus appears not to be critical for incremental learning of odor valences or for recognition of particular odors as familiar or unfamiliar (Eichenbaum et al., 1986; Petrulis and Eichenbaum, 2000; Petrulis et al., 2000). Instead, the ability to retain memories of individual odors requires the parahippocampal region (Otto and Eichenbaum, 1992a;

Orbitofrontal Cortex System

The OFC and the associated mediodorsal thalamus are involved in learning the “rules” that must be applied in different learning tasks. This conceptualization incorporates the observation that animals with OFC or MDthal damage have difficulty acquiring odor discrimination and discrimination reversal learning sets and learning the match/nonmatch rule in DNMTS (Eichenbaum et al., 1980, 1983a; McBride and Slotnick, 1997). Also, OFC is involved in representing associations between odors and other stimuli, particularly those with intrinsic valence, such as taste, nociception, and visceral sensations, and the ability to alter behavior associated with changes in stimulus-reinforcer associations (Rolls, 2000). The OFC does not, however, seem to be a “higher” olfactory center in the sense of supporting more complex discriminations independent of reward (Petrulis et al., 1998). 3.

Amygdala System

The amygdala is also involved in some aspects of olfactory memory formation. Neurons in the basolateral amygdala are primarily active during the initial stages of odor-reward learning (Hess et al., 1997; Schoenbaum et al., 1998, 1999, 2000). Paradoxically, lesions of this area have no effect on the acquisition of odor-reward associations (Eichenbaum et al., 1986; Slotnick, 1985). This discrepancy is probably due to differences in methodology and on the strength and nature of the reinforcers used between studies and remains to be fully elucidated. However, studies of odor-guided fear conditioning suggest requisite amygdala involvement only when there is high motivational content (Otto et al., 2000) or a highly arousing context (Cahill and McGaugh, 1990). 4.

Olfactory Bulb and Cortex

Several models of the piriform cortex and olfactory bulb suggest that the connectional architecture of these areas is sufficient to store self-organized odor memories (Ambros-Ingerson et al., 1990; Haberly and Bower, 1989; Hasselmo et al., 1990). Neural plasticity associated with learning or learning-like contingencies has been demonstrated in PIR (Kanter and Haberly, 1990; Roman et al., 1993a; Saar et al., 1999). Electrophysiological data are consistent with the idea that olfactory memories are

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stored in a sparse and distributed manner within the piriform cortex and the olfactory bulbs (Freeman, 1991; McCollum et al., 1991; Schoenbaum and Eichenbaum, 1995a; Wilson, 2000). Unfortunately, this type of arrangement makes it difficult to interpret the effects of PIR lesions on olfactory memory. Nevertheless, lesions of PIR can selectively impair odor memory formation without rendering the animal anosmic (Slotnick and Schoonover, 1992; Zhang et al., 1998). Ironically, we know much less about the role of PIR in olfactory behavior and memory than about other regions receiving secondary and tertiary olfactory projections. Surprisingly, in some situations memory-related plasticity can be observed in olfactory receptor neurons. For example, in mice with congenitally low sensitivity to particular odorants, repeated exposure can increase responsiveness of the main olfactory epithelium to these odors (Wang et al., 1993). More dramatically, exposure to artificial odors later used for homing increases sensitivity in the olfactory receptor neurons of salmon (Dittman et al., 1997; Hasler et al, 1978; Nevitt et al., 1994), and this odor memory may be triggered by hormonal changes when salmon are learning about their natal stream (Hasler et al., 1978; Morin et al., 1989). This kind of receptor-based olfactory learning is likely limited to very specific and time-delimited contexts in which errors in learning are not susceptible to correction and have extreme fitness consequences. Nevertheless, these results suggest that behaviorally relevant plasticity may be widely distributed in olfactory circuits and even found at the very earliest stages of olfactory processing. B.

Reentrant Loops in the Olfactory System

Although the massive interconnections between olfactory structures (Haberly and Price, 1978a,b), systemwide electrophysiological oscillations (Freeman, 1991), and the modulation of olfactory structures by motivational state (Pager, 1974, 1983) have been known for some time, it is only recently that we have come to appreciate the functional consequences of this interconnectivity. This feedback from higher structures is most apparent in OB and PIR neuronal activity in animals performing olfactory discrimination tasks. The major finding is that most neurons in these “primary sensory” areas are driven more by nonolfactory task demands, such as responding to water reward, trial onset cues, etc., than by the sensory qualities of the odors (Di Prisco and Freeman, 1985; Kay and Laurent, 1999; Schoenbaum and Eichenbaum, 1995a). It seems likely that the sculpting of neural activity in these areas, in compliance with critical features of the task, is due to feedback from central structures.

C.

Neuromodulation

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Petrulis and Eichenbaum olfactory stimulation. Proc. Natl. Acad. Sci. USA 94: 4119–4124. Zhang, Y., Burk, J. A., Glode, B. M., and Mair, R. G. (1998). Effects of thalamic and olfactory cortical lesions on continuous olfactory delayed nonmatching-to-sample and olfactory discrimination in rats (Rattus norvegicus). Behav. Neurosci. 112:39–53. Zola-Morgan, S., Squire, L. R., and Amaral, D. G. (1989). Lesions of the hippocampal formation but not lesions of the fornix or the mammillary nuclei produce long-lasting memory impairment in monkeys. J. Neurosci. 9:898–913. Zyzak, D. R., Otto, T., Eichenbaum, H., and Gallagher, M. (1995). Cognitive decline associated with normal aging in rats: a neuropsychological approach. Learn. Mem. 2:1–16.

21 Nasal Patency and the Aerodynamics of Nasal Airflow: Measurement by Rhinomanometry and Acoustic Rhinometry, and the Influence of Pharmacological Agents Richard E. Frye Children’s Hospital, Boston, Massachusetts, U.S.A.

I.

INTRODUCTION

damaged nasal function. Tonndorf (1939) demonstrated turbulent flow in nasal models, throwing into question Kayser’s laminar flow assumption. The rhinomanometer was further improved upon when Spoor (1965) incorporated electronic pressure transducers, thereby eliminating the Utube water manometer—a device with limited frequency response (Nakano, 1967; Randall, 1962).

A. Recognition of the Nose in Respiratory Function: A Brief History Ebers’ Papyrus, the only complete Egyptian papyrus, mentions the nose as a respiratory organ: “As to the breath which enters into the nose; it enters the heart and lungs; these give to the whole belly” (Ebbell, 1937). The Egyptians knew that the nose secretes mucus, contains arteries and veins, and is responsible for olfaction. Operations to repair nasal bone fractures and remove polyps were recorded (Pahor, 1992; Pahor and Kimura, 1991). Galen was the only Greek physician or philosopher on record to recognize the importance of the nose in respiration (Kimmelman, 1989). In 1844, Piorry espoused the importance of the nose in respiratory function. He divided the etiology of nasal stenosis into those produced by septal deviation or alternating vasomotor stenosis (Williams et al., 1970). Half a century later, Franke (1894) measured nasopharyngeal pressure changes in patients with nasal abnormalities. Soon thereafter, Kayser (1895) created the first anterior rhinomanometer by simultaneously measuring nasal volume flow and nasopharyngeal pressure. Based on measurements from these devices, Franke and Kayser performed operations to enlarge the nasal lumen. However, in many cases the operations failed to improve the underlying condition and

B. Functional Physiology of the Nasal Airway: A Brief Review The nasal airway optimizes gas exchanged by conditioning and filtering inspired air; incoming air is heated, humidified, and filtered of airborne pathogens and environmental pollutants. Thermoregulation and systemic water balance is maintained by recovering heat and water vapor from expired air and is independent of nasal airway resistance (Keck et al., 2000). Thomson and Dudley-Buxton (1923) highlighted the functional significance of nasal morphology by demonstrating that the anthropological cephalometric nasal index varies across race in relation to indigenous climate. Indeed, the major change in nasal morphology from Australopithecus sp. to Homo sapiens supposedly provided a selective advantage by conserving moisture in arid environments (Franciscus and Trinkaus, 1988). Nasal airway patency is functionally coordinated with pulmonary function. By matching lower airway 439

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impedance, the nose assists in the control of breathing frequency and expiration length and provides positive endexpiratory pressure. Turbinate swelling is increased by pulmonary stretch, lower airway irritation, normocapnic progressive hypoxia, and respiratory drive (Lung and Wang, 1991; Maltais et al., 1991; Nishihira and McCaffrey, 1987; Series et al., 1989). Although stimulation of neither the nose nor the larynx influences elastic or resistive lung properties (Jacobs and Dickson, 1986), the nasal airway can influence distal airway function. For example, removal of nasal obstruction improves sleep apnea, and nasal breathing improves exercise tolerance in patients with chronic obstructive pulmonary disease and decreases pulmonary work during exercise in the nonathlete (Lamblin et al., 2000; Lavie, 1987; Morrison et al. 1989; Petruson and Bjuro, 1990, Tanaka et al., 1988). C.

Influence of the Nose on Olfactory Function

The nose contains the olfactory neuroepithelium, a small (~2 cm2) region of the nasal mucosa located on the cribriform plate, upper nasal septum, dorsal superior turbinate, and regions of the middle turbinate. The irregular contour of the nasal cavity, combined with high velocity airflow, produces nonlinear aerodynamics that promote odorant mixing while producing a complicated odorant distribution. Only 15% of the incoming airstream passes near the olfactory epithelium. Subtle alternations in nasal geometry can deflect the airstream away from the olfactory epithelium; in many cases nasal function and patency perception are unaffected. Only 15–20% of patients referred to clinical smell and taste centers have diagnosable obstructive airflow abnormalities (Deems et al., 1991; Mott and Leopold, 1991). II.

AERODYNAMICS OF THE NASAL CAVITY

Nasal aerodynamics are influenced by nasal anatomy and physiology. Functional nasal cavity aerodynamics is evaluated in vivo by rhinomanometry—a technique that measures differential pressure across, and volume flow through, the nasal cavity. Nasal pressure and flow are nonlinearly related in normal and disordered airways due to turbulent flow and the dynamic narrowing of the nasal valve. A.

Turbulent Flow

Nasal cavity aerodynamics is customarily described by the equations of fluid mechanics, particularly the HagenPoiseuille equation. This equation relates the driving-

pressure across a conduit (i.e., the nasal cavity) to the volume airflow rate through the conduit: p


8   lq  r4o

In this equation p is pressure, q is volume flow, l is length of the conduit,  is dynamic viscosity, and ro is the radius of the tube. This equation is easily modified to express Poiseuille resistance, R, an analog to the electrical ohmic-type resistance: 8  l p
R
q  ro4

Poiseuille resistance is used to describe nasal airway resistance (NAR), as well as upper and lower airway resistance. This equation assumes that the airway is circular, straight, rigid, and uniform and that air is homogeneous and noncompressible. Although the latter assumption is rarely violated to any significant extent, the former assumptions are most certainly violated by the complicated shape of the nasal cavity and the compliant nature of the nasal valve. Since the resistance of a conduit is inversely proportional to the fourth power of the radius (Leyton, 1975), small changes in the diameter of the nasal cavity can greatly influence Poiseuille resistance. In straight, rigid, uniform circular conduits, laminar flow predominates at low flow rates, whereas turbulent flow predominates at high flow rates (Rohrer, 1915). Reynolds (1883) developed an index to measure the relative turbulence of fluid flow: Re


2ro   q 

The Reynolds number depends on fluid density () and molecular viscosity (). A Reynolds number below 1500 usually indicates laminar flow, while a Reynolds number between 1500 and 2000 is indicative of mixed turbulent and laminar flow. Turbulence usually develops when the Reynolds number exceeds 2000, but this is dependent on the shape and roughness of the conduit. For example, a bifurcating conduit will develop turbulence at a Reynolds number of 900 (Jones et al., 1969). Although the average velocity through a conduit can be calculated by dividing the volume flow rate by the crosssectional area of the conduit, the velocity at any particular point is a function of distance from the conduit’s wall. Fluid near the wall moves slowly owing to shear stress, while fluid in the center moves most rapidly. The velocity profile gradient depends on the flow type. Laminar flow is organized in concentric layers, with the outermost layer traveling at almost zero velocity and the centermost layer traveling at maximum velocity. This arrangement results in a parabolic flow profile.

Nasal Patency and Nasal Airflow Aerodynamics

Turbulent flow develops at high flow rates or when the geometry of a conduit contains irregular walls, bifurcations, bends, or abrupt changes in cross-sectional area (CSA). Flow becomes disordered and local eddies develop within the jetstream. Once turbulence develops, the force that previously promoted forward fluid motion moves the fluid in directions perpendicular to the forward motion. Efficiency is greatly decreased and a substantial increase in pressure is needed to produce a small increase in flow rate. The turbulent flow profile is flat, with a quick velocity drop near the wall of the conduit. B.

The Collapsible Nasal Valve

The pressure drop across a tube due to airflow depends on the smallest CSA, also know as the minimal cross-sectional area (MCA) (Williams et al., 1970). In the normal nasal airway, this is the nasal valve, which, under normal conditions, accounts for 90% of the pressure drop (Jones et al., 1988). Although Uddstromer recognized the importance of the nasal valve in 1939, its function was not quantitatively described until 1970 (Bridger and Proctor, 1970). The nasal valve is located approximately 2 cm posterior to the entrance of the naris; its CSA is normally about 1.5 cm2. It is composed of compliant tissue, allowing it to partly collapse when the differential pressure reaches a critical value; thus, a collapsible tube model can be applied. The differential pressure across a collapsible tube depends on the fluid flow rate, which, in turn, depends on resistance. Since resistance depends on the MCA, and the MCA depends on the differential pressure, pressure and flow indirectly regulate each other. Collapsible tubes are known as flow regulators, which prevent flow from exceeding an upper limit (Conrad, 1969; Holt, 1969). Critical pressure depends on both upstream and downstream resistance; thus, the dynamics of the nasal valve depend on the nasal airflow direction (i.e., expiration or inspiration). Nasal alar muscle activity reduces nasal valve elasticity, thereby increasing critical pressure (Cole et al., 1985). Indeed, nasal resistance on the paralyzed side of patients with unilateral facial palsy is four times that of the nonparalyzed side owing to the inactivity of the nasal alar muscles (Van Dishoeck, 1964). Many factors influence the onset and magnitude of alar muscle activity, including breathing rate, maximum flow rate, acceleration of airflow, sleep state, CO2 concentration, resistive loading, and negative airway pressure (Mezzanotte et al., 1992; Strohl et al., 1980, 1982). Sagging or depression of the upper lateral nasal cartilage, anterior nasal septum buckling, or inferior turbinate inflammation can narrow the nasal valve area, leading to a

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lower critical pressure (Adamson, 1987; Goode, 1985; Kasperbauer and Kern, 1987) or a static nasal valve (Guillette and Perry, 1990). Obstruction in this region is more critical than any in other region of the nasal chamber (Berkinshaw et al., 1987). C.

Measuring Nasal Airway Properties

Many approaches are used to measure nasal cavity properties. Changes in nasal peak flow and pressures were the first parameters recognized. Simultaneous pressure and flow measurements allow nasal airway resistance (NAR) calculations. Various preset points for pressure or flow values have been selected to standardize NAR. However, no standardized measurement is universally accepted. Despite the development of advanced curve fitting algorithms, mathematical model coefficients are not familiar to many clinicians and documentation of clinical correlation is lacking. NAR is best measured by anterior rhinomanometry, although this method is the most technically difficult, NAR varies significantly among subjects, although withinsubject variation is quite small and physiological alterations in NAR result from the nasal cycle, body position, and nasal decongestion. Acoustic rhinometry is a relatively new technique for quantitatively measuring nasal cavity dimensions. Sound pulse trains are introduced into the nose and the sound reflections are analyzed. Nasal CSA is graphed as a function of distance from the naris. Rigid nasal endoscopy has correlated anatomical landmarks with sequential CSA minima: the first corresponds to the nasal valve, the second corresponds to the anterior end of the inferior turbinate, and the third corresponds to the anterior end of the middle turbinate (Corey et al., 1999). Primary reportable measurements include unilateral or total nose MCA as well as nasal cavity volume (NCV). Acoustic rhinometry was introduced to the otorhinolaryngology community only a decade ago—a relatively short time for a new technology to grow in popularity. Since its introduction the reliability and accuracy has been studied repeatedly, and normative values have been developed for a wide variety of ages and ethnic groups. This technique requires minimal cooperation from the subject and does not require breathing effort, making it particular helpful in evaluating children with nasal obstruction. The utility of acoustic rhinometry is clearly demonstrated in its ability to measure parameters in the nasal airway of infants — a feat rather difficult to perform with rhinomanometry. The advantages and drawbacks of acoustic rhinometry and rhinomanometry must be considered individually, as each method measures a different entity. Both methods are reliable over several weeks, if performed by an experienced

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operator under controlled circumstances (Silkoff et al., 1999). Anatomical nasal cavity parameters, as measured by acoustic rhinometry, provide important information concerning the size and location of the maximum flow velocity. However, only anterior nasal cavity measurements correlate with clinical abnormalities. Indeed, the accuracy of acoustic rhinometric measurements is greatly reduced beyond the MCA. Special caution is required when interpreting posterior CSA and NCV measurements, since the sound reflections depend on nasal cavity shape and the size and location of the MCA. For example, a differential change in the expansion of the first and second minima, after pharmaceutical, operative, or prosthetic manipulation of the nose, may produce apparent movement of the MCA; if the MCA has moved, the change in the MCA value will be based on the CSA of a different anatomical landmark, making the measure’s validity questionable (Tomkinson and Eccles, 1998). A small MCA, as seen during mucosal inflammation or severe septal deviation, underestimates posterior NCV by limiting the transmission and reflection of sound distal to the MCA. For example, NCV measured by coronal high-resolution computed tomography (CT) correlates well with acoustic rhinometry measurements in the anterior, but not the posterior, nasal cavity (Dastidar et al., 1999a,b). Following decongestion or other procedures that expand the MCA, the posterior NCV will be less underestimated, if at all. Thus, NCV expansion will be falsely elevated, albeit not reliably. Indeed, the application of external nasal valve dilator strips causes not only a significant increase in nasal valve CSA but also an increase in NCV (Ng et al., 1998). In addition, turbulent resistance as well as dynamics of the nasal valve cannot be measured with a “snapshot” approach. Despite the potential problems with acoustic rhinomanometry, it may provide the needed information to explain the between-subject variability in rhinomanometric measurements and fill in some of the structural parameters of the aerodynamic equations. Alternatively, the information from both rhinomanometry and acoustic rhinometry can be combined into a unique index. This approach was taken, for example, by Kesavanathan et al. (1995), who determined the characteristics of the nasal pressure-volume ratio relationship in the anterior turbinate and nasal valve regions and their relation to nasal resistance for the normal and decongested nasal mucosa.

III.

NASAL CAVITY ANATOMY

Nasal cavity anatomy influences nasal resistance, aerodynamics, particle deposition, and olfactory function. Since

air takes the path of least resistance, it is easily redistributed if one path is blocked. Thus, airstream distribution can be profoundly influenced without a substantial change in nasal resistance. Indeed, nasal resistance is typically elevated only by severe nasal abnormalities, yet particle distribution can be altered by rather minor abnormalities. Clinical studies have associated certain nasal abnormalities with olfactory dysfunction; however, the nasal abnormalities that are described are rarely localized to a specific nasal area. Several attempts to correlate olfactory function with specific anatomic nasal areas have been made (Hornung and Leopold, 1999; Leopold, 1988). A.

Nasal Anatomy and Patency

1.

Simulated Nasal Abnormalities

Several investigators have introduced artificial obstructions in the human airway. For example, Cole et al. (1988) found that fiberfoam protruding 3–5 mm into the nondecongested airway at the upper lateral cartilage area (i.e., nasal valve area), but not into other areas of the nasal cavity, significantly influenced NAR. When the mucosa was decongested, 4- and 5-mm obstructions in the upper lateral cartilage area were required to meaningfully affect NAR. In the turbinate region of the nasal chamber largersized obstructions blocking an extensive portion of the nondecongested airway were required to significantly alter NAR (Chaban et al., 1988). A simple nasal valve stent was inserted in the noses of patients with a static nasal valve and normal subjects with artificially created midseptal nasal obstructions (Guillette and Perry, 1990). Artificially created obstructions and nasal valve abnormalities both resulted in equivalent NAR. However, a nasal stent only significantly improved the patients with nasal valve disorders. Haight et al. (1985) found that selective decongestion of the nasal valve region, but not the nasal choanal region, markedly influenced NAR. Using a clear acrylic model of the nasal passageway, Levine et al. (1986) showed that moderate and severe anterior septal deviations significantly influenced Rohrer’s K1 and K2 coefficients—values that measure laminar and turbulent airflow, respectively. Moderate and severe posterior septal deviations changed these coefficients to a lesser degree. Septal deviations placed in the turbinate region of the nasal chamber, even when severe, did not influence these coefficients. Simulated mild and moderate enlargement of all turbinates altered the K1 coefficient. These studies indicate that anterior airway obstructions, particularly in the region of the nasal valve, have a much larger impact on NAR than posterior obstructions or flow limitations. Only severe obstructions influence NAR within the turbinate region.

Nasal Patency and Nasal Airflow Aerodynamics

2.

Clinical Abnormalities

Grymer et al. (1997) studied 230 randomly selected adults, 14% of whom had a subjective feeling of nasal obstruction. Patients with a small anterior CSA or symptoms of sinusitis or rhinitis, mostly as a result of anterior septal deviations or severe mucosa swelling, were most likely to have subjective nasal obstruction. The importance of the anterior nasal chamber and the nasal valve in producing NAR has been repeatedly demonstrated. Roithmann et al. (1994) showed a significant, negative, nonlinear relationship between NAR and MCA area in 78 patients suffering from nasal obstruction—small intrusions into the nasal lumen produced unusually large increases in NAR when located in the valve region. Patients with postrhinoplasty nasal obstruction had a significantly smaller nasal valve CSA. In a later study these same authors found that symptoms, as well as CSA anomalies, were mitigated by external nasal dilation (Roithmann et al., 1997a). Acoustic rhinometry and anterior rhinomanometry are most sensitive in revealing severe deviations in the anterior nasal cavity, but are less sensitive in demonstrating middle and posterior deviations (Szucs and Clement, 1998). Dinis et al. (1997) examined 45 consecutive adult subjects with complaints of nasal obstruction; CT scans delineated the specific etiology. Rhinomanometry uncovered abnormalities in patients with anterior septal deviation, but not posterior septal deviation or sinusitis. Although disorders in the anterior nasal cavity disproportionately influence NAR, general nasal airway obstruction also causes airflow and symptomatic changes. Adenoid hypertrophy decreases nasopharyngeal CSA (Cho et al., 1999; Mostafa, 1997). Nasal provocation with allergins results in a dose-dependent change in MCA and NAR (Austin and Foreman, 1994; Roithmann et al., 1997b; Zweiman et al., 1997). 3.

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NAR, anterior CSA, and symptoms (Kamami, 1997, Kamami et al., 2000). Increasing the anterior nasal cavity size to a critical level may mitigate nasal valve dysfunction. For example, lateral rhinotomy with medial maxillectomy improves NAR and nasal valve CSA despite interrupting nasal valve support. This is presumably due to increased CSA in the anterior nasal chamber by concomitant resection of the anterior inferior turbinate (Leug et al., 1998). Nasal valve suspension in patients with nasal obstruction due to nasal valve collapse improves subject nasal patency and NAR without a significant increase in MCA (Paniello, 1996). Septoplasty with or without turbinoplasty, inferior turbinate cauterization, rhinoplasty, or uvulopalatopharyngoplasty improves objective nasal patency and acoustic rhinometric measurements (Reber et al., 1998; Shemen and Hamburg, 1997). However, acoustic rhinometric measurements vary widely and do not correlate well with subjective nasal patency. Inferior turbinoplasty and endoscopic sinus surgery improved symptoms and acoustic rhinometric measurements for patients with chronic nasal obstruction not caused by septal deviation. Radical trimming of the inferior turbinate reduced total NAR (Wight et al., 1988a). Acoustic rhinometric patency measures and subjective symptoms improved after bilateral inferior turbinoplasty, although the degree of subjective and objective improvement did not correlate well (Grymer et al., 1996). Although several procedures for improving inferior turbinate hypertrophy improve nasal function, Passali et al. (1999) found that submucosal resection without lateral displacement is superior to electrocautery, cryotherapy, laser cautery, or turbinectomy. Illum (1997) showed that compensatory anterior inferior turbinectomy with septoplasty does not improve symptoms beyond septoplasty alone. Functional endoscopic sinus surgery supposedly improves chronic sinusitis by reducing mucosal edema without changing structural anatomy (Keles et al., 1998).

Nasal Surgery

The influence of corrective surgery on subjective and objective nasal patency varies from report to report. This is probably due to the lumping of heterogeneous disorders together and the failure to employ standardized outcome measures. However, studies that use well-validated outcome measures and study populations consisting of relative specific disorders or operations do show some positive effects of surgery. The studies confirm that the anterior nose, in the region of the vestibule and nasal valve, is particularly important for improving nasal patency. Enlarging the anterior nasal cavity improves objective and subjective nasal patency. Laser-assisted outpatient septoplasty for moderate anterior septal deviation improves

4.

Pharmaceutical Treatments

Pharmaceuticals used in the treatment of rhinnitis, specifically topical decongestants, antihistamines, and steroids, have been evaluated by objective nasal patency measurements. Rhinomanometry is commonly used to monitor NAR and airflow, whereas acoustic rhinometry can measure mucosal swelling. Caveats mentioned above concerning changes in NCV with large changes in mucosal swelling should be carefully considered when examining the results of these studies. A number of recent studies have quantitatively examined the effects of the topical decongestant oxymetazoline on airway patency. Most of these studies have employed

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placebo and double-blind controls. Oxymetazoline is efficacious when evaluated by NCV, MCA, NAR, or rhinostereometry (Bickford et al., 1999; Graf et al., 1999) and appears to be the most effective imidazoline derivative (Hochban et al., 1999). Many studies do not show doseresponse effect beyond an effective dose. For example, although 0.25 and 0.50 mg/mL significantly increased NCV, the effect was not different between the two doses (Hummel et al., 1998). Twenty-five and 50 g, but not 6.25 or 12.5 of oxymetazoline, significantly reduced NAR. Although total NCV increased in a dose-dependent manner, MCA did significantly increase beyond the lowest dose (Taverner et al., 1999). Although rebound swelling is not seen in several studies (Graf et al., 1999; Hochban et al., 1999), other studies have demonstrated its dynamics (Morris et al., 1997). However, it may be related to dosage. Few other nasal decongestants have been objectively evaluated. One dose of pseudoephedrine (60 mg) significantly improved symptoms and increased total MCA and NCV, but did not change NAR, in healthy patients with moderate to severe acquired NAR as a complication of the common cold (Taverner et al., 1999). Phenylpropanolamine (100 mg twice) a day had effects similar to topical oxymetazoline, as measured by rhinostereometry and MCA (Graf et al., 1999). Inhaled furosemide significantly decreased NAR in 12 patients with perennial nonallergic rhinitis (Masieri et al., 1997). Intranasal steroid efficacy has also been studied with quantitative nasal patency measurements. Fluticasone and beclomethasone aqueous nasal spray increased NCV in 32 patients with severe polyposis as compared to placebo, although fluticasone but not beclomethasone significantly improved morning peak inspiratory flow rate after the first week of treatment (Lund et al., 1998). Beclomethasone and budesonide improved nasal airflow in patients with nasal polyposis, although beclomethasone but not flunisolide improved NAR in nasal polyposis patients following surgery. The efficacy of intranasal steroids for perennial rhinitis in children and adults and in seasonal allergic rhinitis has been confirmed using rhinomanometric measurements (see Mygind et al., 1997). Nasal provocation is used to determine the efficacy of specific pharmaceutical treatments for allergic rhinitis. Spaeth et al. (1996) documented the efficacy of azelastine using rhinomanometry, acoustic rhinometry, rhinoscopy, and symptom scores after histamine and allergen provocation. Astemizole improves NAR and symptom scores after long-term provocation in subjects allergic to grass pollen (Horak et al., 1993). Nielsen et al. (1996) confirmed the utility of steroids for treating seasonal allergic rhinitis by demonstrating a difference in NCV and symptom scores after metacholine challenge in patient’s with grass pollen

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allergy. Hilberg (1995) demonstrated that pretreatment with beclomethasone is better than terfenadine in preventing the allergen-induced increase in NAR. Intranasal steroids also reduce the histamine-induced provocation response in perennial rhinitis (Mygind et al., 1997). The efficacy of the thromboxane A2 receptor antagonist ramatroban was demonstrated after allergen challenge with house dust; treatment prevented significant change in NCV and MCA from baseline (Terada et al., 1998).

5.

Nasal Valve Dilation

The nasal valve and anterior vestibule are important contributors to NAR. As noted earlier in this review, the nasal valve is dynamic and collapsible, allowing it to change its resistance in proportion to airflow rate. A smaller resting nasal valve CSA results in a higher baseline valvular resistance and a relatively lower maximum airflow rate. Anterior septal deviations may deform the nasal valve, making its baseline resistance higher, thereby causing nasal obstruction. External nasal dilator strips, such as the The BreathRightTM strip (CNS. Inc., Minneapolis, MN), can effectively relieve most anterior nasal abnormalities. Application of such strips increased nasal valve CSA in the pre- and postdecongested nose (Ng et al., 1998). Strip application improved MCA and NAR in normal subjects, patients with septal deviation in the nasal valve area, and patients with mucosal congestion, although the most marked change occurred in the septal deviation group (Roithmann et al., 1998). NAR and CSA are improved in normal Caucasian subjects after the application of nasal strips (Gosepath et al., 1997). However, similar improvement was not reliably seen in African American subjects (Portugal et al., 1997). Application of other nasal dilators (Airplus, Prevancure AB, Vastra Frolunda, Sweden; Improved Mechanical Therapeutic Nasal Dilator, Breath EEZ Corp., Brooklyn, New York) increases MCA and decreases NAR (Chaudhry et al., 1996; Nielsen et al., 1997). In a comparative study, Lorino et al. (1998) showed that the internal nasal mechanical dilator Nozovent (Prevancure AB, Vastra Frolunda, Sweden), but not the external nasal strip device Respir (Kentia Diffusion, Boulogne, France) decreased NAR. Nasal strips have physiological effects. Breathe-Right nasal strips improved the respiratory disturbance index for patients with obstructive sleep apnea and snoring if the patient also suffered from hyperplasia or hypertrophy of the lower turbinates, septal deviation or allergic rhinitis, only minor pharyngeal obstruction, or was less than 55 years in age (Gosepath et al., 1999). Nasal strips also

Nasal Patency and Nasal Airflow Aerodynamics

significantly decrease heart rate, ventilation, and VO2 in athletes during submaximal exercise (Griffin et al., 1997) B.

Nasal Anatomy and Olfactory Ability

1. Clinical Observations Schneider and Wolf (1960) pioneered the study of nasal anatomy and olfactory function. Unfortunately, the study has a number of pitfalls, including the fact that the clinician that made the nasal cavity measurements was not blind to the threshold data. Most modern studies have not properly measured olfaction, evaluated sufficient sample sizes, or used adequate methodological procedures (for reviews, see Doty and Frye, 1989; Leopold, 1986; Mott and Leopold, 1991). In general, loss of olfactory function is related to allergic rhinitis, polyposis, nasal sinus disease, or adenoid hypertrophy. For example, both allergic and nonallergic rhinitis patients have hyposmia, but nonallergic rhinitis patients have a higher olfactory threshold than allergic rhinitis patients (Simola and Malmberg, 1998). Hyposmia associated with nasal disease may be due to airflow obstruction or olfactory mucosa inflammation. For example, patients with chronic sinusitis who have inflammatory cell influx into the olfactory mucosa are much more likely to have an olfactory deficit (Kern, 2000). A reduction in the N1 component of olfactory and trigeminal chemosensory event–related potentials are correlated with acute rhinitis symptoms. Only the trigeminal N1 component normalizes when mucus secretion subsides; olfactory chemosensory event–related potentials returned towards normal over a one-month period (Hummel et al., 1998a). This protracted hyposmia lends evidence to a nonobstructive etiology. In some cases, olfactory function can be improved by endoscopic operative procedures (Seiden and Smith, 1988) or adenoidectomy (Ghorbanian et al., 1983). Septoplasty can improve olfaction and nasal resistance (Stevens and Stevens, 1985), and the degree of septal curving before surgery may be correlated with the degree of olfactory improvement (Kittle and Waller, 1973; Shevrygin, 1973). Although total removal of the inferior turbinate is related to improvement of olfactory function (Ophir et al., 1986), such improvement varies with the technique used (Elwany and Harrison, 1990). Even though studies suggest that surgical or medical intervention can improve olfaction, success is limited. Indeed, many patients do not experience restoration of olfactory function following intervention. Nasal sinus disease associated with swelling of the superior nasal cavity likely obstructs airflow to the olfactory region, while disorders associated with the inferior turbinate or nasal

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septum presumably redistribute the airstream within the nasal cavity. Airflow obstruction is only one mechanism of olfactory dysfunction, and changes in the olfactory epithelium due to, for example, inflammatory processes may also have a significant effect (for review, see Doty and Mishra, 2001). 2.

Nasal Cavity Volume and Olfactory Function

To identify the relationship between nasal anatomy and olfactory dysfunction, Leopold (1988) obtained nasal cavity CT scans of 34 patients with conductive or idiopathic hyposmia. The nasal cavity superior to the middle turbinate was divided into nine sections by placing coronal borders anterior and posterior to the cribriform plate and horizontal borders at 5 and 10 mm below the cribriform plate. The volumes of these regions were measured and entered into a stepwise regression analysis using olfactory test score (as measured by an odor confusion matrix) (see Chapter 9) as the dependent variable. Interestingly, a larger volume in the region 10–15 mm below the cribriform plate and a smaller volume 1–5 mm inferior and anterior to the cribriform plate was associated with higher olfactory scores. A larger volume in the region 10–15 mm below and posterior to the cribriform plates potentiates both effects. Hornung and Leopold (1999) measured unilateral, as opposed to bilateral, olfactory function in 19 patients with static conductive hyposmia due to polyposis or mucosal edema. Like their previous study, the volume between the cribriform plate and middle meatus was divided up into nine sections. In addition, the nasal chamber and vestibule was divided into 12 additional volumetric areas (Fig. 1). Interestingly, a larger volume 5–10 mm below and anterior to the cribriform plate and a smaller nasal vestibule volume correlated with better olfactory function. A larger volume along the septum in the region between the inferior and middle turbinates markedly increased the former effect. These studies suggest that critical areas of the nasal cavity, both near and remote from the olfactory receptors, influence olfactory function, presumably by directing air towards the olfactory cleft. A smaller nasal vestibule may direct air to the olfactory cleft from the anterior aspect, and a larger volume anterior and inferior to the cribriform plate may increase airflow to the olfactory cleft. A larger volume along the septum just below the middle turbinate may direct airflow to the olfactory cleft from a medial aspect. In should be recognized that by excluding patients and noses with anosmia or normosmia, as has been done in the aforementioned studies, the variation in the olfactory data, as well as the degree to which these data can be generalized to normal subjects, is reduced. In addition, even

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Figure 1 Cutaway view of a right nasal cavity showing the subdivided nasal regions whose volume was correlated with olfactory function. (From Hornung and Leopold, 1999).

patients with conductive hyposmia may have some component of inflammation at the olfactory mucosa.

IV.

NASAL AIRFLOW PATTERNS

The complicated shape of the nasal cavity divides the incoming air into jet streams that flow between the nasal turbinates and along the nasal septum. Turbulent flow, vortices, and regions of negative pressure are created. Vortices promote mixing, whereas high-speed flows associated with regions of negative pressure may produce nasal abnormalities. For example, Ogawa (1986) reports that polyps occur more often on the concave side of unilateral septal deviated noses, possibly because of higher flow rates. To localize specific nasal cavity areas that influence the direction and velocity of the airstream, investigators have turned to nasal models. Water or air, along with an indicator substance such as dye or smoke, respectively, is moved through a model in order to identify flow stream dynamics. Most nasal models are qualitative, but some studies have directly measured velocity. Mathematical models of the nasal cavity, which simulate nasal cavity aerodynamics and particle disposition, have also been used.

A.

Nasal Airway Models

Nasal cavity models do not stimulate elastic or dynamic properties of the mucosa or the nasal valve. Since most models are based on a single nasal cavity, finding generalization may be limited. In addition, suspended particles may not simulate molecular dynamics accurately, and induction of Pitot tubes or anemometers into a model may alter aerodynamics. Nevertheless, these studies provide information on general principles of nasal airflow and insight into the particle transport and distribution. 1.

Water Flow Models

Aluminum particle deposition in the olfactory region of a plastic nasal model increases with water velocity (Stuiver, 1958). Masing (1967) injected ink into the naris and nasopharynx during simulated inspiration and expiration, respectively, at a velocity of 0.75 L/min. Injection into the lateral, dorsal, or ventral naris caused ink flow along the inferior nasal cavity, while injection into the medial or central naris resulted in diversion of ink to the superior and middle nasal regions. Injection into the dorsal nasopharynx diverted ink to superior nasal regions, while ink injection into the medial, central, and ventral nasopharynx was diverted into the middle and inferior nasal regions. Ink release in the medial nasopharynx caused the stream to

Nasal Patency and Nasal Airflow Aerodynamics

travel in the inferior nasal region after forming postturbinal vortices. Swift and Proctor (1977) used both water and air in clear polyester resin cadaver casts to observe streamlines and measure airflow rates. With an inspiratory volume rate of 12.5 L/min and a nostril CSA of 0.9 cm2, the air velocity was 138 m/min and laminar. The nasal valve, which had a CSA of 0.32 cm2, had a computed average velocity of 390 m/min and a measured maximum velocity of 1116 m/min. The abrupt increase in nasal chamber CSA posterior to the nasal valve resulted in turbulence and a marked decrease in airflow velocity. Turbulence continued throughout the nasal cavity. Most of the flow passed between the middle meatus and the nasal septum above the inferior turbinate at a rate of 120–180 m/min. A lesser amount of air followed the nasal septum and then changed direction, following the floor of the nasal chamber. A small fraction of the main stream broke off and formed a standing eddy in the olfactory region. Morgan et al. (1991) used a water-dye siphon system to study nasal airflow in the F344 rat and rhesus monkey. Distribution of the dye differed between species for a given release point, indicating that a common distribution mechanism does not exist across species. However, the data suggest that the anterior portion of the nose is important for directing the incoming airstream, regardless of the species. 2. Gas Flow Models Proetz (1951) pumped smoke through models of both normal and abnormal nasal airways. Inspired air was directed toward the septum until it reached the nasal vestibule, at which point it fanned out. The streams reconverged as they approached the choana. The middle turbinate divided the expiratory airstream in half, with half following the inspiratory pathway and the other half forming eddies and vortices. Nasal turbinates were not important for directing inspiratory flow, but were essential for directing expiratory flow. Nasal valve region abnormalities changed the direction of the smoke flow. Swelling of the upper two thirds of the nose directed smoke flow in a straight path toward the nasopharynx and prevented the smoke flow from fanning out inside the nasal cavity. Septal spurs and middle meatus polyps did not alter normal smoke flow, while superior meatus polyps deflected the air currents downward and away from the olfactory meatus. External deformities resulting from the narrowing or collapse of the naris augmented the pressure drop across the nose but did not change the pattern of smoke flow. Girardin et al. (1983) used a laser Doppler device to measure the velocity of a water aerosol introduced into a

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clear bioplastic model at a flow rate of 10 L/min. A total of 100–350 measurements were made at various horizontal and vertical points at five cross sections: the nasal valve, preturbinal, midturbinal, postturbinal, and nasopharynx regions. During inspiration, the nasal valve directed air along the floor and lateral aspects of the nasal cavity at a rate of 63 m/min and along the upper nasal cavity entrance at lower velocities. In the preturbinal region, flow velocity was greatest in the lower portion and, like the nasal valve region, showed a very irregular velocity profile. Airflow in the midturbinal area was more regular, with the highest flow rates along the medial floor and middle nasal cavity areas. At the postturbinal and nasopharyngeal regions, the flow profiles remained uniform but demonstrated low velocities owing to the larger total CSA. Expiratory velocity flow profiles appeared less uniform and, in general, had greater maximum velocities than inspiratory flow profiles. While the majority of the inspiratory flow was shunted through the inferior and middle meatuses, most of the expiratory flow was shunted along the septum. Although Reynolds numbers were indicative of laminar flow, the flow profiles indicated that turbulent flow predominated throughout the nasal cavity. Air velocity in the superior posterior nasal cavity was low during inspiration and high during expiration. Hornung et al. (1987) used a vacuum pump to draw Xenon 133 gas though a plastic nose at 2.5, 7.0, and 20 L/min. A catheter released radioactive gas in four locations within the nostril and at the initial opening of the nasal chamber. Positioning the catheter in the center of the naris distributed radioactivity evenly throughout the nose, with the exception of the superior region, whereas positioning the catheter in the ventral medial portion of the nostril increased the radioactivity in the dorsal middle and inferior meatuses. The greatest superior region radioactivity was observed when the catheter was placed in the initial portion of the nasal chamber. Increasing the flow rate increased radioactivity in the anterior olfactory region. Simmen et al. (1999) pumped aerosolized water particles through an anatomical human model of the choana with a translucent replica of the original nasal septum. Physiological pressures were simulated. Turbulence was present throughout all flow velocities, with turbulence being most prominent during airflow acceleration and deceleration and less prominent at near-steady flow. Under normal conditions the majority of the main flow stream passed through the middle meatus at all rates and the olfactory region was aerated toward the end of inspiration and during the entire expiration phase. Simulated hypertrophic mucosal membranes and turbinates increased the proportion of air passing through the middle meatus, and turbinate decongestion resulted in more even flow

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distribution. Turbinectomy redirected airflow along the floor of the nose. Although this model demonstrates some interesting results, the lack of a simulated nasal valve may have decreased the turbulence and dynamics of the airstream. Using a large-scale mock human nasal cavity and a hot wire anemometer, Scherer et al. (1989) measured nasal air currents at inspiratory flow rates between 15 and 120 L/min. At least 50% of the airflow passed through the inferior and middle meatuses, while 15% passed through the olfactory region. Air velocity was relatively high through the inferior and middle meatuses and lower olfactory slit. Simulated nasal hairs increased turbulence. Although the air velocity profiles were typical of turbulent flow through the inspiratory airflow range, Reynolds numbers were only 400 at lower flow rates. When this model was studied with steady physiological flow rates [1,100 (66), 560 (33.6), and 180 (10.8) mL/sec (L/min)], laminar flow predominated, although moderate turbulence was seen. 3. Computer Simulated Flow Using a mathematical simulation of incompressible, steady, laminar flow through three-dimensional nasal cavity represented by a trapezoid outline and two curved plates as the inferior and middle turbinates, Elad et al. (1993) showed that the majority of air flowed along the nasal cavity floor, while the turbinate structures directed flow in an anteriorposterior direction. These researchers suggest that the turbinates and the nasal cavity shape are responsible for forcing airflow towards the olfactory region. Keyhani et al. (1995) solved the steady-state Navier-Stokes and continuity equations for an anatomically correct finite element mesh designed from a CT scan of a healthy adult nose in order to determine the laminar airflow patterns in the nasal cavity at quiet breathing flow rates (Fig. 2). The highest inspiratory velocities occurred along the nasal floor and between the inferior and middle turbinates with laminar flow predominating as velocity varied between resting breathing rates of 125 (7.5)–200 (12) mL/sec (L/min); about 10% of the airflow passed into the olfactory cleft. These results were found to be consistent with the results of the large-scale mock human nasal cavity developed by Scherer et al. (1989) and Hahn et al. (1993). The lack of representation of the nasal valve in these simulations calls into question the validity of determination of turbulent flow within the nasal cavity. B.

Particle Deposition and Uptake

Determining the filtering efficiency of the nose may be of help in understanding odorant transport. For example, by knowing the proportion of inspired air shunted to the

Figure 2 Nasal model used to calculate concentration field and odorant mass flux in order to investigate inspired odorant molecule transport and uptake. (Top) Three-dimensional finite element mesh of the right nasal cavity with view of septum and nasal floor. (Left) Slice through the three-dimensional mesh at plane 8. (Right) Midsagittal outline of the model nasal cavity and locations of nine coronal planes. Three coronal planes are demonstrated with the olfactory region highlighted with hatch marks. (From Keyhani et al., 1997.)

olfactory region and the amount of particle deposition in the nose, the number of particles reaching the olfactory region can be calculated. However, since particle deposition is a function of such variables as airstream velocity, subject age, and particle size, such calculations are complex. 1.

Theoretical Studies

Scott et al. (1978) developed a theoretic geometric model of the nasal cavity from dimensions reported by Proctor and Swift (1971) and cadaver specimens. Particle deposition was calculated for five nasal regions: the nostril, the nasal valve, the expansion area, the nasal cavity area (further divided into four regions), and the entrance to the nasopharynx. Deposition of particles within the first 0.5 mm of the nostril was due to nasal hairs, whereas particle

Nasal Patency and Nasal Airflow Aerodynamics

deposition in the nasal valve region was found only for particles that crossed the main flow stream into the boundary streamline created by convergence of the lateral wall toward the nasal septum. Particle deposition in the expansion region, which represented the highest region of deposition in the nose, was due to turbulence and vortices. Deposition within the nasal cavity and nasopharynx entrance was caused by bending and sudden constrictions in CSA. A combination of a 15% decrease in the airway CSA and a 15% increase in nostril hair decreased particle deposition. Changes in particle deposition were attributed to changes in air velocity. The olfactory fissure was not modeled in this study, but since its anatomical characteristics are similar to those of the expansion region, a substantial amount of particle deposition would be expected. Although the validity of this model was assessed only by comparing total deposition values to empirical data, identification of the expansion area as the major site of major particle deposition is in accord with other reports (Fry and Black, 1972). Indeed, areas of large smoke particle deposition have been identified just posterior to the nasal valve and in the olfactory fissure (Proetz, 1951). This has been attributed to the “impingement effect,” which causes particle deposition just distal to an abrupt bend or expansion of a tube. 2.

Computer Simulation

Hahn et al. (1994) developed an odorant transport model, which included odorant molecule aerodynamic transport through bulk and lateral flow mechanisms and local odorant molecule movement from the mucosa surface to olfactory receptor by sorption and diffusion, as well as olfactory receptor interaction. The model predicted that increasing the flow rate would increase the perceived odor intensity for highly soluble odorants but decrease odor intensity for insoluble odorants. However, if only a limited sorption surface area is available, perceived odor intensity should decrease for all odorants regardless of the solubility. In a more detailed study, Keyhani et al. (1997) investigated inspired odorant molecule transport and uptake using the simulation designed by Keyhani et al. (1995). The concentration field and odorant mass flux at the nasal walls was calculated by uncoupled steady convective-diffusion equations. Total odorant flux, a measure highly correlated with perceived odor intensity, was a function of several transport parameters including the odorant solubility and diffusivity of the mucosal lining and the thickness of the mucus layer. Odorant flux increased with inlet concentration in a nonlinear fashion and depended on odorant solubility. For example, relative flux decreased for poorly soluble odorants and increased for highly soluble odorants.

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Odorant flux decreased along the olfactory slit from anterior to posterior and from inferior to superior, with this gradient being dependent on odorant mucus solubility. Thus, different odorants generated discernibly different flux patterns across the olfactory mucosa. The nasal valve was included in this model, and this model assumed a steady flow, thereby reducing the contribution of turbulent flow. The significance of this is not known; however, turbulent currents and vortices in the olfactory silt will probable influence the odorant flux pattern. 3.

In Vivo Studies

In vivo particle deposition measurement indicates the number of particles available for transport to the olfactory region, not the specific deposition location, and is calculated in one of two ways. Either the ratio of the particle concentration inhaled through the nose to the concentration exhaled through the nose is subtracted from a similar ratio measured at the mouth or an aerosol is drawn into the nose and out of the mouth during a breath-hold. Becquemin et al. (1991) measured deposition of 1-, 2.05-, and 2.8-m particles in the noses of adults, older children (12–15 years), and younger children (5.5–11.5 years) during rest and exercise. Average inspiratory flow rates were 27.5, 18.5, and 17.0 L/min during rest and 63.4, 31.3, and 28.0 L/min during exercise for adults, older children, and younger children, respectively. Particle deposition was significantly lower in younger children than adults for two particle sizes during rest and for all sizes during exercise. Particle deposition was significantly lower in older children for the smallest particle during exercise. Particle deposition increased with flow rate, implicating an inertial impaction rather than a gravitational sedimentation deposition mechanisum. This observation confirms the conclusion of earlier, less sophisticated, studies (Landahl and Black, 1947; Landahl and Tracewell, 1949). Since average volume flow rate decreased with age, the filtering efficiency of the child’s nose may be lower due to airflow rate or anatomy, or both. Although deposition may decrease with air velocity, a lower volume airflow rate does not necessarily indicate a lower velocity across subjects since velocity is dependent on nasal CSA. Particle deposition is related to particle density and size through the formula d 2Q ( is the particle density, d is the particle diameter, and Q is the volume airflow rate). This value is equivalent to the Stokes equation and correlates well with nasal deposition when it is above 337 g m sec for inspiration or 215 g m sec for expiration, suggesting that diffusional effects are more important than inertial effects for submicrometer particles (see Yu et al., 1981).

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V.

THE NASAL CYCLE

A.

The Nasal Cycle as an Ultradian Rhythm

A periodic simultaneous change in the volume of each nasal cavity has been reported in up to 80% of the adult population (Principato and Ozenberger, 1970). Although this change is classically believed to be opposing across the two sides of the nose, several variations of this cycle exist, and in some patients the direction of volume change may be in the same direction on both sides of the nose or may only involve one nasal cavity. Alteratively, the degree to which the volume and resistance of each nasal cavity changes may not be equal, and a dominant nasal cavity may result. This ultradian rhythm, called the nasal cycle, is observed in a number of animals. When present, its periodicity reportedly ranges from 40 minutes to 4 hours (Bojsen-Muller and Fahrenkrug, 1971; Eccles, 1978). The criterion for defining the nasal cycle is complex and depends on the detection method. For example, autocorrelation analysis assumes a regular periodic signal that is consistent in amplitude and frequency. As discussed above, nasal cavity volume and turbinate swelling is influenced by many physiological and environmental factors, and, as described below, the nasal cycle is highly influenced by physiological and environmental stimuli. Thus, the idea of an idealized nasal cycle with equal and consistent right and left nasal cavity volume changes with a consistent frequency is rather naive. It is not surprising that investigators who use strict criteria (i.e., Gilbert and Rosenwasser, 1987, Mirza et al., 1997) do not find a nasal cycle in a substantial number of subjects evaluated. Total NAR remains relatively constant, while the difference in NAR between the two sides of the nose may be as large as 12 cm H2O/L/sec for the normal airway (Cole and Haight, 1986). Although controversy exists regarding the operational definition of the nasal cycle and its prevalence, much of the discord probably stems from differences in measurement technique (i.e., Flanagan and Eccles, 1997). Recent studies using acoustic rhinometry demonstrate that the cycle is present, in some form, in a majority of adults and in children as young as 3 years. It persists after cessation of nasal airflow and occurs independent of structural abnormalities such as septal deviation (Gungor et al., 1999; Lund, 1996; Sung et al., 2000). The prevalence of the nasal cycle appears to change with age. For example, Fisher et al. (1995) found a “classical” reciprocal alternating pattern in 80%, an “in concert” pattern in 7%, and an “irregular” pattern in 13% of 15 healthy children ranging in age from 3 to 10 years. There is some evidence that the

proportion of subjects exhibiting a “classic” nasal cycle decreases with age (Mirza et al., 1997). Nasal turbinate swelling, which causes changing in NCV and NAR, is controlled by autonomic vascular tonicity of the nasal mucosa. Although the medulla may produce the autonomic changes through an N-methylD-aspartate–mediated system, the production of the underlying rhythm probably originates in the hypothalamus (Eccles, 1978; Galioto et al., 1991; Haxhiu et al., 1987). Sympathetic tonus decreases both turbinate blood flow and size (Malm, 1977) by vasoconstriction of deep capacitance vessels without altering superficial blood vessel flow (Kurita et al., 1988). Parasympathetic tonus causes opposite effects, although to a lesser extent (Anggard, 1977; Haight and Cole, 1986). The cyclical changes in asymmetrical autonomic tonicity are related to the basic rest-activity cycle (BRAC) of Kleitman (1967). Such diverse phenomena as asymmetrical hemispheric electroencephalographic (EEG) activity (Wertz et al., 1983), adrenal gland secretion of catecholamines (Kennedy et al., 1986), performance on visual/spatial psychological tasks (Klein et al., 1986), and sleep stage (Frye and Doty, 1992) are associated with the BRAC. The “rest” phase of the BRAC is associated with proportionately more right hemispheric integrated EEG activity, a spatial cognitive mode, a parasympathetic tonicity in unpaired organs, sympathetic tonicity on the left side of the body, parasympathetic tonicity on the right side of the body, a reduction in spontaneous vigilance, and an increase in napping behavior. Relatively greater airflow through the right nasal chamber is associated with the “activated” phase of the BRAC, during which effects opposite to those noted above are observed. The influence of the nasal cycle on NAR is modulated by many factors. Although emotional state, humidity, temperature, exercise, hyperventilation, anoxia, hypercapnia, environmental pollution, and psychological factors symmetrically alter total NAR, unilateral sensory stimulation asymmetrically alters NAR (Cole, 1982). For example, unilateral pressure to the axilla (Davies and Eccles, 1985) or lateral recumbency (Haight and Cole, 1986) will decrease NAR in the opposite or superior nasal cavity, respectively. Afferent pressure receptors in the pelvic and pectoral girdles and the deep subcutaneous tissue of the thorax are responsible for the latter response. Extended lateral recumbency can result in sustained inhibition of the nasal cycle (Haight and Cole, 1984). Abnormal responsiveness of the nasal turbinates, as seen in nasal sinus disease (e.g., allergic rhinitis, sinusitis) (Hilberg et al., 1995, Ophir et al., 1988), vasomotor rhinitis (Kuening, 1968), and multiple chemical sensitivities (Doty et al., 1988), may influence the nasal cycle. In addition, mucocilliary clearance and reactivity to nasal allergen provo-

Nasal Patency and Nasal Airflow Aerodynamics

cation is enhanced in the congested nasal chamber (Brooks et al., 1991; Littlejohn et al., 1992). Furthermore, the extremes of the nasal cycle can adversely affect patients with obstructed airways by further increasing NAR. B.

The Nasal Cycle and Olfactory Function

From a theoretical perspective, the nasal cycle may influence olfaction, on at least one side of the nose, in four ways. First, a change in turbinate size alters airstream velocity and distribution, thereby altering odorant access to the olfactory epithelium. Second, changes in autonomic tonicity of the mucosa can alter penetration or concentration of the odorant molecules reaching the olfactory receptors by changing the quantity and consistency of nasal secretions. Third, fluctuations in central arousal mechanism may alter olfactory function, especially since the locus coeruleus has direct connections to the anterior olfactory nucleus and olfactory bulb. Finally, if it is assumed that olfactory processing is localized, at least to some degree, to the right hemisphere, variations in relative hemispheric EEG activity correlated with the nasal cycle could alter odor information processing and perception. To determine if a relationship exists between the nasal cycle and olfactory sensitivity, Frye and Doty (1992) measured unilateral 2-butanone olfactory thresholds and NAR for 33 men and 44 women in two sessions separated by 4 hours. In approximately half of the subjects the nostril not sampling the odorant was occluded. If the nasal cycle had not spontaneously changed its phase at the beginning of the second session, an attempt was made to change the phase by applying pressure under the armpit, in the palm of the hand, or by auditory stimulation on the side of the more patent airway. In subjects whose contralateral naris was blocked, low right NAR was associated with decreased olfactory thresholds on both sides of the nose, whereas low left NAR was associated with comparatively increased olfactory thresholds on both sides of the nose. Thus, augmentation of olfactory sensitivity was associated with increased arousal and greater left hemispheric integrated EEG activity, and olfactory sensitivity attenuation was associated with decreased arousal and greater right hemispheric integrated EEG activity. These data suggest that NAR, per se, was not responsible of alterations in olfactory sensitivity and that cerebral EEG or central arousal mechanisms likely are involved. To better define this relationship, NAR was measured in eight right-handed male subjects every 15 minutes over 6 hours (R. E. Frye, A. Valle, and R. L. Doty, unpublished data). Olfactory sensitivity to phenyl ethyl alcohol and subject response bias was measured using a signal detection paradigm. The perithreshold odor con-

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centration was determined by a unilateral olfactory threshold test preceding the study; the contralateral nostril was blocked during odor sampling. The nasal cycle and unilateral nasal resistance were also measured. The left dominant phase of the nasal cycle was associated with an increased sensitivity and decreased subject response bias for odors presented to the right and left sides of the nose, respectively. Since asymmetrical changes in integrated hemispheric EEG activity are correlated with the nasal cycle and the majority of the olfactory bulb’s afferents project unilaterally, interhemispheric alteration in decision processing associated with olfactory recognition may explain this finding. VI. A.

NASAL AIRFLOW PERCEPTION Perception of Changes in Nasal Patency

Self-reported perceived changes in and measurements of nasal patency correlate following large modifications in nasal lumen size. For example, septoplasty (Broms et al., 1982; Larsen and Kristensen, 1990), inferior turbinate submucosal diathermy (Jones et al., 1985a), and aerosol steroid treatment of seasonal allergic rhinnitis and severe polyposis (Lancer et al., 1987, Lund et al., 1998) improve both perceived and measured nasal patency. Changes in perceived nasal patency induced by histamine or nasal allergen challenge are reliably correlated with NAR and rhinostereometry, but not MCA measurements (Graf, 1996; Lane et al., 1996; Zweiman et al., 1997). However, the perceived change in nasal patency after decongestion with 1% phenylephrine did not correlate with changes in MCA or NAR (Kim et al., 1998). Although symptom score does not correlate with NAR (Hardcastle et al., 1988a,b), perceived nasal patency does correlate with preoperative, but not postoperative, NAR (Gordon et al., 1989). The inspiratory turbulent parameter of Rohrer’s equation was found to be related to the severity of nasal obstructive symptoms in 75 patients (Naito et al., 1995). The poor relationship between perceived and measured nasal patency in normal subjects may be due to large intersubject variability. For example, individual correlations between daily nasal inspiratory peak flow rate and perceived nasal patency were strong despite significant differences in individual regression lines (Fairley et al., 1993). Although an overall relationship between perceived nasal patency and MCA or NCV was not found in subjects with nasal obstruction, these variables were correlated when 10 new subjects were studied on an individual level (Tai et al., 1998). Unilateral measurements and higher baseline NAR improve the strength of the relationship between perceived

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Frye

and measured nasal patency. For example, the correlation between NAR and perceived nasal patency in normal subjects was improved when unilateral, rather than total nasal airflow, was evaluated (Sipila et al., 1995) or when only the side of the nose with the greater NAR was studied (Hirschberg and Rezek, 1998). A correlation between unilateral perceived nasal patency and pre- and postdecongestion MCA and NAR was found in symptomatic patients (Roithmann et al., 1994). Small variations in measurable nasal patency changes are not reliably perceived in normal subjects. NAR changes induced by aspirin or allowed to occur spontaneously over a long or short observation period (i.e., single observation to 6 weeks) do not correlate with perceived patency changes (Jones et al., 1985b; Jones et al., 1989a). Gungor et al. (1999) found that nasal cycle related fluctuations in CSA and NCV did not correlate with perceived nasal patency. B.

Nasal Flow Receptors

Nasal flow receptors mediate the sensation of nasal airflow. These receptors are located in both the nasal vestibule and the nasal chamber, although the receptors at each location convey a different sensation. Local anesthesia of the nasal vestibule receptors produces a sensation of nasal obstruction (Jones et al., 1987), whereas local anesthesia of the nasal chamber receptors results in a sensation of increased nasal patency (Jones et al., 1986). Nasal chamber flow receptors are likely localized in the middle and posterior inferior turbinate mucosa (Wight et al., 1988b). Nasal patency receptors are probably predominant since local anesthesia of both the nasal vestibule and chamber produce a sensation of nasal obstruction (Jones et al., 1989b). Indeed, the sensitivity of the nose to an air jet pulse and to temperature changes is greatest at the nasal vestibule (Clarke and Jones, 1992, 1994). Trigeminal nerve afferents have been implicated in the cat (Davis and Eccles, 1987) and rat (Tsubone, 1989). In the human, the palatine nerve (a branch of V2) innervates the turbinates and nasal septum, while the ethmoid nerve (a branch of V1) innervates the vestibule. It is not known whether the different receptors causes different sensations; however, thermoreceptors may mediate airflow sensation since mucosal temperature, as measured by noncontact infrared thermometry, is correlated with subjective nasal patency (Willatt, 1993; Willatt and Jones, 1996). C.

Influence of Odors on Airflow Sensation

Eccles et al. (1987) demonstrated that particular odors influence nasal airflow perception without changing NAR. An aromatic combination (menthol, camphor, oil of pine

needles, and methyl salicylate) but not vanilla produced significantly higher perceived nasal patency ratings. L-Menthol lozenges also increased airflow sensation (Eccles et al., 1990; Naito et al., 1991). However, this effect was not induced by D-menthol, D-isomethol, or D-neomenthol vapors (Eccles et al., 1988). Simulation of trigeminal nasal flow receptors or interference with trigeminal thermoreceptor calcium conductance may cause these findings since L-menthol is a trigeminal stimulant.

VII.

CONCLUSIONS: AIRFLOW DYSNAMICS IN RELATION TO OLFACTION

The contribution of airflow within the olfactory cleft to olfaction has been considered by a large number of investigators. Unfortunately, many studies have significant design limitations and provide only limited insight into the mechanisms involved. Nonetheless, a window of elucidation for some basic principles in this area is provided. Incoming air currents are directed by the nasal vestibule. Turbulent flow is important for both mixing of odorant molecules and proper humidification and filtering of the incoming air. Although Reynolds numbers are not indicative of turbulent flow, such flow develops in the expansion region during inspiration and remains throughout the nose. Disorders within this region may alter vortices and turbulent flow by redirection of airstreams and augmenting nasal resistance. However, turbulence airflow prior to reaching the olfactory cleft may not be particularly important since secondary air currents and vortices are probably produced in the olfactory cleft during inspiration, thereby creating a standing eddy and promoting odorant mixing and inertial deposition. Although the particular portion of the naris responsable for directing particles to the olfactory region is controversial, it is known that the expiratory airstream exhibits higher velocities and a more direct path to the olfactory regions than the inspiratory airstream (Girardin et al., 1983). Inspiratory airflow velocity and volume flow correlates with particle deposition and the proportion of particles transported to the olfactory region. Humans will adjust duration of a sniff to optimize odorant perception in a low-flow nostril (Sobel et al., 2000). Indeed, in humans, airflow rate is positively correlated with odor intensity (Rehn, 1978), the number of odorants identified in a confusion matrix (Schwartz et al., 1987), and the magnitude of the olfactory evoked potential (Kobal and Hummel, 1991). In the clinical setting, a variety of mechanisms related to diseases of the nasal cavity can alter olfaction. Conductive airflow abnormalities definitely alter airflow

Nasal Patency and Nasal Airflow Aerodynamics

patterns. In some cases, endoscopic operative procedures, septoplasty, turbinoplasty, or adenoidectomy can improve olfactory function. However, many patients do not experience restoration of olfactory function following intervention. Nasal sinus disease associated with swelling of the superior nasal cavity likely obstructs airflow to the olfactory region, while disorders associated with the inferior turbinate or nasal septum presumably redistribute the airstream within the nasal cavity. Local changes in the volume of the olfactory meatus more than 1 cm inferior to the cribriform plate influence olfactory function, and polyps located in the superior meatus can deflect incoming air currents away from the olfactory meatus. Since only a small percentage of air flows through this region, abnormalities in the olfactory region most likely have little, if any, influence on nasal resistance, while profoundly affecting the proper delivery of odorant to the olfactory cleft. Airflow obstruction only accounts for a subset of alterations in olfactory perception. Many other nonobstructive mechanisms may result in anosmia or dysosmia. For example, infiltration of inflammatory cells into the olfactory mucosa may alter function of the olfactory receptors. Although direct destruction of olfactory receptors by inflammatory cells has not been shown, patients with inflammatory changes in the olfactory mucosa are at risk for olfactory deficits. In addition, changes in mucosa thickness and mucus consistency as a result of inflammation can alter odorant flux. Changes in olfactory mucosa topography may change olfactory perception as well as intensity. For example, a limited odorant sorption surface area will reduce, rather than increase, odor intensity as airflow increases (Hahn et al., 1994). Thus, decreased available olfactory mucosa will paradoxically decrease olfactory sensation as a patient attempts to use adaptive mechanisms of odor exploration. Odorant flux along the olfactory slit in an anterior to posterior and inferior to superior direction is unique to odorants of different solubility (Keyhani et al., 1997). These flux patterns produce a unique odor signature and contribute to odor identification. Destruction of portions of the olfactory mucosa can change this pattern identification in several ways. For example, a punchedout pattern of olfactory receptor dysfunction randomly disrupts identification of particular portions of the pattern, whereas olfactory receptor dysfunction in the anterior inferior portion of the olfactory slit decreases odor perception at the point of maximum flux. Random disruption of olfactory receptor function could result in perception of a nonunique pattern resulting in a dysosmia, whereas disruption of olfactory receptor function at the point of maximum flux may completely prevent odorant perception.

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Frye Scott, W. R., Taulbee, D. B., and Yu, C. P. (1978). Theoretical study of nasal deposition. Bull. Math. Biol. 40:581–603. Seiden, A. M., and Smith, D. V. (1988). Endoscopic intranasal surgery on olfaction. Chem. Senses 13:736. Series, F., Cormier, Y., Desmeules, M., and LaForge, J. (1989). Influence of respiratory drive on upper airway resistance in normal men. J. Appl. Physiol. 66:1242–1249. Shemen, L., and Hamburg, R. (1997). Preoperative and postoperative nasal septal surgery assessment with acoustic rhinometry. Otolaryngol. Head Neck Surg. 117:338–342. Shevrygin, B. V. (1973). Surgical intervention on the nasal septum for the purpose of improving and preserving olfaction. Zh. Ushn. Nos. Gor. Bolezn. 31:247–249. Silkoff, P. E., Chakravorty, S., Chapnik, J., Cole, P., and Zamel, N. (1999). Reproducibility of acoustic rhinometry and rhinomanometry in normal subjects. Am. J. Rhinol. 13:131–135. Simmen, D., Scherrer, J. L., Moe, K., and Heinz, B. (1999). A dynamic and direct visualization model for the study of nasal airflow. Arch. Otolaryngol. Head Neck Surg. 125:1015–1021. Simola, M., and Malmberg, H. (1998). Sense of smell in allergic and nonallergic rhinitis. Allergy 53:190–194. Sipila, J., Suonpaa, J., Silvoniemi, P., and Laippala, P. (1995). Correlations between subjective sensation of nasal patency and rhinomanometry in both unilateral and total nasal assessment. J. Otorhinolaryngol. Relat. Spec. 57:260–263. Sobel, N., Khan, R. M., Hartley, C. A., Sullivan, E. V., and Gabrieli, J. D. (2000). Sniffing longer rather than stronger to maintain olfactory detection threshold. Chem. Senses 25:1–8. Spaeth, J., Schultze, V., Klimek, L., Lengersdorf, A., and Mosges, R. (1996). Azelastine reduces histamine-induced swelling of nasal mucosa. J. Otorhinolaryngol. Relat. Spec. 58:157–163. Spoor, A. (1965). A new method for measuring nasal conductivity. Rhinol. Int. 3:27–35. Stevens, C. N., and Stevens, M. H. (1985). Quantitative effects of nasal surgery on olfaction. Am. J. Otolaryngol. 6:264–267. Strohl, K. P., Hensley, M. J., Hallett, M., Saunders, N. A., and Ingram, R. H. (1980). Activation of upper airway muscles before onset of inspiration in normal humans. J. Appl. Physiol. 49:638–642. Strohl, K. P., O’Cain, C. F., and Slutsky, A. S. (1982). Alae nasi activation and nasal resistance in healthy subjects. J Appl. Physiol. 52:1432–1437. Stuiver, M. (1958). Biophysics of the sense of smell. Master’s thesis, Groningen, The Netherlands. Sung, Y. W., Lee, M. H., Kim, I. J., Lim, D. W., Rha, K. S., and Park, C. I. (2000). Nasal cycle in patients with septal deviation: evaluation by acoustic rhinometry. Am. J. Rhinol. 14: 171–174. Swift, D. L., and Proctor, D. F. (1977). Access of air to the respiratory tract. In Respiratory Defense Mechanisms, J. D. Brian, D. F. Proctor, and L. M. Reid (Eds.). Marcel Dekker, New York, pp. 63–93. Szucs, E., and Clement, P. A. (1998). Acoustic rhinometry and rhinomanometry in the evaluation of nasal patency of patients with nasal septal deviation. Am. J. Rhinol. 12:345–352.

Nasal Patency and Nasal Airflow Aerodynamics Tai, C. F., Ho, K. Y., and Hasegawa, M. (1998). Evaluating the sensation of nasal obstruction with acoustic rhinometry and rhinomanometry. Kao Hsiung I Hsueh Ko Hsueh Tsa Chih 14: 548–553. Tanaka, Y., Morikawa, T., and Honda, Y. (1988). An assessment of nasal function in control of breathing. J. Appl. Physiol. 65:1520–1524. Taverner, D., Bickford, L., Shakib, S., and Tonkin, A. (1999a). Evaluation of the dose-response relationship for intra-nasal oxymetazoline hydrochloride in normal adults. Eur. J. Clin. Pharmacol. 55:509–513. Taverner, D., Danz, C., and Economos, D. (1999b). The effects of oral pseudoephedrine on nasal patency in the common cold: a double-blind single-dose placebo-controlled trial. Clin. Otolaryngol. 24:47–51. Terada, N., Yamakoshi, T., Hasegawa, M., Tanikawa, H., Maesako, K., Ishikawa, K., and Konno, A. (1998). The effect of ramatroban (BAY u 3405), a thromboxane A2 receptor antagonist, on nasal cavity volume and minimum cross-sectional area and nasal mucosal hemodynamics after nasal mucosal allergen challenge in patients with perennial allergic rhinitis. Acta Otolaryngol. Suppl. 537: 32–37. Tonndorf, J. (1939). Der Weg der Atemluft in der menschlichen Nase. Arch. Ohren. Nasen Kehlkopfhail 146:41–63. Tomkinson, A., and Eccles, R. (1998). Acoustic rhinometry: an explanation of some common artefacts associated with nasal decongestion. Clin. Otolaryngol. 23:20–26. Tsubone, H. (1989). Nasal “flow” receptors of the rat. Respir. Physiol. 75:51–64. Uddstromer, M. (1939). Nasal respiration: a critical survey of some current physiological and clinical aspects on the respiratory mechanism with a description of a new method of diagnosis. Acta Otolaryngol. (Stockh.) S42:1–146.

459 Van Dishoeck, H. (1964). The part of the valve and the turbinate in total nasal resistance. Int. Rhinol. 2:19–26. Werntz, D. A., Bickford, R. G., Bloom, F. E., and ShannahoffKhalsa, D. S. (1983). Alternating cerebral hemispheric activity and the lateralization of autonomic nervous function. Hum. Neurobiol. 2:39–43 Wight, R. G., Jones, A. S., and Beckingham, E. (1988a). Radical trimming of the inferior turbinate and its effect on nasal resistance to airflow. J. Laryngol. Otol. 102:694–696. Wight, R. G., Jones, A. S., and Clegg, R. T. (1988b). Comparison of anterior and radical trimming of the inferior turbinates and the effects on nasal resistance to airflow. Clin. Otolaryngol. 13:223–226. Willatt, D. J. (1993). Continuous infrared thermometry of the nasal mucosa. Rhinology 31:63–67. Willatt, D. J., and Jones, A. S. (1996). The role of the temperature of the nasal lining in the sensation of nasal patency. Clin. Otolaryngol. 21:519–523. Williams, H. L., Banvoetz, J. D., Brewer, D. W., Hinderer, K. H., McLaurin, J. W., Murtagh, J. A., Ogura, J. H., Sauders, S. H., Hyatt, R. E., Semenov, H., and Tonndorf, J. (1970). Report of Committee on Standardization of Definitions, Terms, Symbols in Rhinometry of the American Academy of Ophthalmology and Otolaryngology: A Handbook and Glossary. American Academy of Ophthalmology and Otolaryngology, Rochester, MN. Yu, C. P., Diu, C. K., and Soong, T. K. (1981). Statistical analysis of aerosol deposition in nose and mouth. Am. Ind. Hyg. Assoc. J. 42:726–733. Zweiman, B., Getsy, J., Kalenian, M., Lane, A., Schwartz, L. B., Doty, R., and Lanza, D. (1997). Nasal airway changes assessed by acoustic rhinometry and mediator release during immediate and late reactions to allergen challenge. J. Allerg. Clin. Immunol. 100:624–631.

22 Clinical Disorders of Olfaction Claire Murphy San Diego State University and University of California, San Diego, School of Medicine San Diego, California, U.S.A.

Richard L. Doty University of Pennsylvania, Philadelphia, Pennsylvania, U.S.A.

Heather J. Duncan University of Cincinnati College of Medicine, Cincinnati, Ohio, U.S.A.

I.

INTRODUCTION

these topics are not specifically addressed here. The reader is referred to Chapter 44 for disorders of taste function.

Olfactory dysfunction can arise from a variety of reasons and can profoundly influence a patient’s quality of life. Such problems are not uncommon, being present in at least 1% of the population under the age of 65 years, and in well over 50% of the population older than 65 years (Doty et al., 1984, 1986; Hoffman et al., 1998; Murphy et al., 2001; Schiffman, 1983). We now know that decrements in olfactory function are among the first clinical signs of Alzheimer’s disease and idiopathic Parkinson’s disease, and are commonly present in epilepsy, multiple sclerosis, and schizophrenia (see Chapter 23). Although some patients initially present with a frank complaint of a smell disturbance, others are unaware of their dysfunction, pointing out the need for routine quantitative olfactory assessment, which is now easily performed in the office (see Chapter 10). In this chapter, we describe the major olfactory disorders, how they are classified, and how they are evaluated and treated. Since other chapters focus, in detail, on olfactory dysfunction observed as a result of head trauma (Chapter 30), epilepsy (Chapter 23), toxic chemical exposure (Chapter 27), nutritional disturbances (Chapter 42), and neurodegenerative diseases and schizophrenia (Chapter 23),

II. CLASSIFICATION OF OLFACTORY DISORDERS Olfactory disorders can be reliably classified as follows: (a) (b) (c) (d) (e)

(f)

(g)

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Anosmia: inability to detect qualitative olfactory sensations (i.e., absence of smell function) Partial anosmia: ability to perceive some, but not all, odorants Hyposmia or microsmia: decreased sensitivity to odorants Hyperosmia: abnormally acute smell function Dysosmia (sometimes termed cacosmia or parosmia): distorted or perverted smell perception to odorant stimulation Phantosmia: a dysosmic sensation perceived in the absence of an odor stimulus (a.k.a. olfactory hallucination) Olfactory agnosia: inability to recognize an odor sensation, even though olfactory processing, language, and general intellectual functions are essentially intact, as in some stroke patients.

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Presbyosmia is sometimes used to describe smell loss due to aging, but this term is less specific than those noted above (e.g., it does not distinguish between anosmia and hyposmia) and is laden, by definition, with the notion that it is age, per se, that is causing the age-related deficit. When possible, it is useful to classify olfactory impairments into three general classes: (1) conductive or transport impairments from obstruction of the nasal passages (e.g., by chronic nasal inflammation, polyposis, etc.); (2) sensorineural impairment from damage to the olfactory neuroepithelium (e.g., by viruses, airborne toxins, etc.); and (3) central olfactory neural impairment from central nervous system (CNS) damage (e.g., tumors, masses impacting on olfactory tract, neurodegenerative disease, etc.). However, definitive classification of a given patient’s disorder into a given class is often not feasible, and these categories are not mutually exclusive. For example, both damage and blockage of airflow to the receptors can occur from chronic rhinosinusitis, and some viruses that damage the olfactory neuroepithelium also are transported into the CNS via the olfactory nerves, subsequently damaging central elements of the system as well (see Chapter 26).

III. CLINICAL EVALUATION OF OLFACTORY DISORDERS

TestTM (PST)(Doty et al., 1995), the 12-odor Brief-Smell Identification TestTM (B-SIT; also known as the CrossCultural Smell Identification TestTM) (Doty et al., 1996), the 12-item Odor Memory TestTM (OMT) (Bromley and Doty, 1995; Doty et al., 1995), the 40-odor University of Pennsylvania Smell Identification Test (UPSIT; known commercially as the Smell Identification TestTM or SIT) (Doty, 1995), an Odor Confusion Matrix Test (Wright, 1987), the Scandinavian Odor Identification Test (SOIT) (Nordin et al., 1999), the ‘Sniffin’ Sticks’ test (Hummel et al., 1997), the Viennese Olfactory Test Battery (WOTB) (Lehrner and Deecke, 1999), the Jet Stream Olfactometer Test (Ikeda et al., 1999), and an 8-odor identification test (Simmen et al., 1999). Electrophysiological tests (reviewed in detail in Chapter 11) are available in some specialized medical centers and can aid in the detection of malingering. Normative odor event-related potential (OERP) data have recently been published (Murphy et al., 2000b). Like psychophysical tests, such measures are sensitive to aging, gender, and a number of diseases. Unlike their visual and auditory counterparts, however, OERPs are presently unable to discern where in the olfactory pathway an anomaly exists. Although recent studies employing source localization analyses can roughly locate the source of the generator potentials involved, such localization is dependent upon the assumptions made in the underlying models and does not necessarily provide localization of pathology.

A.

B.

Quantitative Olfactory Testing

A common error made on the part of clinicians is to accept a patient’s report of sensory dysfunction and not to objectively verify the presence or magnitude of the problem. Many persons, particularly the elderly and those with dementia, are unaware of their dysfunction or are inaccurate in assessing its magnitude (Doty et al., 1987; Nordin et al., 1995). Standardized quantitative olfactory testing allows, in most instances, for (1) the characterization of the nature and degree of the chemosensory problem, (2) establishing the validity of the patient’s complaint, including the detection of malingering, (3) monitoring changes in function over time, and (4) providing objective data for establishing disability compensation. Fortunately, easy-toadminister tests of smell function have been developed, a number of which are commercially available. Such tests, which vary considerably in terms of reliability (see Chapter 10), include the T&T olfactometer test (Takagi, 1989), the San Diego Odor Identification Test (Anderson et al., 1992; Murphy et al., 1994), the Smell Threshold TestTM (Doty, 2000), the Alcohol Sniff Test (AST) (Davidson and Murphy, 1997), the 3-odor Pocket Smell

Medical History

The etiology of most cases of olfactory dysfunction can be ascertained from carefully questioning the patient about the nature, timing, onset, duration, and pattern of their symptoms, as well as a historical determination of antecedent events (e.g., head trauma, upper respiratory infections, toxic exposures, nasal surgeries). Fluctuations in function usually reflect obstructive, rather than neural, factors. Subtle symptoms of central tumors, dementia, tremor, and seizure activity (e.g., automatisms, occurrence of blackouts, auras, and déjà vu) should be sought, given the frequent association between smell dysfunction and not only brain tumors, but such disorders as epilepsy, idiopathic Parkinson’s disease, Alzheimer’s disease, and multiple sclerosis (see Chapter 23). Delayed puberty in association with anosmia, with or without midline craniofacial abnormalities, deafness, and renal anomalies, suggests the possibility of Kallmann’s syndrome or one of its variants. Medications being used prior to or at the time of the symptom onset should be determined, as some can profoundly influence olfaction [e.g., antifungal agents, angiotensin-converting enzyme (ACE) inhibitors].

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Medical conditions potentially associated with smell impairment should also be identified (e.g., liver disease, hypothyroidism, or diabetes). A history of epistaxis, discharge (clear, purulent, or bloody), nasal obstruction, and somatic symptoms, including headache or irritation, as well as a report as to whether the problem seems to be more prevalent on one side of the nose or the other, may be of localizing value. Idiopathic cases that present during winter months (which is more common than not) suggest the possibility of a viral origin, even if other elements of an upper respiratory infection were not present or recognized. It is critical for the clinician to be aware that while patients often present with the complaint of taste loss, quantitative testing usually reveals only an olfactory problem, reflecting decreased retronasal stimulation of the olfactory receptors during deglutition (Burdach and Doty, 1987; Murphy et al., 1977) (see Chapter 44). Importantly, the clinician should be cognizant of the fact that combinations of causal factors may be present that need to be considered. For example, persons with allergies or older persons may be more susceptible than others to smell loss from viral and other causes because of prior cumulative damage to the olfactory epithelium. Today, nasal endoscopy is the method of choice in assessing the health of the nasal cavity, and visualization of the olfactory meatal region is now possible through the use of both flexible and rigid rhinoscopes (Davidson et al., 1995). Modern imaging techniques can detect inflammatory processes within the nose and sinuses, as well as brain lesions and the integrity of the olfactory bulbs, tracts, and cortical parenchyma (see Chapter 28). For example, patients complaining of never having a sense of smell typically lack normal olfactory bulbs or tracts upon appropriate magnetic resonance imaging (MRI). Some laboratory tests (e.g., blood serum tests) are helpful in detecting underlying medical conditions suggested by history and physical examinations, such as infection, nutritional deficiencies (e.g., vitamins B6, B12), allergy, diabetes mellitus, and thyroid, liver, and kidney disease. Visual acuity, visual field, and optic disc examinations can aid in the detection of possible intracranial mass lesions that, in addition to producing visual deficits, impinge upon the olfactory tract.

IV.

CAUSES OF OLFACTORY DYSFUNCTION

As can be seen in Table 1, there are many reported etiologies for olfactory disturbance. Approximately two thirds of cases of chronic anosmia or hyposmia (i.e., those which are presumably permanent) that present to a clinic are likely due to prior upper respiratory infections, head trauma (Chapter 30), and nasal and paranasal sinus dis-

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ease, and most can be expected to reflect significant damage to the olfactory neuroepithelium (Deems et al., 1991; Goodspeed et al., 1987; Mott and Leopold, 1991). Listed below are major causes of olfactory dysfunction not discussed in detail elsewhere in the volume, along with an overview of findings for each of the disorders involved. A.

Upper Respiratory Infections

Although rarely appreciated, the most frequent cause of smell loss in the adult is an upper respiratory infection (URI), such as is associated with the common cold, influenza, pneumonia, or human immunodeficiency virus (HIV) (Akerlund et al., 1995; Deems et al., 1991; Hummel et al., 1998; Murphy et al., 2000a). Often the respiratory illness is described as being more severe than usual, and in many cases a dysosmia or phantosmia is present. The latter phenomena typically subside over time, leaving the patient with a noticeable olfactory deficit. Exactly what predisposes someone to viral- or bacterial-induced smell dysfunction or the mechanisms underlying it remains unclear, although most such losses become manifest in middle or older age, suggesting the potential importance of cumulative insult and the challenge of regeneration of the neuroepithelium in advancing age when proliferation of basal cells and immature neurons is significantly reduced (Loo et al., 1996). Direct insult to the olfactory neuroepithelium is presumably the primary basis of the problem in URIs, as biopsy studies of olfactory epithelia from patients with post-URI anosmia evidence extensive cicatrization, decreases in receptor cell number, absent or decreased numbers of cilia on remaining receptor cells, and replacement of sensory epithelium with respiratory epithelium (Douek et al., 1975; Jafek et al., 1990b; Yamagishi et al., 1988, 1994). However, as noted in Chapter 26, many viruses invade the CNS via the olfactory neuroepithelium, and the possibility exists that some viruses may influence central structures in addition to, or independent of, peripheral damage. Even though spontaneous recovery in some of these patients is theoretically possible, meaningful recovery is rare when marked loss has been present for a period of time. As with the case of cessation of smoking (Frye et al., 1990), however, there may be some moderate return of function over relatively long periods of time, depending upon the magnitude of the insult to the underlying basal cell membrane. For example, one study followed up 21 patients with URI-related smell loss longitudinally (average duration
3 years), noting that 19 evidenced significantly higher UPSIT scores on retest and that 13 reported subjective improvement (Duncan and Seiden, 1995). However, it should be noted that, according to standardized norms (Doty, 1995), the

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Table 1 Reported Agents, Diseases, Drugs, Interventions, and Other Etiological Categories Associated in the Medical or Toxicological Literature with Olfactory Dysfunctiona Drugs Adrenal steroids (chronic use) Amino acids (excess) Cysteine Histidine Analgesics Antipyrine Anesthetics, local Cocaine HCl Procaine HCl Tetracaine HCl Anticancer agents (e.g., methotrexate) Antihistamines (e.g., chlorpheniramine malate) Antimicrobials Griseofulvin Lincomycin Macrolides Neomycin Pencillins Streptomycin Tetracyclines Tyrothricin Antirheumatics Mercury/gold salts D-Penicillamine Antithyroids Methimazole Propylthiouracil Thiouracil Antivirals Cardiovascular/hypertensives Gastric medications Cimetidine Hyperlipoproteinemia medications Artovastatin calcium (Lipitor) Cholestyramine Clofibrate Intranasal saline solutions with: Acetylcholine Acetyl, -methylcholine Menthol Strychnine Zinc sulfate Local vasoconstrictors Opiates Codeine Hydromophone HCl Morphine Psychopharmaceuticals (e.g., LSD, psilocybin) Sympathomimetics Amphetamine sulfate Fenbutrazate HCI Phenmetrazine theoclate a

Categories are not mutually exclusive.

Endocrine/Metabolic Addison’s disease Congenital adrenal hyperplasia Cushing’s syndrome Diabetes mellitus Froelich’s syndrome Gigantism Hypergonadotropic hypogonadism Hypothyroidism Kallmann’s syndrome Pregnancy Panhypopituitarism Pseudohypoparathyroidism Sjögren’s syndrome Turner’s syndrome Industrial Dusts, Metals, Volatiles Acetone Acids (e.g., sulfuric) Ashes Benzene Benzol Butyl acetate Cadmium Carbon disulfide Cement Chalk Chlorine Chromium Coke/coal Cotton Cresol Ethyl acetate Ethyl and methyl acrylate Flour Formaldehyde Grain Hydrazine Hydrogen selenide Hydrogen sulfide Iron carboxyl Lead Mercury Nickel Nitrous gases Paint solvents Paper Pepper Peppermint oil Phosphorus oxychloride Potash Silicone dioxide Spices Trichloroethylene

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Table 1 (continued) Infections — Viral/Bacterial Acquired immunodeficiency syndrome (AIDS) Acute viral rhinitis Bacterial rhinosinusitis Bronchiectasis Fungal Influenza Rickettsial Microfilarial Lesions of the nose/Airway blockage Adenoid hypertrophy Allergic rhinitis Perennial Seasonal Atrophic rhinitis Chronic inflammatory rhinitis Hypertrophic rhinitis Nasal polyposis Rhinitis medicamentosa Structural abnormality Deviated septum Weakness of alae nasi Vasomotor rhinitis Medical Interventions Adrenalectomy Anesthesia Anterior craniotomy Arteriography Chemotherapy Frontal lobe resection Gastrectomy Hemodialysis Hypophysectomy Influenza vaccination Laryngectomy Oophorectomy Paranasal sinus exenteration Radiation therapy Rhinoplasty Temporal lobe resection Thyroidectomy Neoplasms—Intracranial Frontal lobe gliomas and other tumors Midline cranial tumors Parasagital meningiomas Tumors of the corpus callosum Olfactory groove/cribriform plate meningiomas Osteomas Paraoptic chiasma tumors Aneurysms Craniopharyngioma Pituitary tumors (esp. adenomas) Suprasellar cholesteatoma Suprasellar meningioma

Temporal lobe tumors Neoplasms—Intranasal Neuro-olfactory tumors Esthesioepithelioma Esthesioneuroblastoma Esthesioneurocytoma Esthesioneuroepithelioma Other benign or malignant nasal tumors Adenocarcinoma Leukemic infiltration Nasopharyngeal tumors with extension Neurofibroma Paranasal tumors with extension Schwannoma Neoplasms—Extranasal and Extracranial Breast Gastrointestinal tract Laryngeal Lung Ovary Testicular Neurological Amyotrophic lateral sclerosis Alzheimer’s disease Cerebral abscess (esp. frontal or ethmoidal regions) Down syndrome Familial dysautonomia Guam ALS/PD/dementia Head trauma Huntington’s disease Hydrocephalus Korsakoff’s psychosis Migraine Meningitis Multiple sclerosis Myesthenia gravis Paget’s disease Parkinson’s disease Refsum’s syndrome Restless leg syndrome Syphilis Syringomyelia Temporal lobe epilepsy Hamartomas Mesial temporal sclerosis Scars/previous infarcts Vascular insufficiency/anoxia Small multiple cerebrovascular accidents Subclavian steal syndrome Transient ischemic attacks Nutritional/metabolic Abetalipoproteinemia Chronic alcoholism Chronic renal failure (continued)

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Table 1 (continued) Cirrhosis of liver Gout Protein-calorie malnutition Total parenteral nutrition w/o adequate replacement Trace metal deficiencies Copper Zinc Whipple’s disease Vitamin deficiency Vitamin A Vitamin B6 Vitamin B12

average patient of this study was not anosmic on the first test occasion [mean (SEM) UPSIT score
21.2 (1.7)] and that the magnitude of improvement was modest, with the mean score still indicative of borderline severe/moderate microsmia [mean (SEM)
26.2 (1.5)]. Nonetheless, a positive correlation (r
0.55; p  0.001) was found between the change in UPSIT scores and the time between the two test administrations, implying that the longer the post-URI interval, the greater the recovery. More studies are needed to establish whether such recovery continues over longer time periods and whether an asymptote in performance occurs at some point.

Psychiatric Anorexia nervosa (severe stage) Attention deficit disorder Depressive disorders Hysteria Malingering Olfactory reference syndrome Schizophrenia Schizotypy Seasonal affective disorder Pulmonary Chronic obstructive pulmonary disease

of whom exhibit a generally more pathological epithelium (e.g., disordered arrangement of cells, more islands of respiratory-like epithelium) (Lee et al., 2000). This hypothesis is further supported by findings of weak or no associations between olfactory test scores and measures of nasal airway patency (save severe blockage), whether measured by rhinoscopy, rhinomanometry, or acoustic rhinometry (Apter et al., 1999; Cowart et al., 1993; Nordin et al., 1998; Scott et al., 1988).

B. Nasal and Sinus Disease Olfactory impairment that accompanies nasal or sinus disease has been traditionally viewed as being solely conductive (Fig. 1). Although marked airflow blockage undoubtedly alters olfactory sensitivity in some patients, empirical data suggest that surgical (e.g., excision of polyps) or medical (e.g., administration of topical or systemic steroids) treatment rarely returns function to normal, implying that blockage alone cannot completely explain the olfactory loss (for review, see Doty and Mishra, 2001). While, in general, olfactory dysfunction is related to the severity of rhinosinusitis [e.g., in one study, the mean UPSIT scores were 35, 31, 26, and 23 for Kennedy Stages I to IV of the disease, respectively (Downey et al., 1996)], the defining factor may, in fact, be the severity of histopathological changes within the olfactory mucosa (Jafek et al., 1987). Thus, Kern (2000) has reported that such severity is related to olfactory test scores in patients with chronic rhinosinusitis. Furthermore, biopsies from the neuroepithelial region of patients with nasal disease are less likely to yield olfactory-related tissue than biopsies from controls (Feron et al., 1998). The same is true for anosmic vs. nonanosmic rhinosinusitis patients, the former

Figure 1 Schematic drawing (coronal) of the nasal cavity and paranasal sinuses. The left side is typical of chronic rhinosinusitis, with pansinus obstruction. Airflow to the nasal vault (and olfactory epithelium) is obstructed by congestion of the ostiomeatal complex (i, infundibulum; up, uncinate process; eb, ethmoidal bulla; fr, frontal recess; mt, middle turbinate). The right side shows the situation following endoscopic surgery, which opens up the middle meatus, allowing freer air flow to the olfactory mucosa. The superimposed rectangle indicates the general location of the olfactory epithelium, which is actually posterior to this plane of section, on the upper lateral wall of the nasal septum, on the cribriform plate, and on the medial wall of the superior turbinate. (From Smith and Duncan, 1992.)

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1. Hypertrophied Adenoids

et al., 1996; Kondo et al., 1998; Mott et al., 1997; Rydzewski et al., 2000; Simola and Malmberg, 1998). Overall, there appears to be general correspondence between a patient’s self-report of olfactory loss and objective test measures. Golding-Wood et al. (1996), for example, evaluated odor identification ability before and after 6 weeks of betamethasone treatment in 25 well-documented perennial rhinitis patients. The patient group was initially divided into two groups: those who affirmatively answered the question “Is your sense of smell impaired?” (n
15) and those who did not (n
10). The UPSIT scores of each of the 15 members of the former group were higher after the betamethasone treatment [respective group means (SD)
18.93 (9.4) and 33.4 (4.01)]. This was not the case for those who initially felt that they had no problems smelling [respective pre-/posttreatment means (SD)
33.40 (4.01) and 32.8 (4.94)]. Moderate correlations between the UPSIT scores and the self-ratings of olfactory function were found both before (r
0.52) and after (r
0.58) treatment. As has been noted by others, however, the average posttreatment UPSIT score was still indicative of a mild hyposmic condition. The UPSIT scores retained, among the patients, a similar rank order before and after treatment (Spearman r
0.75). Several studies have examined olfactory function before and after allergen challenges (e.g., Hilberg, 1995; Hinriksdottir et al., 1997; Klimek and Eggers, 1997; Lane et al., 1996; Moll et al., 1998). In all cases, smell function decreased as a result of the challenges, although in the few cases that examined measures of airway patency, no association was noted between the degree of smell dysfunction and the patency measure. Hilberg (1995) assessed the comparative effects of the oral antihistamine terfenadine (an H1-blocker) and the topical steroid, budesonide, on an allergen challenge in subjects with nasal allergy uncomplicated by polyposis on olfactory dysfunction and various other hay fever symptoms. Although both drugs had an effect on the nonolfactory hay fever symptoms during the nasal pollen challenge, only the budesonide improved the challenge-related decrement in olfactory sensitivity. This steroid also was more effective in increasing nasal volume. Unfortunately, the improvement in olfactory function occurred in less than half of the patients (7/17; 41%).

Hypertrophied adenoid tissue can significantly block the nasal airflow of children whose airways are otherwise patent. In general, nasal resistance decreases by ~20–40% following adenoidectomy (Crysdale et al., 1985; Fielder, 1985). Olfactory thresholds to phenyl ethyl alcohol, determined in children with varying degrees of nasal obstruction, are related directly to clinical ratings of nasal obstruction (Fig. 2) (Delank, 1992; Ghorbanian et al., 1983). 2. Acute Viral-Related Rhinitis or Rhinosinusitis Two studies have quantitatively assessed olfaction longitudinally following the onset of the common cold. Akerlund et al. (1995) measured 1-butanol odor-detection thresholds in a group of student volunteers before and 4 days after nasal inoculation with the coronavirus 229E. The nine individuals who developed a cold had impaired olfactory thresholds on the postinoculation test relative to the controls—impairment that correlated with nasal congestion, but not nasal discharge. Hummel et al. (1998) evaluated smell function and airway patency in 18 women and 18 men at the time of onset of a natural cold, as well as 2, 4, 6, and 35 days later. The cold produced a decrease in the volume of the anterior nasal cavity, an increase in mucus secretion, an increase in olfactory thresholds, a decrease in intensity ratings, and a decrease in odorevoked potential amplitudes (N1) to both olfactory and trigeminal stimuli. Even when the airway was patent and mucus secretion was minimal, evoked potential amplitudes to olfactory stimuli were still depressed, indicating to the authors that URIs may influence olfactory function independent of nasal congestion. 3. Acute (e.g., Allergic) and Chronic Rhinitis and Rhinosinusitis The first large-scale empirical study of olfaction in allergic rhinitis was that by Cowart et al. (1993). Phenyl ethyl alcohol detection threshold measures were obtained from 91 patients with symptoms of allergic rhinitis and from 80 nonatopic controls. The allergy patients exhibited greater dysfunction than the controls, with 23.1% having a threshold at or above the 2.5 percentile of the controls. Clinical or radiographic evidence of rhinosinusitis or nasal polyps or both was associated with hyposmia in the allergy patients: 14.3% with no associated rhinosinusitis exhibited hyposmia, whereas 42.9% with associated rhinosinusitis did so. These general observations have been noted by others for rhinitis or rhinosinusitis patients employing a variety of olfactory test measures (Apter et al., 1999; Golding-Wood

C.

Nasal Surgery

Gross-Isseroff et al. (1989) obtained detection threshold and UPSIT measures in children with choanal atresia before and after surgical repair at relatively advanced ages (8–31 years). The three patients who had suffered from bilateral atresia had permanent olfactory deficits, whereas the one patient who had suffered from unilateral atresia

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Figure 2 (A) Nasal obstruction ratings, based upon assessment of mouth breathing and hyponasality, in 28 children before and after adenoidectomy. (B) Phenyl ethyl alcohol odor-detection thresholds before and after adenoidectomy in the same study population. Each line joins preoperative and postoperative values for an individual subject. (From Ghorbanian et al., 1983).

appeared to have normal function. These findings suggested to the authors the question as to whether early sensory exposure is needed for normal development of olfactory function (see Chapter 29). The limited empirical data available suggest that septoplasty and rhinoplasty have no adverse effects on olfactory function and may possibly improve function very slightly if the airway is significantly constricted (Kimmelman, 1994; Stevens and Stevens, 1985). Olfactory function has been evaluated before and after common operative procedures for chronic rhinosinusitis and/or polyposis unresponsive to more conservative treatments (e.g., allergen avoidance, nasal corticosteroids). Among such operations are middle turbinate medialization, polypectomy, uncinectomy, anterior ethmoidectomy, posterior ethmoidectomy, and sphenoidectomy, alone or in combination. With rare exception (e.g., Klimek et al., 1997), olfactory function improves considerably following such surgeries (Delank and Stoll, 1994, 1998; Downey et al., 1996; Hoseman et al., 2000; Kimmelman, 1994; Leonard et al., 1988; Seiden and Smith, 1988), even though the proportion of patients regaining normal olfactory function is typically less than 40% (e.g., Delank and Stoll, 1994, 1998; Leonard et al., 1988; Min et al., 1995) and many cases likely regresses within a year to preoperative levels (Klimek et al., 1997).

The degree of improvement is well exemplified by examining the median pre- and postoperative UPSIT scores calculated across six studies in which five or more patients were tested (Eichel, 1994; el Naggar et al., 1995; Friedman et al., 1999; Kimmelman, 1994; Lund and Scadding, 1994; Seiden and Smith, 1988). These values, 17.0 and 25.5, respectively, are indicative of an average change in function from total anosmia to a borderline moderate/severe microsmia.

D.

Tumors

Olfactory dysfunction can result from a variety of intranasal and intracranial tumors. McCormack and Harris (1955) reported on five cases in which neurogenic tumors arising in the lateral nasal wall were accompanied by anosmia and noted that over 100 such cases had been cited in the literature up to that time. Around 20% of the tumors of the temporal lobe or lesions of the uncinate convolution are said to produce some form of olfactory disturbance, most typically the hallucination of a bad smell (Furstenberg et al., 1943). The olfactory bulbs and tracts are very sensitive to pressure from meningiomas from the dura of the cribriform plate and surrounding regions (e.g., olfactory groove

Clinical Disorders of Olfaction

meningiomas, suprasellar ridge meningiomas), pituitary growths that extend above the diaphram of the sella turcica, and tumors inside or on the floor of the third ventricle. Thus, both unilateral and bilateral hyposmia and anosmia have been reported in cases of frontal lobe glioma, suprasellar meningioma, and sphenoidal ridge meningioma, as well as in cases involving nonneoplastic space–filling lesions, such as large internal carotid aneurysms extending over the pituitary fossa, aneurysms of the anterior communicating bifurcation, and hydrocephalus that pushes the floor of the third ventricle downward (Graff-Radford et al., 1997). Although signs other than olfactory ones are typically present in such cases, olfactory dysfunction can, in fact, be the sole sign (Barraquer-Berre and Fargus, 1950; McCormack and Harris, 1955; FitzSimon et al., 1997). According to Finelli and Mair (1991), the single most egregious error of neurologists is failure to recognize the symptom of anosmia as the principal or sole feature of an olfactory groove neoplasm. Unfortunately, the classical surgical approach to anterior skull base tumors — bifrontal craniotomy — is almost always associated with postoperative anosmia and other complications. For this reason, a number of surgical approaches that better spare the olfactory system have been devised (e.g., unilateral frontal craniotomy with orbital osteotomy; the transglabellar/subcranial approach)(Babu et al., 1995; Jung et al., 1997). It is of interest that olfactory tests were employed over 60 years ago to help diagnose cribriform plate meningiomas and to localize other tumors impinging upon the olfactory nerve (Elsberg, 1935a,b; Elsberg and Levy, 1935). Despite the limitations of the olfactory test procedure used [i.e., the so-called blast injection technique (see Chapter 10)], the olfactory recognition threshold was generally elevated on the side where a neoplasm exerted pressure on one olfactory nerve. When both nerves were involved, bilateral threshold elevation was noted, with greater elevation on the most affected side. Such changes were noted for expanding lesions on the ventral surfaces of the frontal cerebral lobes, as well as for suprasellar meningiomas and aneurysms of the internal carotid artery or the anterior part of the circle of Willis. Threshold values were not influenced by pituitary adenomas confined to the region of the sella turcica, but were heightened if growth occurred above the sella. Although most intracerebral tumors were not associated with altered recognition thresholds, they were associated with prolonged duration of olfactory adaptation or fatigue. Thus, tumors in or near the midline of the cranial cavity produced long-lasting adaptation (e.g., parasagital meningiomas, tumors of the corpus callosum, and infiltrating growths extending to the medial surface of a cerebral hemi-

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sphere). When generalized intracranial pressure was observed, decreased recognition threshold sensitivity was sometimes present (Elsberg, 1935b). Although modern imaging techniques may make such testing, in the general sense, seem rather archaic, it is conceivable that olfactory testing could still be of value today in the early detection of some brain tumors. Recently, Daniels et al. (2001) examined odor discrimination performance and OERPs in 20 subjects with unilateral frontal or temporal lobe brain tumors. Patients with right-side lesions exhibited deficits in odor discrimination in both the left and right nares. Patients with left-side lesions only exhibited attenuated function when the odorant was presented to the left side. The amplitude of the OERPs was decreased after left-side stimulation, but not after right-side stimulation. Interestingly, a correlation was present between the olfactory and acoustic event–related potentials in patients with right-side lesions after right-side stimulation. E.

Congenital Anosmia

A number of patients report never having experienced smell function, with the phenotype ranging from being unable to detect only one or a few compounds to total anosmia.* In *

Most persons are said to be constitutionally unable to detect a few specific odorants, a phenomenon termed specific anosmia. First described by Blakeslee for the fragrance of verbena flowers (Blakeslee, 1918), dozens of specific anosmias have been reported in the literature, including ones for musks, trimethylamine, hydrogen cyanide,n-butyl mercaptan, and isovaleric acid (for review, see Takagi, 1989). However, like the blind spot of the retina, specific anosmias are not typically recognized by a patient and are rarely reasons for seeking clinical help. Moreover, some specific anosmics can, in fact, detect high concentrations of odorants to which they are said to be anosmic, implying hyposmia rather than anosmia, and not all specific anosmias are really that specific. Amoore (1971), for example, tested the thresholds of 10 subjects who exhibited specific anosmia to isobutyric acid to 17 other compounds, finding heightened thresholds for straight chain fatty acids with four to seven carbon atoms. Importantly, exposure to, or repeated testing with, some odorants, including ones associated with specific anosmias, can result in an increase in their sensitivity in both humans and rats (Doty and FergusonSegall, 1989; Doty et al., 1981). For example, Wysocki et al. (1989) found that the ability to perceive androstenone could be induced in 10 of 20 initially insensitive subjects by systematically exposing them to this odorant, an observation confirmed by others (Moller et al., 1999). This finding led Wysocki et al. to suggest that three categories of humans subjects may exist in regards to responsiveness to this substance; namely, the truly anosmic, the inducible, and those who are either constitutionally sensitive or have experienced incidental exposure to the agent.

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the vast majority of cases of total anosmia, appropriate MRI evaluation of the cribriform and gyrus rectus regions reveals agenesis or dysgenesis of the olfactory bulbs and stalks (Yousem et al., 1996). While some of these cases may represent long-term degeneration from viral or traumatic insults to the olfactory epithelium or fila (reflecting the withdrawal of trophic influences on the bulb from intact olfactory neurons), most are presently assumed to be hereditary. Lygonis (1969) reported total anosmia (without any apparent associated disorders) in members of four generations of a family living in an isolated island community and suggested that the pattern of inheritance was likely autosomal dominant. Singh et al. (1970) described a familial anosmia loosely associated with premature baldness and vascular headaches, which appeared to be inherited as dominant with varying penetrance. Several congenital olfactory syndromes are accompanied by endocrine dysfunction (e.g., Kallmann’s syndrome), and are discussed in the next section on endocrine disorders. F.

Endocrine Disorders

Although a number of endocrine disorders have been associated with smell dysfunction, many more have not even been assessed for this problem. Of those that have been examined, the mechanisms responsible for the smell loss — aside from obvious anatomical alterations in the primary olfactory pathways—are poorly understood. Listed below are disorders for which at least some quantitative olfactory test data are available. 1. Adrenocortical Insufficiency (Addison’s Disease) Henkin and Bartter (1966) reported that patients with untreated adrenocortical insufficiency exhibited greater odor detection threshold sensitivity, relative to normal controls, not only for the odorants pyridine, thiophene, and nitrobenzene, but for the vapors above aqueous solutions of six tastants (NaCl, KCl, HCl, NaHCO3, sucrose, and urea). These effects were remarkable, with none of distributions of scores from the patient and control groups showing any overlap. In the case of the volatiles arising from the tastants, the patients detected differences between the water and the vapors of the tastants at 1/10,000 the concentration of that noted for normals. The increased sensitivity did not return to normal following daily 20 mg injections of deoxycorticosterone acetate (DOCA) for periods up to 10 days. In contrast, treatment of the subjects with 20 mg of the carbohydrate-active steroid prednisolone returned sensitivity to normal within 24 hours. These phenomena, however, appear quite general, as similar alterations in taste sensitivity were noted for all of the

aforementioned tastants, as well as for pure-tone auditory thresholds, and for filtered and nonfiltered speech discrimination tests performed at 40 and 60 dB above threshold (Henkin and Daly, 1968; Henkin et al., 1967). Attempts to replicate or extend these findings have apparently not been forthcoming. Such efforts are sorely needed, however, since such marked hypersensitivity is unprecedented in the chemical senses literature, and numerous tests of auditory, gustatory, and olfactory function in rats before and after adrenalectomy have found no hypersensitivity of any sort. Indeed, the available data suggest that adrenalectomy may, in fact, produce decrements in chemosensory function (e.g., Brosvic and Rowe, 1992; Conn and Mast, 1973; Doty et al., 1991; Kosten and Contreras, 1985; Weigel et al., 1989). 2.

Chromatin Negative Gonadal Dysgenesis (Turner’s Syndrome)

Turner’s syndrome (TS) is a form of gonadal dysgenesis resulting from a 45, X karyotype (X-chromosomal monosomy). TS is characterized by a female phenotype, a shield-like chest, short stature, short and sometimes webbed neck, low-set ears, small mandible, a higharched palate, and sexual infantilism. Other problems in some cases include congenital lymphedema, skeletal anomalies, abnormalities of the nails, and cardiac and renal deficits. In the sole olfactory study published on TS, nine patients were found to have elevated detection and recognition thresholds to the three odorants assessed (pyridine, thiophene, nitrobenzene) (Henkin, 1967). Gonadal hormone therapy reversed the chemosensory deficits. Interestingly, the mothers of the patients exhibited similar olfactory abnormalities, which the author suggests is in accord with a potential genetic basis for the chemosensory disorder. 3.

Cushing’s Syndrome

Cushing’s syndrome (CS) is a disease characterized by chronic excessive secretion of adrenal corticosteroids (i.e., hypercortisolism). Approximately 80% of such cases reflect excessive secretion of adrenocorticotropic hormone (ACTH) (e.g., ACTH-producing pituitary tumors), whereas the remainder reflect ACTH-independent etiologies (e.g., adrenal gland adenomas and carcinomas, cortisol-secreting tumors) (Ferrante, 1999). According to Henkin (1975), patients with CS exhibit a decrease in sensory detection acuity for olfaction, gestation, hearing, and proprioception. Dogs given chronic dexamethasone (an animal model of Cushing’s syndrome) do appear to have increased detection thresholds

Clinical Disorders of Olfaction

for benzaldehyde and eugenol, in accord with this notion (Ezeh et al., 1992). 4.

Hypothyroidism

It is well established that many hypothyroid patients complain of chemosensory problems (Deems et al., 1991). Thus, Lewitt et al. (1989) reported that 11 of 16 (69%) hypothyroid patients they evaluated complained of alterations in taste and smell function, and McConnell et al. (1975) reported that 7 of 18 patients with untreated primary hypothyroidism (39%) were cognizant of some alteration in their sense of smell, with 3 (17%) experiencing dysosmia. Intuitively one might expect hypothyroidism to be associated with chemosensory deficits, since it is generally assumed that this disease produces alterations in other sensory systems. Thus, 36–83% of hypothyroid patients are said to experience somesthetic disturbances, and 25–45% auditory or visual dysfunction, with night blindness reportedly being a common feature of the disorder (Mattes et al., 1986). Nonetheless, this literature is somewhat limited, and there is controversy as to whether objective measures of olfactory function are, in fact, altered in this disease. In a pioneering study on this topic, McConnell et al. (1975) reported that odor-detection thresholds for pyridine and nitrobenezene, as well as various taste threshold measures, were strikingly elevated in patients with untreated hypothyroidism—a problem that readily resolved following thyroxine treatment. In contrast, Lewitt et al. (1989) found no detection threshold differences between 16 patients and 17 controls for the odorant phenyl ethyl alcohol and only a slight, but statistically significant, difference on a suprathreshold test of odor identification. Various measures of suprathreshold taste function, as well as auditory and visual evoked potentials, did not differ between the controls and patients, and no pre-/post-thyroxin differences were observed for any measure. Similar negative findings have been reported by others for various taste measures (Pittman and Beschi, 1967). The reason for these discrepancies is not clear, although common test substances and procedures were not used across studies, and it is not apparent whether the patients were comparable in terms of the nature, duration, and severity of their hypothyroid problems among the studies. It is of interest that well-controlled psychophysical studies employing rats have found no differences in either odor- or taste-detection performance before or after the induction of hypothyroidism (Brosvic et al., 1992, 1996) but have shown large differences in taste preference measures for NaCl, HCl, and quinine, but not for sucrose, following functional thyroidectomy (Brosvic et al., 1996). Although

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an earlier mouse study linked hypothyroidism to anosmia, the paradigm employed was, in fact, a two-bottle preference paradigm, leaving open the possibility that dysosmia, rather than anosmia, induced the behavioral changes (Beard and Mackay-Sim, 1987). 5.

Pseudohypoparathyroidism

Pseudohypoparathyroidism (PHP) is a rare endocrine disorder characterized by deficiencies in responsiveness to, but not in the production of, parathyroid hormone. Individuals with Type Ia PHP exhibit a generalized hormone resistance, a deficiency of the alpha chain of the stimulatory guanine nucleotide-binding protein (Gs) of adenylyl cyclase, and an unusual constellation of skeletal and developmental abnormalities termed Albright hereditary osteodystrophy (AHO). Among the abnormalities expressed in AHO are obesity, short stature, brachydactyly, round faces, and subcutaneous ossifications. Type Ib PHP patients, in contrast, exhibit a specific hormone resistance to parathyroid hormone, do not express a deficiency in Gs protein activity, and do not have AHO. PHP was first reported to be associated with olfactory dysfunction in 1968 (Henkin, 1968). Subsequent studies reported that Type Ia PHP, but not Type Ib PHP, is accompanied by decreased olfactory function (Ikeda et al., 1988; Weinstock et al., 1986). The olfactory problem was believed to reflect the Gs protein deficiency of the Type Ia patients, since Gs proteins are involved in the first stages of olfactory transduction (Ikeda et al., 1988; Weinstock et al., 1986). However, a more recent comprehensive study, while confirming that Type Ib PHP patients exhibit altered olfactory function on three types of tests (odor identification, detection threshold, and discrimination/memory), also found Type 1b patients to have similar olfactory dysfunction, making it unlikely that the olfactory dysfunction is related to the proposed Gs protein deficiency (Doty et al., 1997). In this same study, patients with pseudopseudohypoparathyroidism (PPHP), a disorder found in some relatives of patients with Type Ia PHP, were found to have relatively normal smell function. Since, like PHP Type Ia, PPHP is accompanied by a Gs protein deficiency and by AHO, but not by a generalized end organ hormone insensitivity, the smell loss of PHP Type Ia patients clearly cannot be explained on the basis of Gs. Thus, while we now know that several forms of PHP exhibit olfactory loss, the mechanism for the loss is obscure. 6.

Kallmann’s Syndrome

While there are reports of smell loss in cases of hypergonadotrophic hypogonadism (Males and Schneider, 1972),

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cases of anosmia in association with idiopathic hypogonadotrophic hypogonadism (IHH) (Kallmann’s syndrome, or KS) are much more common. KS is generally believed to reflect an autosomal dominant mode of inheritance with incomplete expressivity (Quinton et al., 2001). Anomalies in addition to anosmia that are sometimes seen in KS patients are cryptorchidism, midline craniofacial abnormalities, tooth agenesis, deafness, and renal anomalies. Although women can have KS, it is much more prevalent in men. Interestingly, family members related to individuals with this syndrome may have both hypogonadism and anosmia; others may have anosmia but normal gonadal function, whereas others may be entirely normal. Although MRI studies generally reveal bilateral agenesis or dysgenesis of the olfactory bulbs and tracts (Bajaj et al., 1993; Klingmuller et al., 1987; Yousem et al., 1993), there is one report of a IHH patient with aplasia of the left, but not the right, olfactory tract and bulb (Wustenberg et al., 2001). The limited data available suggest that the olfactory epithelia of patients with KS have pathological changes analogous to those of mammals whose connections between the olfactory fila and the olfactory bulb are severed; namely, morphologically immature receptor neurons lacking cilia, a decrease in the number of olfactory receptor cells, and the formation of intraepithelial neuromas (Schwob et al., 1993). In one nasal biopsy study, the total lack of an olfactory neuroepithelium in a KS subject was reported (Jafek et al., 1990a), although this observation must be viewed conservatively in light of sampling problems endemic to such biopsy studies (Paik et al., 1992). Probably because of its association with altered olfactory pathway anatomy, the anosmia noted in Kallmann’s syndrome is not reversed by either gonadotropin or gonadal steroid therapy. However, because the testicular derangement can be largely reversed by hormones when administered at the appropriate time, it is important that attempts be made to detect the endocrine problem as early as possible. Sparkes et al. (1968) stress that anosmia serves as an important diagnostic marker in the early detection of such problems, and Mroueh and Kase (1968) suggest that perhaps all pediatric patients presenting with anosmia should undergo a specific gonadal evaluation to prevent the possible development of irreversible atrophic changes. Unfortunately many individuals lacking smell ability (particularly youngsters who do not recognize the deficit or who are too shy to mention it) do not seek help for this difficulty, and routine pediatric examinations rarely test olfactory function. G.

Psychiatric Disorders

A number of psychiatric disorders are reportedly associated with altered smell function, including schizophrenia and

chronic hallucinatory psychoses (Chapter 23), seasonal affective disorder (Postolache et al., 1999, 2001), and severe stage anorexia nervosa (Fedoroff et al., 1995). Among the more interesting of such syndromes is “olfactory reference syndrome,” a condition generally viewed as distinct from schizophrenic or affective disorders (Bishop, 1980; Pryse-Phillips, 1971). In this disorder, the patient believes that smells emanate either from his or her own body (intrinsic hallucinations) or from elsewhere (extrinsic hallucinations). The hallucinations can be “minimal,” where the odor is complained about but the patient does not take steps to remove it, or the “reasonable” reaction, in which the patient takes steps to eliminate the odor (e.g., complaining to authorities or plugging up the chimney with newspapers). In intrinsic hallucinatory cases, compulsive washing behavior, changing clothes, and restriction of social activity is frequently seen. In many cases, this syndrome is closely linked with an obsessive-compulsive disorder and is amenable to treatment with serotonin-uptake inhibitors (Dominguez and Puig, 1997; O’Sullivan et al., 2000; Stein et al., 1998).

V.

TREATMENT OF OLFACTORY DISORDERS

Meaningful treatments are available for some, but not all, patients whose olfactory dysfunction is conductive, i.e., resulting from blockage of airflow to the olfactory neuroepithelium. Obstruction may arise from several causes, including allergic rhinitis, polyps, chronic sinusitis, or rhinitis. Effective therapies for olfactory loss secondary to allergic rhinitis include allergy management, topical cromolyn, topical and systemic corticosteroid therapies, and surgical procedures to reduce inflammation or obstructions. A brief course of systemic steroid therapy can be useful in distinguishing between conductive and sensorineural olfactory loss, as patients with the former will often respond positively to some extent to the treatment (Davidson et al., 1987), although longer-term systemic steroid therapy is not advised. Topical nasal steroids are often ineffectual in returning smell function because the steroid fails to reach the affected regions in the upper nasal passages. Increased efficacy presumably occurs when the nasal drops or spray are administered in the headdown Moffett’s position (Mott et al., 1997). In patients with obstructive inflammatory disease, swelling of the ostiomeatal complex can prevent drainage from the sinuses, causing chronic sinusitis. Antibiotic therapy in combination with control of the allergic symptoms underlying the inflammation is effective in many of these cases. Resistant cases typically require surgery to improve drainage and clear infection.

Clinical Disorders of Olfaction

Patients with nasal polyps typically present with longstanding history of inflammation and congestion, and many have a previous history of medical or surgical treatment for the polyps. Though relief may be time limited in many of these cases, surgery is indicated both to treat the obstructed breathing and to improve smell function. Even a moderate degree of ostiomeatal disease without accompanying polyps can result in significant olfactory impairment (Smith and Seiden, 1991). Septal deviation can interfere with drainage at the osteomeatal complex, contributing to the development of sinusitis. Septal deviation can also obstruct airflow, and thus odorant flow, to the olfactory receptors. In patients with sinusitis secondary to inflammation of the ostiomeatal complex and septal deviation, endoscopic sinus surgery (ESS) combined with septoplasty (which also improves the approach for ESS) will improve many of the symptoms related to the underlying disease (Davidson et al. 1995; Murphy et al., 2000). Surgical procedures that reduce nasal obstruction have been demonstrated to improve olfactory function (Ghorbanian et al., 1983; Ophir et al., 1986). The alleviation of allergic disease in the ostiomeatal complex by endoscopic ethmoidectomy can improve or restore olfactory sensitivity (Hosemann et al., 2000; Seiden and Smith, 1988). Thus, olfactory disorders caused by obstruction are often amenable to treatment (Davidson et al., 1995; Doty and Snow, 1987). Surgical patients may require ancillary treatment. In patients who remained anosmic after surgery for nasal and sinus polyps, Stevens (2001) reported that oral but not intranasal steriods were effective in restoring smell function to normal in most patients. Tomooka et al. (2000) reported improved function with nasal irrigation after endoscopic sinus surgery. There is no treatment for smell loss secondary to congenital or other malformations of the olfactory bulbs or stalks. In general, the olfactory dysfunction due to sensorineural causes is difficult to manage, and the prognosis for patients suffering from long-standing total loss due to upper respiratory illness or head trauma is poor. Most patients who recover smell function subsequent to head trauma do so within 12 weeks of injury (Costanzo et al., 1995). Although there are no verified treatments for trauma-related olfactory loss, anti-inflammatory agents may minimize posttraumatic sequelae in some cases. Recent rat research suggests that application of nerve growth factor onto the olfactory epithelium may alleviate axotomy-induced degenerative changes in the olfactory receptor neurons, although it is not known whether this has any functional consequence or if such a procedure in humans would be efficacious (Kasuno et al., 2000). Tobacco smoking by itself rarely causes complete loss of the sense of smell, although patients who quit smoking

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typically have dose-related improvement in olfactory function and flavor sensation over time (Frye et al., 1990). Central lesions, such as CNS tumors that impinge on olfactory bulbs and tracts and epileptogenic foci within the medial temporal lobe that result in olfactory seizures, can often be resected in a manner that allows for some restoration of olfactory function, as mentioned earlier in the chapter. Medications that induce distortions of olfaction can often be discontinued and replaced with other types of medications or modes of therapy. Despite the fact there are advocates for zinc and vitamin therapies, there is no compelling evidence that these therapies work except in cases where frank zinc or vitamin deficiencies exist. Similarly, the employment of aminophylline, in attempts to increase the level of cAMP within the olfactory receptor cells, have no sound empirical basis for improving olfaction and have never been assessed relative to a placebo. Prognosis for recovery seems to be better for patients with less severe hyposmia or microsmia than for those with anosmia or severe microsmia. In some etiologies, this reflects the less extensive damage into the basal cell layer of the epithelia and possibly less fibrosis around the foramina of the cribriform plate through which the olfactory nerve axons pass. An important component of therapy for many patients is the quantitative establishment of the true degree of olfactory loss. This places the patient’s problem into overall perspective; thus, it can be therapeutic for an older person to learn that, while his or her smell function is not what it used to be, it still falls above the average of his or her peer group. ACKNOWLEDGMENTS This paper was supported, in part, by the following grants from the National Institutes of Health, Bethesda, MD: RO1 AG 04085, RO1 AG 27496, RO1 DC02064, RO1 DC 02974, RO1 DC 04278, and PO1 DC 00161. REFERENCES Akerlund, A., Bende, M., and Murphy, C. (1995). Olfactory threshold and nasal mucosal changes in experimentally induced common cold. Acta Oto-Laryngol. 115:88–92. Amoore, J. E. (1971). Olfactory genetics and anosmia. In Handbook of Sensory Physiology. Vol. IV. Chemical Senses. Part I, Beidler, L. M. (Ed.). Springer-Verlag, Berlin, pp. 145–156. Anderson, J., Maxwell, L., and Murphy, C. (1992). Odorant identification testing in the young child. Chem Senses 17:590. Apter, A. J., Gent, J. F., and Frank, M. E. (1999). Fluctuating olfactory sensitivity and distorted odor perception in allergic rhinitis. Arch. Otolaryngol. Head Neck Surg. 125:1005–1010.

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477 Scott, A. E., Cain, W. S., and Clavet, G. (1988). Topical corticosteroids can alleviate olfactory dysfunction. Chem. Senses 13:735 Seiden, A. M., and Smith, D. V. (1998). Endroscopic intranasal surgery as an approach to restoring olfactory function. Chem. Senses 13:736. Simmen, D., Briner, H. R., and Hess, K. (1999). Screeningtest des Geruchssinness mit Riechdisketten. Laryngo-RhinoOtologie 78:125–130. Simola, M., and Malmberg, H. (1998). Sense of smell in allergic and nonallergic rhinitis. Allergy 53:190–194. Singh, N., Grewal, M. S., and Austin, J. H. (1970). Familial anosmia. Arch. Neurol. 22:40–44. Smith, D. V., and Seiden, A. M. (1991). Olfactory dysfunction. In The Human Sense of Smell, D. G. Laing, R. L. Doty, and W. Breipohl, (Eds.). Springer-Verlag, New York, pp. 283–305. Sparkes, R. S., Simpson, R. W., and Paulsen, C. A. (1968). Familial hypogonadotropic hypogonadism with anosmia. Arch. Intern. Med. 121:534–538. Stein, D. J., Le Roux, L., Bouwer, C., and Van Heerden, B. (1998). Is olfactory reference syndrome an obsessive-compulse spectrum disorder?: two cases and a discussion. J. Neuropsychiat. Clin. Neurosci. 10:96–99. Stevens, C. N., and Stevens, M. H. (1985). Quantitative effects of nasal surgery on olfaction. Am. J. Otolaryngol. 6: 264–267. Takagi, S. F. (1989). Human Olfaction. University of Tokyo Press, Tokyo. Tomooka, L. T., Murphy, C., and Davidson, T. M. (2000). Clinical study and literature review of nasal irrigation. Laryngoscope 110:1189–1193. Weigel, M. T., Prazma, G., and Pillsbury, H. C. (1989). Auditory acuity in adrenocorticoid insufficiency. Am. J. Otol. 10: 267–271. Weinstock, R. S., Wright, H. N., Spiegel, A. M., Levine, M. A., and Moses, A. M. (1986). Olfactory dysfunction in humans with deficient guanine nucleotide-binding protein. Nature 322:635–636. Wright, H. N. (1987). Characterization of olfactory dysfunction. Arch. Otolaryngol. Head Neck Surg. 113:163–168. Wustenberg, E. G., Fleischer, A., Gerbert, B., Abolmaali, N., Huttenbrink, K. B., and Hummel, T. (2001). Normal sense of smell in Kallmann syndrome. A case report [in German]. Laryngo-Rhino-Otologie 80:85–89. Wysocki, C. J., Dorries, K. M., and Beauchamp, G. K. (1989). Ability to perceive androstenone can be acquired by ostensibly anosmic people. Proc. Natl. Acad. Sci. USA 86: 7976–7978. Yamagishi, M., Hasegawa, S., and Nakano, Y. (1988). Examination and classification of human olfactory mucosa in patients with clinical olfactory disturbances. Arch. Oto-RhinoLaryngol. 245:316–320. Yamagishi, M., Fujiwara, M., and Nakamura, H. (1994). Olfactory mucosal findings and clinical course in patients with olfactory disorders following upper respiratory viral infection. Rhinology 32:113–118.

478 Yousem, D. M., Turner, W. J., Li, C., Snyder, P. J., and Doty, R. L. (1993). Kallmann syndrome: MR evaluation of olfactory system. Am. J. Neuroradiol. 14:839–843.

Murphy et al. Yousem, D. M., Geckle, R. J., Bilker, W., McKeown, D. A., and Doty, R. L. (1996). MR evaluation of patients with congenital hyposmia or anosmia. Am. J. Roentgenol. 166:439–443.

23 Odor Perception in Neurodegenerative Diseases Richard L. Doty University of Pennsylvania, Philadelphia, Pennsylvania, U.S.A.

I.

INTRODUCTION

with PD. Third, in some neurodegenerative diseases, such as PD, scores on most olfactory tests are unrelated to disease stage or progression, whereas in others, such as AD, this appears not to be the case. And finally, it is now well established that in MS the number of plaques within olfactory-related CNS structures, but not in other brain regions, is strongly correlated with the degree of olfactory dysfunction. The goal of this chapter is to review the empirical data related to smell dysfunction in major neurological disorders, most of which are viewed as degenerative. Greater attention is paid to the those disorders for which the largest literature has evolved (e.g., AD, PD, and SZ) and for which olfactory dysfunction is relatively well characterized. Although some information on potential neuropathological correlates is presented, the bulk of this information is provided in Chapter 24.

Since the pioneering studies of Ansari and Johnson (1975) and Waldton (1974), it has become apparent that the ability to smell is compromised in a number of neurodegenerative diseases, including Alzheimer’s disease (AD), Down syndrome (DS), Huntington’s disease (HD), idiopathic Parkinson’s disease (PD), multiple sclerosis (MS), and the parkinsonism-dementia complex of Guam (PDC) (for reviews, see Doty, 1991, 2001; Ferreyra-Moyano and Barragan, 1989; Hawkes et al., 1999; Mesholam et al., 1998; Moberg et al., 1999; Murphy, 1999; Serby, 1987). Alterations in olfaction in such a wide range of disorders— along with the findings of olfactory dysfunction in the normal elderly, epilepsy, multi-infarct dementia (MID), schizophrenia (SZ), and brain surgery cases—raises the possibility that such olfactory anomalies simply reflect nonspecific general disruption of central nervous system (CNS) pathways. However, this is unlikely for several reasons. First, in some diseases, such as AD and PD, the olfactory deficit presents very early in the disease process, long before significant brain deterioration is evident. Second, the degree of olfactory dysfunction differs, on average, among most of these disorders. For example, AD, PD, and PDC are accompanied by marked alterations in the ability to smell, whereas HD, MID, and SZ are accompanied by more moderate alterations. Progressive supranuclear palsy (PSP) and 1-methyl-4-phenyl-1, 2, 3, 6-tetrahydropyridine–induced parkinsonism (MPTP-P) are associated with only minor changes in the ability to smell, in spite of the fact that they share major clinical features

II. OLFACTORY FUNCTION IN ALZHEIMER’S DISEASE The diagnosis of AD is a pathological diagnosis possible only at autopsy. Probable AD, a diagnosis based upon a set of well-defined clinical criteria (e.g., an idiopathic, slowly developing memory loss), is what is typically referred to as AD in living persons (McKhann et al., 1984). Early diagnosis of neurodegenerative diseases such as AD is important, not only for long-term care, but for increasing the efficacy of medications that may thwart, to some degree, the progression of the symptoms. Hence, markers for early AD are of significant value to the physician and patient. 479

480

Figure 1 (A). University of Pennsylvania Smell Identification Test (UPSIT) scores for patients with Alzheimer’s disease (AD) and for age-, gender, and race-matched controls. (B). Detection threshold values for phenyl ethyl alcohol for patients with AD and for matched controls. Each dot signifies an individual subject’s data point. Although some overlap appears between the AD and control subject data when plotted in this manner, very few of the AD subjects performed better than their matched controls. (From Doty et al., 1987.)

A considerable amount is known about the olfactory dysfunction of AD. First, the loss—which is usually not total—is present in both nasal chambers and in the earliest stages of the disease, including some cases of questionable AD (Doty et al., 1987, 1991a; Morgan et al., 1995; Schiffman et al., 1990) (Fig. 1). Second, the smell deficit is robust, present in a large proportion of patients (85–90%), and detected by a wide range of olfactory tests, including tests of odor identification, detection threshold sensitivity, discrimination, and memory; however, suprathreshold intensity ratings may be less sensitive to the deficit (Table 1). In a recent meta-analysis of 11 olfactory/AD studies, effect sizes ranging from 0.98 to 12.15 (median
2.17) were noted (Mesholam et al., 1998). Third, the olfactory dysfunction is reflected by decreased

Doty

odor-related activation of central structures (e.g., subfrontal temporal lobe), as measured by functional imaging procedures such as positron emission tomography (PET) (Buchsbaum et al., 1991; Kareken et al., 2001). Fourth, the chemosensory loss is relatively specific to olfaction, as most measures of taste function seem not to be involved (Murphy et al., 1990) (see, however, Schiffman et al., 1990). Fifth, many AD patients are unaware of their olfactory deficit until formal testing (Doty et al., 1987; Nordin et al., 1995a). For example, Doty et al. (1987) reported that only 2 of 34 early-stage AD patients (6%) responded affirmatively to the question, posed before olfactory testing, “Do you suffer from smell and/or taste problems?,” despite the fact that over 90% of the patients exhibited lower scores on the standardized 40-item University of Pennsylvania Smell Identification Test (UPSIT) than their age-matched controls. Sixth, the olfactory loss of AD appears, on average, to be equivalent to that observed in PD and PDC, suggesting these disorders may share a common pathological substrate (Doty et al., 1991a). Seventh, as briefly noted earlier, despite the possibility of such a substrate, the olfactory dysfunction of AD appears to progress with time (Corwin and Serby, 1985; Knupfer and Spiegel, 1986; Murphy et al., 1990; Nordin et al., 1997; Richard and Bizzini, 1981; Serby et al., 1985), unlike the situation with PD, where the smell loss seems to be more stable (Doty, 1991) (see, however, Tissingh et al., 2001). Nonetheless, the reliability of psychophysical testing in significantly demented patients must be questioned. Eighth, olfactory testing is useful in the differential diagnosis of AD from disorders commonly misdiagnosed as AD, such as depression. Indeed, even a three-item odor identification test accurately discriminates patients with AD from patients with major affective disorder, being superior to the widely used 30-item MiniMental State Examination (MMSE) in this regard (McCaffrey et al., 2000; Solomon et al., 1998). Ninth, there is evidence that smell loss is present in some inherited forms of AD (e.g., Nee and Lippa, 2001) and, moreover, that the loss in idiopathic AD correlates with a family history of dementia (Schiffman et al., 1990), implying some genetic linkage, a point discussed below. Interestingly, in light of evidence that postmenopausal estrogen may mitigate some forms of AD-related cognitive decline (e.g., in verbal memory) (Henderson et al., 1996), there is circumstantial, but yet to be confirmed, evidence that estrogens may also lessen to some degree postmenopausal olfactory loss (Deems et al., 1991; Dhong et al., 1999). Olfactory dysfunction—particularly in conjunction with other risk factors—is a predictor of subsequent development of AD in older persons. Five recent studies attest to

Odor Perception in Neurodegenerative Diseases

481

Table 1 Procedural Details of Alzheimer’s Disease Olfactory Function Studies Ref. Waldton, 1974 Richard and Bizzini, 1981 Corwin and Serby, 1985 Peabody and Tinklenberg, 1985 Serby et al., 1985 (note: same data as Corwin and Serby, 1985) Knupfer and Spiegel, 1986

No. of subjects and sex

Mean age (SD or range)

Type of test

Odorant types

Findings (p-value)

66 F

72.2 (nr)

Ability to “apprehend”

6 types

D (nst)

2 M, 6 F

74.3 (9.5)

Detection threshold

n-propanol

D (nst)

11 nr

nr

Odor ID

10 pairs

D (0.02)

14 M, 4 F

66 (53–79)

Odor ID

5 types

D (nst)a

11 nr

nr

Odor ID

10 pairs

D (0.02)

6 M, 12 F

81.6 (5.8)

Koss et al., 1987

12 M, 5 F 14 M, 11 F 6 M, 9 F 10 nr

Koss et al., 1988

10 M

66.7 (nr) 69.0 (8.5) 68.9 (9.3) nr nr 61.7 (8.7)

Moberg et al., 1987 Rezek, 1987

42 nr 18 nr

72.5 (7.8) 70.0 (4.8)

Kesslak et al., 1988

8 M, 10 F

64.2 (1.7)

Green et al., 1989

5 M, 7 F

68.4 (6.3)

Detection threshold Detection threshold Detection threshold Odor memory Odor ID, naming UPSITb UPSIT Detection threshold UPSIT Detection threshold UPSIT Detection threshold Odor Memory Noncued ID Cued ID Detection threshold Detection threshold UPSIT Odor matching Odor intensity ratings Odor intensity ratings Odor matching

Eucalyptol Citral Prunolide Multiple Multiple 40 types 40 types Phenylethanol 40 types Pyridine 40 types Pyridine Multiple 5 types 5 types Pentanol Cinnamon oil 40 types 15 sets Phenylethanol Eugenol 10 types

D (0.001) D (0.001) D (0.001) D (0.001) D (0.001) D (0.001) D (0.001) D (0.001) D (0.001) D (ns) D (0.001) D (ns) D (0.001) D (0.002) D (0.01) D (ns) D (0.001) D (0.05) D (0.05) Normal Normal D (0.05)

10 M, 11 F

72.8 (5.4)

Detection threshold

n-butanol

D (0.001)

30 (nr)

69.7 (6.9)

Detection of suprathreshold odorants

14 types

D (nst)

Warner et al., 1986 Doty et al., 1987

Murphy et al., 1990 Schiffman et al., 1990 Buchsbaum et al., 1991) Serby et al., 1991 Almkvist et al., 1992 Perl et al., 1992 Morgan et al., 1995

Nordin et al., 1995a

4 M, 2 F 55 nr

72.1 (3.5) (60–79)

Odor matching UPSIT

30 trials 40 types

D (0.001) D (0.004)

3 M, 11 F 2 M, 18 F

72.6 (7.0) (67–91)

Detection threshold Facial reaction

Pyridine 6 types

D (0.01) D (0.04–0.002)c

15 M, 3 F

73.5 (9.5)

UPSIT SDCOIDd Detection threshold

40 types 8 types n-butanol

D (0.001) D (0.001) D (0.01)

42 M, 38 F

74.0 (6.7)

Detection threshold

n-butanol

D (0.01) (continued)

482

Doty

Table 1 (continued)

Ref. Nordin and Murphy, 1996 Nordin et al., 1997 Bacon et al., 1998 Hawkes and Shephard, 1998 Solomon et al., 1998 Lehrner and Deecke, 1999 Larsson et al., 1999 Bacon Moore et al., 1999 Niccoli-Waller et al., 1999 McCaffrey et al., 2000 Broggio et al., 2001

No. of subjects and sex

Mean age (SD or range)

Type of test

Odorant types

Findings (p-value)

70.6 (8.7) 78.5 (8.5)

Odor memory Detection threshold Detection threshold Detection threshold

10–15 types n-butanol pyridine n-butanol

D (0.05)e D (0.05) D (0.007) D (0.03)

8 nr 8 nr

nr nr

UPSIT OERP

40 types 2 types

D (0.001) D (nst)f

12 M, 8 F

74.5 (7.7)

PSTg

3 types

D (0.001)h

20 nr 11 F

63–94 69.7 (8.2)

WOTBi Odor identification Detection threshold

20 types 20 types 1-butanol

D (0.001) D (0.001) D (ns)

22 M, 18 F

72.83 (7.61)

Detection threshold Odor fluency

n-butanol 10 types

D (0.001) D (0.05)

15 M, 17 F

76.0 (9.1)

Detection threshold

n-butanol

D (0.001)

7 M, 13 F

74.2 (7.9)

PST

3 types

D (0.001)j

8 M, 12 F

75 [66–80]

Discrimination and identification of orally presented stimuli UPSIT

multiple 40 types

D (0.001) D (0.001)

4 M, 12 F

76.4 (10.4)

4 M, 14 F 8 nr

Gray et al., 2001 Kareken et al., 2001

4M, 9F

75.4 [53–79]

4 M, 3 F

73.1 (8.3)

UPSIT Detection threshold

40 types Phenylethanol

D (0.001) D (ns)

McShane et al., 2001

39 M, 53 F

75.6 (8.0)

Ability to “perceive”

1 odorant: lavender water

Royet et al., 2001

3 M, 12 F

68.4 (8.4)

Identification Odor intensity ratings Pleasantness ratings Familiarity ratings Edibility ratings

12 items 12 items 12 items 12 items 12 items

No differences in “anosmia” D (0.005) ? (ns) D (0.025)k D (0.05) D (ns)

Nearly all studies employed controls of the same age [and/or in the case of the University of Pennsylvania Smell Identification Test (UPSIT), standardized test norms]; nst, no statistical test applied; nr, not reported; ns, not statistically significant; D, decreased performance in values relative to age-matched controls (note that for detection thresholds, this means elevated threshold, not decreased thresholds, and that this is independent of whether effect is statistically significant); I, increased performance relative to controls. aDepends upon odorant and measure employed. bUniversity of Pennsylvania Smell Identification Test. c8 of 18 AD patients were abnormal on test (44%) compared to 1 of 26 controls (4%). dSan Diego Children’s Odor Identification Test. e“Questionable AD subjects,” which requires absence of reported functional changes by significant others. fFour of the 8 patients had normal OERPs, 4 had abnormal OERPs. gPocket Smell Test™. hComparison was against depressed patients who have no dysfunction. iWiener Olfaktorischen Testbatterie. jComparison was made against depressed normals. kOnly for 2 of 12 odorants.

Odor Perception in Neurodegenerative Diseases

this fact. In the first, Bacon et al. (1998) administered an nbutanol detection threshold test to 70 normal volunteers at risk for AD (i.e., older individuals who had memory impairment). The sensitivity of those who subsequently progressed to a diagnosis of AD (n
8) was lower than that of those who did not, implying that increased olfactory thresholds may be an indicator of incipient disease. Importantly, of 15 patients who were diagnosed as having questionable AD, 9 with at least one APOE-4 allele were less sensitive than the remaining 6, who had no such allele, suggesting an association between this genetic risk factor for AD and smell dysfunction.* In the second, Graves et al. (1999) administered the 12-item version of the UPSIT (the Brief Smell Identification TestTM or B-SIT) to 1604 nondemented community-dwelling senior citizens 65 years of age or older. Over a subsequent 2-year time period, the BSIT scores were a better predictor of cognitive decline than scores on a global cognitive test. Persons who were anosmic and possessed at least one APOE-4 allele had 4.9 times the risk of having cognitive decline than normosmic persons not possessing this allele. This is in contrast to the 1.23 times greater risk for cognitive decline in normosmic individuals possessing at least one such allele. When the data were stratified by sex, women who were anosmic and possessed at least one APOE-4 allele had an odds ratio of 9.71, compared to an odds ratio of 1.90 for women who were normosmic and possessed at least one allele. The respective odds ratios for men were 3.18 and 0.67. The third study evaluated, at 6-month intervals, the cognitive and olfactory function of 90 outpatients with mild cognitive impairment (Devanand et al., 2000). UPSIT scores were lower in patients with mild cognitive impairment than in controls. Of 77 patients followed over a 2-year period, those with mild, moderate, or severe smell loss, as well as those with low UPSIT scores who reported no problems smelling, were more likely to develop AD. Low UPSIT scores accompanied by lack of awareness of olfactory problems predicted the time until development of AD. Even in patients with relatively high MMSE scores, low UPSIT scores with lack of deficit awareness remained a significant predictor of AD. UPSIT scores of 30–35 *Apoliopoprotein E is a widely distributed cholesterol transport protein that circulates in the plasma after synthesis by the liver, spleen, and kidneys. In the CNS it is synthesized by macrophages, neurons, and glia. In the PNS it is synthesized by macrophages, nonmyelinating Schwann cells, and ganglionic satellite cells. Common isoforms include E2, E3, and E4, with E2 seemingly having a neuroprotectant action and E4 being associated with a higher risk of not only developing some neurodegenerative diseases, such as AD, but also having a more malignant course of degeneration (Bedlack et al., 2000).

483

showed a moderate to strong sensitivity and specificity for diagnosis of AD at follow-up. In the fourth study, Swan and Carmelli (2002) administered the B-SIT to 359 nondemented elderly persons. Testing 41/2 yrs later found low test scores to be associated with declines in verbal memory, independent of APOE-4 status, but not in executive control or global function. In the fifth study, Royal et al. (2002) found that, in 173 initially nondemented independent retirement community residents, decreased UPSIT scores predicted cognitive decline and an AD-like memory impairment 3 years later. In light of such findings and the association of smell loss with the APOE-4 allele, it is of interest that some close relatives of AD patients may exhibit smell dysfunction. Serby et al. (1996) administered the UPSIT to 28 first-degree relatives of 28 AD patients and 28 healthy controls. Despite similar global cognitive scores (MMSE), the relatives exhibited lower UPSIT scores than those of the controls ( p  0.01). Although olfactory testing was suggested by Nee and Lippa (2001) to not be useful in predicting the symptom onset of one relatively rare genetically determined form of AD (presenilin-1 AD), the aforementioned data, along with evidence of poorer odor detection or identification performance in individuals having one or more APOE alleles (Bacon et al., 1998) and a delayed latency to an olfactory event–related potential in individuals with the APOE-4 allele (Wetter and Murphy, 2001), are in accord with the concept of a genetic vulnerability to olfactory dysfunction in this disease.

III. OLFACTORY FUNCTION IN DOWN SYNDROME Down syndrome, a trisomy 21 disorder, is the most common clinical syndrome associated with mental retardation, accounting for ~17% of the retarded population. In support of early clinical observations (e.g., Brousseau and Brainerd, 1928), empirial studies have found adult DS patients to have difficulty smelling, as measured by tests of odor identification, detection, memory, and EEG responses to odorants (Hemdal et al., 1993; Murphy and Jinich, 1996; Warner et al., 1988; Wetter and Murphy, 1999; Zucco and Negrin, 1994). Such observations are of significance, given that the average smell loss observed in DS is very close to that observed in AD (i.e., UPSIT scores ~20) (McKeown et al., 1996; Warner et al., 1988), and DS patients who live into early adulthood inevitably develop the clinical and neuropathological features of AD (Oliver and Holland, 1986). Importantly, deposition of amyloid in the form of senile plaques or diffuse amyloid deposits occurs in cortical brain regions associated with olfactory processing (e.g., entorhinal cortex) as early as the age of 19 years in DS (Hof et al., 1995).

484

At what age do individuals with DS begin exhibiting olfactory loss? In the sole study designed to shed light on this question, McKeown et al. (1996) administered the UPSIT and a 16-item odor discrimination test to 20 adolescents with DS [mean age (SD)
13.89 years (1.98)], to 20 non-DS retarded children matched on the basis of mental age [Peabody Picture Vocabulary Test-Revised (PPVT-R)] (Dunn, 1981), and to 20 nonretarded children also matched on mental age. Although no meaningful differences in olfactory function were found among the three study groups, the test scores of both the DS and non-DS retarded subjects were markedly lower than those of the nonretarded children of their own chronological age. Moreover, the UPSIT scores were similar in magnitude to those of adult DS subjects (~20). According to published norms (Doty, 1995), a 14-year-old nonretarded boy or girl would be expected to have an UPSIT score within the range of 34–38. The respective average UPSIT scores of the DS and non-DS retarded subjects fell well below this range (18.65 and 21.35, respectively). However, UPSIT scores of this magnitude fall within the low normal range for persons 6 years of age (the average mental age of the subjects). Thus, an UPSIT score of 19 falls at the 14th percentile of a group of 57 6-year-olds available from our Center’s database, whereas a score of 20 falls at the 17th percentile. Given the fact, however, that the average UPSIT scores of these adolescents were equivalent to those of adult DS subjects, it is possible that the smell loss observed in the latter group appears quite early in life—before significant AD-like brain pathology appears. Alternatively, it is conceivable that smell function improves somewhat during the late teen years, only to fall again as the AD-like neuropathology becomes more salient.

IV. OLFACTORY FUNCTION IN IDIOPATHIC PARKINSON’S DISEASE PD, since its first description by James Parkinson in 1817 (Parkinson, 1817), has generally been considered to be a purely motor disease. However, within the last two decades or so it has become apparent that a number of sensory changes are present in this disorder, including subtle alterations in vision and hearing (Gawel et al., 1981; Rodnitzky, 1998). Importantly, it is now clear that smell loss is a major component of this disease, with its prevalence (~90% of all tested cases) being greater than tremor, one of the cardinal signs of the disorder (Doty et al., 1988a). Much is now known about the smell dysfunction of PD. First, as with the case of AD, the loss is generally bilateral and robust, being detected by a wide range of olfactory tests, including tests of odor identification, threshold detection,

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and discrimination (Table 2). A study examining the sensitivity and specificity of the UPSIT in differentiating between clinically diagnosed PD patients and normal controls found these values to be quite high (0.91 and 0.88, respectively, in males 60 years of age) (Doty et al., 1995a). However, as is also true for AD, total anosmia is not the rule. Thus, in one study only 13% of 38 patients who received an odor detection threshold test were unable to detect the highest odorant concentration presented, and only 38.3% of 81 patients had UPSIT scores suggestive of anosmia (Doty et al., 1988a). Moreover, all but one of 41 PD patients asked if an odor was present on each of 40 UPSIT items answered affirmatively to 35 or more of the items, even though the majority were unable to identify most of the odors or felt that the perceived sensation did not correspond to the response alternatives. Second, female PD patients generally have less dysfunction than male PD patients (Stern et al., 1994), as is also the case in AD. Third, while the loss is generally bilateral, there can be slight individual differences in the degree to which the left and right sides of the nose are involved. No association exists, however, between the side of relatively greater involvement and the side of hemiparkinsonism, as might be expected if asymmetrical damage to striatal dopamine systems were involved in the olfactory problem (Doty et al., 1992b). Fourth, the smell loss is indistinguishable from that of AD and the PDC, with UPSIT scores averaging around 20 (Doty et al., 1991a,b). Fifth, the smell loss is unrelated to the magnitude of the motor symptoms (Doty et al., 1988a, 1989, 1992b), although subtle variations among so-called benign vs. malignant forms may be present (Stern et al., 1994). Sixth, the smell loss is unrelated to numerous neuropsychological measures, such as the Randt memory test, reaction time, a finger-tapping test, and selected verbal and performance subsets of the Wechsler Adult Intelligence Scale— Revised (Doty et al., 1989). Seventh, the smell loss appears in both familial and sporadic forms of parkinsonism and may be a sign of the preclinical state of the disease (Berendse, 2001; Markopoulou et al., 1997). Eighth, unlike AD, there is no apparent longitudinal progression in olfactory dysfunction as occurs in other elements of the disease process (Doty et al., 1988a). Ninth, anti-PD medications (e.g., L-dopa, dopamine agonists, anticholinergic compounds) have absolutely no influence on the smell deficit, which occurs as severely in nonmediated or never-medicated patients as in medicated ones (Doty et al., 1992b; Quinn et al., 1987; Roth et al., 1998). Tenth, also like AD, many PD patients are unaware of their olfactory deficit until formal testing (Doty et al., 1988a). Eleventh, olfactory testing is useful in the differential diagnosis of idiopathic PD from a number of other neurodegenerative diseases with motor symptoms, including disorders often misdiagnosed as PD (e.g., PSP, MPTPinduced PD, and essential tremor) (Busenbark et al., 1992;

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Table 2 Procedural Details of Parkinson’s Disease Olfactory Function Studies No. of subjects

Mean age (SD or range)

Type of test

22 M

58 (41 to 67)

Detection threshold

Pentyl acetate

D (0.05)

39 M, 41 M 45 M, 27 F

61.5 (nr) 60 (nr)

Questionnaire Odor detection Detection threshold Odor discrimination

Unknown or NA PEMECa Pentyl acetate 4 types

D (0.001) D (0.01) D (0.03) D (nst)

Corwin and Serby, 1985 Serby et al., 1985 Doty et al., 1988a

5 (nr) 5 (nr) 46 M, 35 F

nr nr 67.4 (8.17)

Quinn et al., 1987 Kesslak et al., 1988

50 M, 28 F 8 M, 10 F

61.5 (10.3) 65.4 (3.2)

Odor identification Odor identification UPSITc Detection threshold Detection threshold UPSIT Odor matching task

10 pairs 10 pairs 40 types Phenethanol Pentyl acetate 40 types 15 sets

D (nst) D (0.05)b D (0.0001) D (0.0001) D (0.001) D (0.05) D (nst)

Bostantjopoulou et al., 1991

22M, 22F

61.9 (9.4)

Odor detection Odor naming task

D (0.001) D (0.01)

Murofushi et al., 1991

11 M, 7 F

59.6 (41–76)

Detection threshold recognition threshold UPSIT

Pentyl acetate Fresh coffee, candy 5 types 5 types 40 types

D (0.001)

UPSIT UPSIT UPSIT

40 types 40 types 40 types

D (0.0001) D (0.001) D (0.001)

Detection threshold Odor identification Odor memory Odor discrimination Odor identification OERPf UPSIT OERP UPSIT—Modified UPSIT OERP SSi Detection threshold

n-butanol 20 types 20 types 8 pairs 8 types vanillin, H2S 40 types H2S 20 types 40 types vanillin, H2S 16 types n-butanol

D (nr) D (nr) D (nr) D (ns) D (0.001) D (0.05)g D (0.0001) D (0.001)h D (0.01) D (0.0001) D (nst) D (0.001)j D (.001)

Ref. Ansari and Johnson, 1975 Korten and Meulstee, 1980 Ward et al., 1983

Doty et al., 1992b Hawkes and Shephard, 1993 Stern et al., 1994 Doty et al., 1995a

30 M, 10 F

61.9 (10.0)

49 M, 47 F 68 M, 50 F 109 M, 71 F

Lehrner et al., 1995

13 (nr)

57 (27–81) 64.3 (9.34) 64 Ss  60 yr 55 Ss: 61–71 yr 47 Ss  71 yr 64.7 (11.4)

Barz et al., 1997

13 M, 18 F

66.8 (44–81)

Hawkes et al., 1997

49 M, 47 F 66 (sex nr) 7 M, 2 F 78 M, 77 F 19 M, 17 F 28 M, 12 F

57 (27–81)

Ahlskog et al., 1998 Hawkes and Shephard, 1998 Daum et al., 2000

66.1 (6.4) 61 (nr) 62 (27–77) 63.6 (8.7)

Odorant types

Findings (p-value)

D (0.01, 0.05)d D (0.01, 0.05)e

(continued)

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Table 2 (continued)

Ref

No. of subjects

Mean age (SD or range)

Type of test

Odorant types

Findings ( p-value)

Montgomery, Jr. et al., 2000a Montgomery, Jr. et al., 2000b Tissingh et al., 2001

9 M, 9 F

64 (45–79)

UPSIT

40 types

D (0.001)

32 M, 26 F, 1 U 25 M, 16 F

69 (38–83) 56.4 (8.9)

Zucco et al., 2002

3 M, 3 F

65 (4.6)

35 M, 15 F 10 M, 10 F

58 (38–80) 67.1 (9.9)

40 types 12 items 32 trials Phenylethanol 12 types 10 types 16 types 40 types Vanillin Propionic Acid

D (0.001) D (0.001) D (0.001) D (0.001) D (0.001)k

Müller et al., 2002 Sobel et al., 2002

UPSIT B-SIT Odor discrimination Detection threshold Odor Matching Identification SSi UPSIT Detection threshold Detection threshold

D (0.0001) D (0.0001) D (0.007) D (0.003)

Nearly all studies have employed controls of the same age (and/or, in the case of UPSIT, standardized test norms); nst, no statistical test applied; nr, not reported; ns, not statistically significant; D, decreased performance in values relative to age-matched controls (note that for detection thresholds, this means elevated threshold, not decreased thresholds, and that this is independent of whether effect is statistically significant); I, increased performance relative to control; U, unknown. aBased upon comparison of combined AD and PD groups vs. 5 other groups. bPhenyl ethyl methyl ethyl carbinol. cUniversity of Pennsylvania Smell Identification Test. dSignificant effects were observed in detection for phenylethanol and methyl cyclopentenolone at the 0.01 level, and for isovaleric acid, gamma-undecalactone, and skatole at the 0.05 level. eSignificant effects were observed in recognition for methyl cyclopentenolone and undecalactone at the 0.01 level. Significant effects were observed for phenylethanol at the 0.05 level. No significant effects were noted for isovaleric acid or skatole. fOdor event related potential. gReflects prolongation of latencies. hThe OERP responses were somewhat equivocal. Of 66 PD patients, 5 had unclear recordings and 1 had none. Of the remaining 60 patients, 37 had OERPs to both CO2 and H2S. Of 10 PD patients with normal UPSIT scores, one had absent H2S responses and 3 had prolonged latencies to this agent. All had intact CO2 responses. In some cases, OERPs obtained from right naris stimulation were significantly delayed relative to controls. iSniffin’ Sticks test, which includes tests of odor identification, discrimination, and detection. jAll p-values 0.001, except for discrimination on the left, which was 0.05. kValues based on combination of both types of tests in an analysis of variance.

Doty et al., 1992a, 1993, 1995a). Finally, as in the case with AD, some asymptomatic first-degree relatives of patients with either familial or sporadic forms of PD appear to exhibit olfactory dysfunction (Montgomery et al., 1999, 2000b; Wolters et al., 2000). Recently, Berendse (2001) administered tests of odor detection, identification, and discrimination to 250 relatives of PD patients (~84% children, ~16% siblings, 1 parent). In 25 hyposmic and 23 normosmic individuals sampled from this group, nigrostriatal dopaminergic function was assessed using single photon emission computer tomography (SPECT) with [125I]-CIT as a dopamine transporter ligand. An abnormal reduction in striatal dopamine transporter binding was present in 4 of the 25 (16%) hyposmic relatives, 2 of whom subsequently developed clinical parkinsonism, and in none of the 23 normosmic relatives. These authors noted, “The observation in the present study that significant reductions in dopamine transporter binding were found only in hyposmic relatives of PD patients suggests that olfactory dysfunction may precede

clinical motor signs in PD.” Such findings reiterate the point that olfactory testing, in conjunction with other measures, is likely useful in the early detection of PD. There is controversy as to whether some odorants are more useful than others in discerning PD patients from controls. An early study from my laboratory found no evidence that any of the UPSIT items were more useful than others in making this distinction (R. L. Doty, unpublished). However, Hawkes and Shephard (1993) administered the UPSIT to 70 PD patients and 70 age-matched controls. The difference between the percentage of subjects in each group correctly identifying each odor was computed. Significant differences were noted for the UPSIT items of pizza and wintergreen, leading these authors to conclude that “these observations raise the possibility that there is a congenital or acquired selective hyposmia in Parkinson’s disease, comparable with the smell blindness to androstenone, for example, which affects 20–47% otherwise healthy individuals and is probably genetically determined.” Additional research is

Odor Perception in Neurodegenerative Diseases

needed to determine whether this interesting observation is repeatable and, if so, whether it reflects differences in intensity among UPSIT items or true odorant-related factors. V. OLFACTORY FUNCTION IN 1-METHYL-4-PHENYL-1, 2, 3, 6-TETRAHYDROPYRIDINE–INDUCED PARKINSONISM If the hypothesis is true that the smell loss of most cases of idiopathic PD is caused by an environmental agent (e.g., a virus) that enters the nose and directly or indirectly damages elements of the olfactory system (the olfactory vector hypothesis) (see Chapter 24), then parkinsonian syndromes induced intravenously by known toxins might be expected to be spared of olfactory dysfunction. One such syndrome, discovered in the early 1980s, is that induced by intravenously administered MPTP. Although MPTP does not readily cross the blood-brain barrier, its toxic metabolite, 1-methyl-4phenylpyridinium, or MPP, does so and produces a syndrome in humans and nonhuman primates remarkably similar to that of idiopathic PD (Langston et al., 1983). In the sole study addressing olfactory function in patients with this rare form of parkinsonism, the UPSIT and a detection threshold test for phenyl ethyl alcohol was administered to six young persons suffering from this disorder (Doty et al., 1992a). Thirteen rare young PD patients and 10 normal subjects served as comparison groups. Despite their major motoric deficits, the MPTP-P patients evidenced no major decrements in cognitive function, could sniff ade-

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quately, and could respond verbally to the questions of the examiner without difficulty. Neither the UPSIT nor the detection threshold scores differed between the MPTP-P and normal groups, even though the patients with MPTP-P had more advanced parkinsonism and contained a higher proportion of cigarette smokers. As expected, the UPSIT and PEA threshold scores of the young PD patients differed significantly from those of the controls (Fig. 2). These data suggest that the functional integrity of the olfactory system of MPTP-P patients is greater than that of PD patients and provided early evidence that olfactory dysfunction is not a concomitant element of all parkinsonian syndromes. VI. OLFACTORY FUNCTION IN THE PARKINSONISM-DEMENTIA COMPLEX OF GUAM Amyotrophic lateral sclerosis and parkinsonism-dementia accounted for at least 15% of adult deaths among the Chamorro populations of Guam and Rota between 1957 and 1965 (Reed and Brody, 1975). Epidemiological studies, including case-control comparisons and extensive pedigree analyses, have failed to identify a genetic cause of these disorders (Kurland, 1988). However, more recent data suggest that genetic susceptibility may be involved at some level. Thus, one study genotyped 12 patients with PDC and 12 disease-free Chamorros for APOE alleles. Although no differences in APOE-4 frequencies were found, the PDC group had a lower frequency of APOE-2 (8.3%) than did the controls (33.3%) (Waring et al., 1994).

Figure 2 University of Pennsylvania Smell Identification Test (UPSIT) and phenyl ethyl alcohol odor detection threshold test scores for patient with MPTP-induced parkinsonism (MPTP-P), young patients with idiopathic Parkinson’s disease (PD), and matched normal controls. (From Doty et al., 1992a.)

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These general observations were confirmed in a subsequent study in which an additional 17 PDC Chamorros were tested (Buee et al., 1996). It is now apparent that Guamanian Chamorros with PDC have deficits in their ability to identify odors that are similar to those of AD and PD patients. In the first of two studies on this topic, 24 Chamorros with PDC were administered the UPSIT (Doty et al., 1991a). Half were from Umatac and Merizo, two southern villages associated with a high prevalence of PDC. The others were from other Guamanian villages with lower PDC prevalence rates. Even though these subjects were ambulatory and living with their families, all evidenced some degree of rigidity and bradykinesia at the time of testing. For comparison, UPSIT data from 24 AD and 24 PD North American patients who had similar levels of smoking behavior were matched to the PDC data on the basis of gender and age. All subjects received a picture test analogous to the UPSIT to control for cultural and cognitive differences that might influence the UPSIT scores (termed the Picture Identification Test, or PIT). The UPSIT scores of the three groups did not differ significantly from one another, despite the fact that they were markedly lower than those obtained for normal persons of the same age and gender. As was done in an earlier PD study (Doty et al., 1988a) each participant was asked whether or not he or she suffered from any smell or taste problems prior to olfactory testing. Three of the PDC patients reported such problems (13%), as compared to two of the AD (8%) and three of the PD (13%) patients, indicating that the level of awareness of the problem is similar, if not identical, in these three disorders. More recently, Ahlskog et al. (1998) administered an abbreviated version of the UPSIT to 9 Chamorros with symptoms of ALS, 9 with symptoms of pure parkinsonism, 11 patients with pure dementia, and 31 patients with PDC, as well as to neurologically normal Chamorro Guamanians and 25 North American controls. The UPSIT scores were markedly depressed in the four disease groups relative to the controls and did not differentiate among the four syndromes of Guamanian neurodegenerative disease. Some of the control subjects had lower scores than their North American counterparts, implying the possibility of a subclinical neurogenerative disease process in nonsymptomatic individuals.

VII. OLFACTORY FUNCTION IN PROGRESSIVE SUPRANUCLEAR PALSY Progressive supranuclear palsy (also termed the Steele, Richardson, and Olszewski syndrome) accounts for ~4% of patients who exhibit parkinsonian symptoms. Tremor is rarely present in this disorder, although rigidity and

Doty

bradykinesia frequently appear relatively early in its progression. The hallmark feature is vertical-gaze paresis (especially down-gaze paresis), which can be overcome by the oculocephalic maneuver and is of supranuclear origin. PSP is commonly misdiagnosed as PD, because it shares so many motor features with PD. Unlike PD, however, the parkinsonian features are less responsive to anti-PD medications (Jackson et al., 1983), and PSP is typically characterized by comparatively more frontal lobe dysfunction, more neuronal degeneration within the basal ganglia and upper brain stem, and less involvement of mesolimbic and mesocortical dopamine systems than PD (Cambier et al., 1985; Jankovic, 1989). Unlike patients with PD or PDC, most patients with PSP tend to have a relatively normal sense of smell, although slight to moderate losses are present in some individuals. In an early study on this topic, the UPSIT and a phenyl ethyl alcohol odor-detection threshold test were administered to 22 patients with PSP who scored well on the PIT. The test scores of these individuals were compared to those from 22 PD patients and 22 neurologically normal age-, gender-, and race-matched controls. The performance of the PSP patients was clearly superior to that of the PD patients [respective UPSIT means (SD): 31.59 (7.18) and 18.82 (6.94)], with approximately half scoring within the normal range. However, the PSP patients did exhibit moderate deficits relative to the controls, whose mean UPSIT score was 35.60 (4.06). VIII. OLFACTORY FUNCTION IN MULTIPLE SYSTEM ATROPHY Wenning et al. (1996) administered the UPSIT to 29 patients with multiple system atrophy (MSA), 15 PSP patients, 118 patients with idiopathic PD, 7 patients with corticobasal degeneration (CBD), and 123 healthy controls. Normal test scores were noted for the PSP and CBD patients and mild impairment for the MSA patients relative to the controls. The authors noted that “preserved or mildly impaired olfactory function in a parkinsonian patient is more likely to be related to atypical parkinsonism such as MSA, PSP or CBD, whereas markedly reduced olfaction is more suggestive of IPD PD.” These authors found that an UPSIT score of 25 was associated with a sensitivity of 77% and a specificity of 85% in differentiating PD from atypical parkinsonism. IX. OLFACTORY FUNCTION IN HUNTINGTON’S DISEASE Huntington’s disease (also termed Huntington’s chorea) is a genetic disorder of dysfunctional movement, cognitive

Odor Perception in Neurodegenerative Diseases

deterioration, and altered behavior with autosomal dominant transmission that becomes phenotypically expressed progressively relatively late in life. As functional capacity worsens, chorea generally lessens and dystonia intensifies. Among its primary motor symptoms are hyperkinesias that take, initially, the form of chorea, being characterized by fleeting movements, which, in some cases, appear semipurposively within the context of overall heightened activity and motor restlessness (Shoulson, 1986). The first olfactory study of HD patients of which I am aware employed 38 HD patients and 38 controls, and found HD to be associated with a deficit in the ability to remember odor qualities (Moberg et al., 1987). This problem was noted in early-affected patients with minimal chorea or cognitive dysfunction and with normal verbal and visual recognition memory performance. Subsequent work has shown that HD patients have decrements in the general ability to smell that could explain such deficits. For example, Nordin et al. (1995b) found performance decrements in HD subjects on tests of odor detection threshold, intensity discrimination, quality discrimination, and identification, with the greatest impairment occurring for odor identification. These authors suggested these effects may be secondary to the odordetection deficits. More recently, this same group compared the n-butanol detection threshold sensitivity of 7 mildly affected HD patients to that of 7 age- and education-matched healthy controls (Hamilton et al., 1999). The subjects were also administered the California Odor Learning Test and the California Verbal Learning Test. Odor threshold sensitivity, but not group membership, accounted for significant variance in total olfactory learning. Both groups learned fewer items in the olfactory modality than in the verbal modality, but retained a similar amount following a delay. The question arises as to whether patients who carry the HD mutation and, hence, are at risk for expressing its symptoms have decreased smell function. Moberg and Doty (1997) administered the UPSIT and a PEA detection threshold test to 25 probands with HD, 12 at-risk offspring, and 37 unrelated controls. Decreased olfaction was noted only in the HD group, with a mean UPSIT score of 24.8 (SD
8.7) and a PEA threshold score of 4.4 log vol/vol (SD
1.4). Since HD is an autosomal dominant genetic disorder with 100% penetrance, approximately half of the at-risk offspring would have been expected to have the mutation and to evidence smell dysfunction if it was a very early marker of HD. Bylsma et al. (1997) extended these findings, testing 20 HD patients who had the disease for a mean of 8.0 years (range: 4–14 years), 20 normal subjects with the genetic mutation that causes HD, and 20 mutation-negative

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adults. Again, only the patients with clinical signs of HD exhibited depressed olfaction (mean UPSIT score
27.4; SD
6.5). At what time in the disease process olfactory deficits appear is not known, but if analogous to other neurodegenerative diseases in which olfaction is compromised, presumably the loss occurs at a relatively early stage of clinical progression. X. OLFACTORY FUNCTION IN MULTIPLE SCLEROSIS For a number of years it was assumed that little olfactory dysfunction was present in multiple sclerosis, since the primary olfactory neurons are unmyelinated. Moreover, relatively early psychophysical studies, including one employing 40 subjects (Ansari, 1976), found no olfactory deficits in this disorder. Indeed, even a comparatively recent study that administered the UPSIT and a match-tosample odor discrimination test to 14 MS patients reported no deficits (Kesslak et al., 1988). In 1984, our group found that 23% of 31 MS patients evaluated evidenced some degree of olfactory dysfunction on the UPSIT (Doty et al., 1984). A decade later we presented case studies in which olfactory dysfunction was the presenting symptom of MS (Constantinescu et al., 1994). More recently we have demonstrated a strong inverse correlation (r
 0.94) between UPSIT scores and the number of MS-related plaques within the subfrontal and subtemporal lobes of patients, providing a physiological basis for the dysfunction (Fig. 3a) (Doty et al., 1997b, 1998b). No such relationship was present between UPSIT scores and plaques in other brain regions (Fig. 3b). These observations have been subsequently replicated by others (Zorzon et al., 2000). It would seem safe to assume that the reason why some workers have not observed olfactory dysfunction in patients with MS is due to the dynamic nature of the disease. Recently, in a study of 5 MS patients tested three or four times across an 18- to 20-month period, we demonstrated that UPSIT scores wax and wane longitudinally in concert with the waxing and waning of the number of MSrelated plaques within the subtemporal and subfrontal lobes (Doty et al., 1999). XI. OLFACTORY FUNCTION IN AMYOTROPHIC LATERAL SCLEROSIS As with the case of PD, amyotrophic lateral sclerosis (ALS) has been traditionally considered a motor neuron disease (MND). However, today we know that this is an oversimplification of this disorder. In 1991, Elian reported

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observed in AD or PD, with the UPSIT scores falling around 30. In a subsequent study, Sajjadian et al. (1994) administered the UPSIT bilaterally to 17 female and 20 male ALS patients and unilaterally to 7 male and 7 female ALS patients. Age-, gender-, smoking habit-, and race-matched controls were also evaluated. While the UPSIT scores of the ALS patients were significantly lower than those of the controls, the degree of dysfunction was similar to that seen by Elian. Thus, only 11% of the ALS patients had UPSIT scores indicative of total or near total anosmia. Despite the fact that nearly half of the ALS patients had UPSIT scores that fell within the normal range, 75.7% of the patients scored below their individually matched controls. Although no sex differences or laterality in the ALSrelated test scores were observed, an age-related decrement was present and, interestingly, significant correlations were found between UPSIT scores and neurophysiological measures of peripheral nerve conductance. If the latter observation proves to be true, it is conceivable that a common pathophysiological process influences the motor neuron responses and segments of the afferent olfactory pathway. Recently, Hawkes et al. (1998) administered the UPSIT to 58 patients with MND and 135 controls. Additionally, olfactory event–related potentials (OERPs) in response to H2S were recorded in 15 patients, and the olfactory bulbs of 8 MND cadavers were histologically examined. Although UPSIT scores were slightly worse in the MND patients, only the bulbar patients exhibited significant decrements. OERPs were normal in 9 patients and delayed in one. OERPs from the remaining 5 subjects were not able to be recorded. Analysis of the olfactory bulb tissue revealed excessive lipofuscin deposition in all 8 cases examined, indicating subclinical neuronal damage. The authors concluded that the olfactory dysfunction in this disorder is relatively mild. Figure 3 (A) Relationship, in patients with multiple sclerosis (MS), between number of plaques in subtemporal and subfrontal lobes and scores on the University of Pennsylvania Smell Identification Test (UPSIT). (B) No such relationship was present between UPSIT scores and plaques in brain regions outside of the primary olfactory cortical areas. (From Doty et al., 1997b.)

that patients with MND exhibit decreased UPSIT scores bilaterally. Specifically, UPSIT scores from 9 male and 6 female MND patients of varying severity were compared with those of age- and sex-matched controls and proved to be significantly depressed. However, the magnitude of the deficit was more similar to that observed in SZ than that

XII. OLFACTORY FUNCTION IN SCHIZOPHRENIA Although SZ is not generally considered a neurodegenerative disease, it is included in this chapter because of evidence that elements of this disease may have progressive components and because a large literature exists regarding olfactory function in this disorder. Interest in olfactory function in SZ has grown exponentially since Campbell and Gregson (1972) first attempted to test odor recognition memory in SZ patients. Indeed, there are now more olfaction-related studies in patients with SZ than in any other neurological disorder. This explosion in research reflects

Odor Perception in Neurodegenerative Diseases

the commercial availability of easy-to-use quantitative olfactory tests (e.g., the UPSIT) and an acute awareness that temporal-limbic brain systems are markedly altered in schizophrenia, making olfactory testing a unique functional probe of these brain regions (Seidman et al., 1992) (for reviews, see Harrison and Pearson, 1989; Martzke et al., 1997; Moberg et al., 1999; Pantelis and Brewer, 1995; Serby et al., 1992). There is suggestion, for example, that odor-identification deficits may be indicative of right orbitofrontal lobe dysfunction (Purdon, 1998). The following summarizes what is known about the olfactory dysfunction of this disease. First, as in the case of AD and PD, the sensory alterations appear to be largely bilateral, although recent data suggest that some left:right differences may be present that could reflect subtypes of SZ (Good et al., 2002). Second, the degree of dysfunction, on average, is considerably less in SZ than in AD or PD (although this may not be the case in elderly individuals) (Moberg et al., 1997a). Thus, the median UPSIT score of 13 SZ studies reporting such data in a recent meta-analysis was 32.5 (Moberg et al., 1999), compared to average UPSIT scores in AD and PD studies of ~20. The median effect size (i.e., the mean difference between patient and control groups expressed in SD units) for 17 SZ studies employing the UPSIT was 0.92 (range: 0.56–3.18). Analogous medians for 8 AD and 4 PD studies were 2.01 (range: 0.98–12.15) and 1.66 (range: 1.27–2.38), respectively. The median effect size for the SZ studies was significantly less than the median effect sizes of the AD and PD studies ( p
0.006 and 0.06, respectively), which did not differ significantly from one another ( p
0.73). Such differentiation is due, in part, to a lower proportion of patients with SZ exhibiting smell loss. Thus, in recent study from our center, 23% of a group of 41 patients with SZ exhibited UPSIT scores indicative of microsmia or anosmia, in contrast to 94.4% of a group of 18 AD patients and 96.3% of a group of 54 PD patients (P. J. Moberg et al., unpublished). Third, the lower olfactory test scores of SZ cannot be explained on the basis of cigarette smoking, which is typically higher in SZ than in non-SZ study populations (Brewer et al., 1996; Houlihan et al., 1994). Fourth, UPSIT scores in patients with SZ appear to be more strongly related to neuropsychological measures of frontal lobe function (e.g., problems in executive function) than to measures of medial temporal lobe function (e.g., memory) (Pantelis and Brewer, 1995; Purdon, 1998; see, however, Good et al., 2002). Fifth, UPSIT scores are correlated with negative, but not positive, symptoms of the disorder, reflecting associations with neuropsychological tasks of prefrontal cortical function (Brewer et al., 1996). Sixth, some subgroups of SZ patients (e.g., those with deficit syndrome), may exhibit greater olfactory dysfunction than oth-

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ers (Malaspina et al., 2002). Seventh, the olfactory loss is discernible to some degree by functional imaging studies (e.g., PET, SPECT), with hypometabolism or decreased activation occurring within central brain structures associated with smell function (e.g., subtemporal and subfrontal lobes), particularly on the right (Bertollo et al., 1996; Buchsbaum et al., 1991; Clark et al., 1991; Crespo-Facorro et al., 2001; Malaspina et al., 1998; Wu et al., 1993). Eighth, the chemosensory decrement appears to occur early in the disease process and, in fact, is found in many patients who may be prone to SZ, such as those with schizotypy or with family histories positive for significant mental illness (Becker et al., 1993; Kopala et al., 1998b, 2001; Park and Schoppe, 1997). Ninth, there is some evidence that at least some forms of the disorder may have a genetic basis (Kopala et al., 1991). Tenth, UPSIT scores in SZ patients are inversely and significantly correlated with disease duration, suggesting that some progression of pathology may be occurring somewhere in the olfactory pathways (Moberg et al., 1997b). This phenomenon appears to be independent of medication history and gender. Presently, odor-identification ability appears to be the only neuropsychological marker known to correlate with disease duration in SZ. In the aforementioned meta-analysis (Moberg et al., 1999), studies tapping the domains of odor identification, detection threshold sensitivity, discrimination, and memory were assessed. Potential moderator variables such as age, gender, medication status, and smoking history were also examined. It is noteworthy that substantial olfactory deficits, across all sensory domains, were documented in patients with SZ. Indeed, no differential deficits were observed for odor identification, detection threshold sensitivity, discrimination, and memory (Table 3).* These data support the view that olfactory functions, as a whole, are deficient in patients suffering from SZ. Outlier analysis did identify two significant outlier studies, in which both the patients and controls were much older than those in the other studies. Calculation of effect sizes for these two studies found the composite effect size for the elderly SZ patients to be over twice that seen in younger SZ patients. Notably, these effect sizes reflect the differences between *Although

there is widespread consensus that identification ability, as measured by the UPSIT, is compromised in SZ (Table 3), there has been some debate regarding other measures, for which fewer studies exist. Bradley (1984) reported that psychotic patients, most notably men with SZ, were hypersensitive to 5-16androsten-3-one. Other work has not confirmed this finding (Gross-Isseroff et al., 1987). As can be seen in Table 3, either heightened thresholds (i.e., decreased sensitivity) or no significant threshold deficits at all are the rule, the latter often reflecting small sample sizes, threshold measures with low reliability, and compromised statistical power.

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Table 3 Procedural Details of Studies of Olfactory Function in Patients with Schizophrenia Ref.

Sex and no. of subjects

Mean age (SD or range)

Campbell and Gregson, 1972 Bradley, 1984 Sreenivasan et al., 1987 Gross-Isseroff et al., 1987

20 (sex nr) 5 M, 6 F 32 (sex nr) 22 M, 20 F

nr (20–50) (15–45) 30.1 (6.4)

Hurwitz et al., 1988 Dunn and Weller, 1989 Kopala et al., 1989 Serby et al., 1990

15 M, 3 F 13 M, 2 F 26 M, 15 F 14 M

23.9 (17–41) 54.2 (28–71) (40–49)

Warner et al., 1990 Geddes et al., 1991 Kopala et al., 1992

12 M 16 M, 8 F 30 M, 10 F

34 (20–42) 38.5 (20–66) 26.25 (7.41)

Seidman et al., 1992 Wu et al., 1993

10 (nr) 10 (nr) 15 M, 1 F 19 M, 1 F

nr nr 36.5 (8.1) 32.1 (9.3)

Houlihan et al., 1994 Kopala et al., 1994 Malaspina et al., 1994 Kopala et al., 1995a Kopala et al., 1995b Seidman et al., 1995 Brewer et al., 1996 Moberg et al., 1997b Moberg et al., 1997b Seidman et al., 1997 Good et al., 1998

23 M, 19 F 131 (nr) 5 M, 6 F 49 M, 16 F 27 F 14 M, 4 F 27 M 4 M, 12 F 18 M, 20 F 24 M, 16 F 65 M

33.25 (6.8) 34.6 (5.0) 34.6 (14.8) 28.3 (16–48) 42.85 (7.04) 39.8 (8.3) 31.8 (8.5) 77.9 (6.5) 50.6 (25.5) 38.5 (6.5) nr

Kopala et al., 1998a

18 (sex nr)

nr

Kopala et al., 1998b Malaspina et al., 1998 Purdon, 1998

12 (sex nr) 5 M, 1 F 18 M, 3 F

36.8 (4.9) 34.6 (14.8) 37.1 (9.6)

Detection threshold UPSIT UPSIT Odor memory UPSIT UPSIT UPSIT UPSIT UPSIT UPSIT UPSIT UPSIT UPSIT UPSIT UPSIT (unilateral) Detection threshold (unilateral) UPSIT detection threshold UPSIT UPSIT UPSIT

Stedman and Clair, 1998 Purdon and FlorHenry, 2000)

37 M, 9 F

36.39 (8.05)

UPSIT

11 M, 6 F

27.7 (7.8)

Sirota et al., 1999

19 M

36.5 (11.9)

12 M

34.2 (11.9)

Purdon and FlorHenry, 2000

11 M, 6 F

27.65 (7.75)

Brewer et al., 2001

55 M, 19 F

22.3 (3.67)

Type of testing Memory for odor positions Detection threshold Odor matching task Detection threshold Detection threshold UPSIT Discrimination test UPSIT UPSIT Detection threshold UPSIT Detection threshold UPSIT detection threshold

Odorant types

Direction of effect and p-value

12 types Androstenone nr Androsteone Pentyl acetate 40 types 4 odor sets 40 types 40 types Geraniol 40 types Androstenone

D (nst) I (0.05)a D (nst) D (ns) D (0.01) D (0.02) D (ns) D (0.005)b D (0.001) D (0.001) D (0.03)c D (ns)d

40 types n-Butanol Phenylethanol 40 types 40 types 10 sets 40 types 40 types 40 types 40 types 40 types 40 types 40 types 40 types 40 types 40 types 40 types Phenylethanol

D (0.01) I (ns) D (ns)

40 types n-Butanol 40 types 40 types 40 types

40 types

D (0.001) D (0.05)e D (0.02)f D (0.05) D (in microsmic range of norms) D (0.001)

UPSIT (Unilateral) Detection threshold (unilateral) Detection threshold Detection threshold Detection threshold Detection threshold

40 types n-Butanol

D (0.05)g D

Pentyl acetate Androstenone Pentyl acetate Androstenone

D (0.0001) D (ns) I (0.04)h I (0.02)i

UPSIT Detection threshold UPSIT

40 types n-Butanol 40 types

D (0.05) D (0.05)j D (0.001)

D (0.01) D (0.001) D (0.004) D (0.001) D (0.05) D (0.02) D (0.001) D (0.006) D (0.001) D (0.001) D (0.001) D (0.02) D (0.05) D (0.10)

Odor Perception in Neurodegenerative Diseases

493

Table 3 (continued)

Ref.

Sex and no. of subjects

Clark et al., 2001 Kohler et al., 2001

20 M, 6 F 22 M, 18 F

Mean age (SD or range) 26.6 (7.9) 31.8 (9.3)

Type of testing

Odorant types

UPSIT UPSIT Detection threshold

40 types 40 types Phenylethanol

Direction of effect and p-value D (nst)k D (0.008) D (ns)

Nearly all studies have employed controls of the same age (and/or, in the case of UPSIT, standardized test norms); nst, no statistical test applied; nr, not reported; ns, not statistically significant; D, decreased performance in values relative to age-matched controls (note that for detection thresholds, this means elevated threshold, not decreased thresholds, and that this is independent of whether effect is statistically significant); I, increased performance relative to control; aSignificance found only for males, not females. bBased upon t-tests (two-tailed) computed by present author from Table 1 of their study. Although the authors note a sex difference in this work, both males and females differed significantly from their sex-matched control groups. cBased upon reanalysis of data performed by Hurowitz and Clark (1990). dThe negative symptom group was less sensitive than the positive symptom group (p  0.01). eThis was between a SZ group with severe polydipsia (n = 5) and a control group (n = 9). No significant difference was noted between a nonpolydipsic SZ group (n = 5) and the controls (n
9). fBased upon t-test (two-tailed) computed by present author from Table 1 of their study between affect twins and normal comparison group. gThis was a unilateral test and the main group effect was diminished by a nostril by group interaction. hBased upon t-test (one-tailed) between controls and a combined map of schizophrenic and brief psychotic disorders patients. iBased upon t-test (one-tailed) between controls and first episode schizophrenic groups. jBased upon t-test (two-tailed) computed by present author from Table 1 of their study. kSeven of 26 subjects score in the norm-based microsmic range.

patients in each age group and their own age-matched controls. As such, the observed differences in effect size between young and elderly SZ patients do not simply reflect normal aging effects, but rather some specific decline in olfactory function presumably due to the disease process itself. These findings, in conjunction with the inverse relationship observed between the duration of illness and UPSIT scores, are in accord with the aforementioned hypothesis that some degenerative component is present within olfaction-related brain regions of patients with SZ. The issue of whether antipsychotic medications influence smell function in SZ has received considerable interest, although the vast majority of studies find no obvious influence (e.g., Brewer et al., 2001; Hurwitz et al., 1988; Kopala et al., 1992, 1998a; Malaspina et al., 1994; Moberg et al., 1997b; Wu et al., 1993). If drug effects are present, they are subtle and present for only some types of psychophysical measures. Sirota et al. (1999) reported that threshold sensitivity to two odorants, isoamyl acetate and androstenone, was decreased in 19 male SZ patients during a drug-free period and even more after initiation of antipsychotic drug treatment. Twelve male SZ patients naïve to antipsychotic drugs reportedly had marginally increased threshold sensitivity, leading these authors to conclude that SZ-related olfactory deficits are due to antipsychotic medications. Considerable variation was present, however, in the threshold measures (e.g., in the controls a 4-log2-step difference was present from one

study to the next), and a variety of drugs with diverse pharmacological actions were combined in the drug analysis, precluding an assessment of the specific pharmacological mechanisms potentially involved. More recently, Purdon and Flor-Henry (2000) obtained n-butanol thresholds from each side of the nose of 17 nonmedicated SZ patients and 17 matched healthy controls. The patients exhibited an asymmetrical impairment of the left naris that was not present in the controls. In a second experiment, thresholds were measured in a new sample of 10 SZ patients before and after neuroleptic treatment. The asymmetry noted in the first study was replicated, and the relative advantage of the right naris shifted to a relative advantage of the left naris over the course of the 8-week-long treatment period. These authors suggested that neuroleptic drugs may benefit left hemisphere functions at a cost to right hemisphere functions. There is some precedent from animal studies for the idea that drugs that alter dopaminergic pathways can influence the ability to smell. For example, d-amphetamine [a drug that releases norepinephrine (N E) and dopamine (DA) and blocks their reuptake and degradation at nerve terminals] enhances the odor-detection performance of rats for ethyl acetate (EA) at low doses and depresses such performance at somewhat higher, yet relatively low, doses (Doty and Ferguson-Segall, 1987). Since (1) marked attenuation or elimination of olfactory bulb NE (but not DA) by intrabulbar injection of 6-hydroxydopamine (6-OHDA) has no significant influence on such detection performance

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(Doty et al., 1988b) and (2) depletion of cortical and olfactory bulb NE by injection of 6-OHDA into the dorsal noradrenergic bundle influences neither the acquisition or maintenance of a two-choice odor discrimination problem (Mair and Harrision, 1990), amphetamine’s effects probably depend upon dopaminergic, rather than noradrenergic, pathways. Direct support for potential dopaminergic regulation of olfactory sensitivity comes from a study using various doses of the dopamine D2 receptor agonist quinpirole (Doty and Risser, 1989). A dose-dependent decrease in the odor-detection performance for ethyl acetate was found. Notably, pretreatment with the D2 receptor antagonist spiperone, which also is a potent 5HT2A antagonist, eliminated these effects. In subsequent work (Doty et al., 1998a), the influence of a D1-selective partial agonist SKF 38393 on the odor-detection performance of rats was similarly evaluated. In a series of four experiments, it was found that, in contrast to quinpirole, SKF 38393 improved odor-detection performance in rats and that this phenomenon could be attenuated by the D1 receptor blocker SCH 23390. This effect was found to be neither sex-specific nor confined to the odor EA. These experiments suggest that D1 and D2 agonists can have differential influences on odor-detection abilities in rats. Despite the aforementioned animal studies, however, the influence of dopaminergic drugs on olfactory performance in humans has received little attention, particularly in SZ. It would be of interest, for example, to know whether Risperidone improves olfactory function. This drug is a novel benzisoxazole antipsychotic agent with potent combined serotonin 5HT2A and dopamine D2 antagonistic properties.

XIII.

OLFACTION IN EPILEPSY

Although early threshold studies reported heightened overall bilateral sensitivity in patients with epilepsy, particularly prior to an ictal event (e.g., Campanella et al., 1978; De et al., 1976; Dimov, 1973; Santorelli and Marotta, 1964), this phenomenon has not been subsequently observed. Whether this reflects mitigating influences of modern antiseizure medications is not clear, but possible, since the vast majority of epileptic patients tested in more recent years have been on medications. In the first study to test patients with respect to side of epileptogenesis, Eskenazi et al. (1986) reported normal bilaterally determined n-butanol odor detection thresholds in 18 epileptic patients relative to 17 controls, an observation confirmed for this odorant for each side of the nose by Martinez et al. (1993) in 21 epileptic patients. West et al.

(1993) similarly found, in 16 patients with left foci and 14 patients with right foci, no detection threshold deficits on either side of the nose, relative to controls, to the odorant phenyl ethyl alcohol. In contrast to detection threshold studies, a number of workers have reported suprathreshold deficits in epileptic patients. For example, Abraham and Mathai (1983) noted decreased performance on an odor-matching task in 28 epileptic patients with right-sided foci. Interestingly, patients with left-sided foci did not exhibit the same difficulty in performing the bilaterally administered olfactory matching task. Eskenazi et al. (1986) reported a small bilateral odor identification deficit in 18 epileptic patients relative to 17 normal controls, which was apparently independent of the side of the seizure focus. More recently, Carroll et al. (1993) found, relative to controls, a decrease in immediate odor memory to common odorants (e.g., vinegar, coconut, coffee, nail varnish, and garlic) in 10 patients with right-sided temporal lobe epilepsy. Ten patients with left-sided epilepsy, as well as 10 epileptics with a nontemporal lobe epileptic focus, did not show this deficit. This difference was seen only for nonnameable, uncommon odors. Three other studies have appeared on this general topic. In the first, Martinez et al. (1993) found deficits in odor discrimination, short- and long-term odor memory, and odor naming in 21 epileptics relative to controls. No differences between the left and right sides of the nose were present in these patients, who subsequently underwent temporal lobe resection. In the second, West et al. (1993) also found, in 20 patients with left foci and 20 with right foci, decrements on each side of the nose on tests of odor identification and memory relative to matched controls. In the third, prolonged OERP latencies were noted in 10 patients with right-side foci after right-side odorant stimulation, and in 12 patients with left-side foci after left-side odorant stimulation (Hummel et al., 1995). Recently, Kohler et al. (2001) administered the UPSIT and a detection threshold test to 14 patients with righttemporal lobe epilepsy, 18 patients with left-temporal lobe epilepsy, 40 patients with SZ, and 25 healthy controls. While the detection threshold measure did not differ among the four groups of subjects, the UPSIT scores of the right temporal lobe epilepsy patients and of the SZ patients were similar to one another and were significantly lower than the UPSIT scores of the other two groups. These findings are in accord with the hypothesis that the right hemisphere may play a special role in the processing of odors, as suggested from some neuroimaging studies (see Chapter 12). The reader is referred elsewhere to the influences of seizure-controlling operations,

Odor Perception in Neurodegenerative Diseases

including commissurectomy, on measures of smell function (Doty et al., 1997a).

XIV.

CONCLUSIONS AND CONSIDERATIONS

In this chapter, I have reviewed a number of studies demonstrating olfactory dysfunction in a wide range of neurological disorders. While the focus has been on those for which the most olfactory data are available, it should be noted that olfactory dysfunction has been observed in a number of disorders not discussed in detail in this chapter. Thus, smell loss is present in Pick’s disease (Richard and Bizzini, 1981), Kallmann’s syndrome (e.g., Yousem et al., 1996), multi-infarct dementia (Knupfer and Spiegel, 1986; Gray et al., 2001), restless leg syndrome (Adler et al., 1998), Korsakoff’s psychosis (e.g., Mair et al., 1983), alcoholism (e.g., DiTraglia et al., 1991), seasonal affective disorder (Postolache et al., 1999), head trauma (e.g., Doty et al., 1997c), attention-deficit hyperractivity disorder (Gansler et al., 1998), stroke (e.g., Rousseaux et al., 1996), and human immunodeficiency or acquired immunodeficiency syndrome (e.g., Brody et al., 1991), to name a few (for review, see Doty, 2001). It is noteworthy that a number of neurological or neurodegenerative disorders are accompanied by no, or only modest, alterations in smell function. In addition to those mentioned earlier in this chapter (e.g., MPTP-P), such disorders include essential tremor (Busenbark et al., 1992), depression (Amsterdam et al., 1987), and panic disorder (Kopala and Good, 1996). However, lack of involvement in neurological diseases seems to be the exception rather than the rule, as most neurodegenerative diseases appear to be associated with at least some degree of olfactory dysfunction. The reasons for the apparent predilection of the olfactory pathways for damage in such disorders are not clear (see Chapter 24). It is important to note that several studies of AD and SZ patients have reported lack of significant effects on odordetection threshold tests, in contrast to highly significant effects on such tests as the UPSIT. In some such cases, the lack of a threshold effect has been assumed to signify that the olfactory system is, in effect, functioning normally and that the deficit on identification reflects a central olfactory agnosia. A case in point for AD is work by Koss et al. These investigators reported, in two publications based upon the same set of patients, significant UPSIT, but not pyridine threshold, deficits in AD patients relative to controls (Koss et al., 1987, 1988). They concluded that the olfactory deficit of AD is associated with a “central” rather than a “peripheral” anomaly. While, ultimately, their overall conclusion may be correct (i.e., that the cause of olfactory deficits in AD reflects damage in structures more

495

central than the olfactory neuroepithelium), the basis upon which they arrive at this conclusion is suspect for a number of reasons. First, the vast majority of studies find threshold deficits in AD patients, and even their own data point to this conclusion. Thus, three of their subjects (30%) were either anosmic or hyposmic by their own criteria, and, in fact, the average threshold value for the AD patients was higher (i.e., indicative of less sensitivity) than that of the controls, even though statistical significance at the 0.05 a level was not achieved. Second, compared to other studies on this topic (see Table 1), their sample size was very small, compromising statistical power. In general, single ascending series detection threshold tests such as administered by them have test-retest reliability values below 0.40, compared to those of the UPSIT, which are uniformly above 0.90 (Doty et al., 1995b). Hence, the power of such a threshold test to detect a deficit is much less than that of a 40-item odor-identification test and is particularly compromised with small subject samples. Third, the general assumption that threshold tests reflect peripheral damage and identification tests reflect central damage is untenable. Thus, central lesions, in fact, have been associated with threshold deficits by some authors (e.g., Rousseaux et al., 1996; Shelley and Shelley, 2000), and peripheral (i.e., neuroepithelial) lesions are known to influence both tests of odor-detection threshold and odor identification (Deems et al., 1991). ACKNOWLEDGMENTS This work was supported, in part, by Grants PO1 DC 00161, RO1 DC 04278, RO1 DC 02974, RO1 AG 08148, and RO1 AG 27496 from the National Institutes of Health, Bethesda, MD. I thank Dr. Paul Moberg for providing some of the information employed is this review. REFERENCES Abraham, A., and Mathai, K. V. (1983). The effect of right temporal lobe lesions on matching of smells. Neuropsychologia 21:277–281. Adler, C. H., Gwinn, K. A., and Newman, S. (1998). Olfactory function in restless legs syndrome. Movement Dis. 13:563–565. Ahlskog, J. E., Waring, S. C., Petersen, R. C., Esteban-Santillan, C., Craig, U. K., O’Brien, P. C., et al. (1998). Olfactory dysfunction in Guamanian ALS, parkinsonism, and dementia. Neurology 51:1672–1677. Almkvist, O., Berglund, B., Nordin, S., and Wahlund, L. O. (1992). Is pyridine odor deficit related to progression of dementia in Alzheimer’s disease? :. Dept. Psychol. Stockholm Univ. 748:1–15.

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24 Olfactory System Neuropathology in Alzheimer’s Disease, Parkinson’s Disease, and Schizophrenia Gregory S. Smutzer, Richard L. Doty, Steven E. Arnold, and John Q. Trojanowski University of Pennsylvania, Philadelphia, Pennsylvania, U.S.A.

I.

receptor cells susceptible to damage from environmental pathogens and toxins, as well as a potential conduit for viruses and other exogenous agents from the environment into the brain (see Chapters 26, 27, and Introduction). This pathway is rather direct, in that only one synapse occurs between an olfactory receptor neuron and olfactory information-processing centers such as the piriform, perirhinal, and entorhinal cortices. Only two synapses occur between the receptor cell and secondary olfactory centers such as the hippocampus. It is noteworthy that these brain regions often exhibit the most abnormalities in AD and schizophrenia, albeit with very different neuropathologies between the diseases. Whether the olfactory pathways are, in fact, somehow involved in the etiology of neurodegenerative diseases is a source of current controversy (see Chapters 26 and 27).

INTRODUCTION

As noted in the previous chapter, the ability to smell is decreased in many, but not all, neurodegenerative diseases. Moreover, the proportion of affected individuals typically varies from disease to disease, as does the severity of the affliction. What are the reasons for such selective differences? While in the case of multiple sclerosis (MS), active plaques in olfaction-related central nervous system regions are known to correlate highly with olfactory deficits (Doty et al., 1997), such clear-cut causal associations have not been made in other neurodegenerative disorders. Furthermore, the problem is compounded by the observation that smell loss occurs early in the progression of several neurological disorders, long before rampant neuronal death or other measurable events of later-stage disease appear. In this chapter we examine the neuropathology of the olfactory circuitry of three neurological disorders for which the most histopathological data are available; namely, Alzheimer’s disease (AD), Parkinson’s disease (PD), and schizophrenia. Our goal is to explore the possible neurobiological causes of the olfactory losses associated with these disorders. Environmental factors, as well as genetic factors, conceivably play a role in the neurogenesis of such sensory decrements. Thus, the ciliated dendritic endings of olfactory receptor neurons — cells whose axons pass from the nasal cavity into the brain without an intervening synapse — are rather directly exposed to the external milieu. This exposure to the outside world makes the

II.

ALZHEIMER’S DISEASE

Alzheimer’s disease is the most common neurodegenerative disorder in humans, being the major cause of dementia in the elderly. As noted in the previous chapter, smell loss is prevalent (~90%) and marked (mean UPSIT scores ~20 out of 40) in this disorder and is present even in its earliest “questionable” diagnostic stages. AD’s classical defining histopathology includes neurofibrillary tangles (comprised of abnormal filaments containing tau, neurofilament (NF) proteins, and ubiquitin) and neuritic plaques (comprised of degenerating neurites interlaced with ab503

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normal filaments and extracellular -amyloid fibers). To what degree such pathology is causally associated with olfactory dysfunction is not currently known. It should be stressed, as was done in the previous chapter, that the frequency and severity of olfactory dysfunction in AD is very similar to that seen in Parkinson’s disease. This observation, and the finding of marked olfactory dysfunction early in the disease process, makes it difficult to ascribe the olfactory dysfunction of AD solely to large numbers of such classical neuropathological elements. A. AD-Related Pathology Within the Olfactory Epithelium As noted elsewhere in this volume, the pseudostratified columnar olfactory epithelium (OE) of humans lies deep

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within the recesses of the superior nasal cavity. At any point in time, a mixture of neuronal cells at different stages of development is present within this epithelium (see Chapters 2, 5, and 6). Among such cells are neuronal progenitor cells, immature sensory neurons, and mature sensory neurons. The marker proteins expressed by the three major cell types of the sensory epithelium are illustrated in Figure 1. In adults, the OE consists of clusters of sensory neurons interspersed among intervening expanses of metaplastic respiratory epithelium. These patches of nonsensory respiratory epithelia likely result from the replacement of sensory neurons by respiratory cells during normal aging and by atrophy of the OE through environmental insult or inflammation (Talamo et al., 1994). In addition, the OE of elderly human subjects is characterized by convolutions of

Figure 1 Schematic illustration showing the molecular phenotype for each of the three important cell types of the adult human olfactory epithelium: (A) olfactory receptor neuron cell body, (B) axon of olfactory receptor neuron, (C) sustentacular cells, (D) basal cells, (E) peripheral nervous system axons, and (F) dystrophic neurites. Abbreviations: GAP-43, growth-associated protein-43; KER-8, keratin 8; MAP 2 and 5, microtubule-associated protein 2 and 5 respectively; N-CAM, neural-cell adhesion molecule; NF-H, M, L, high, middle, and low-molecular weight (respectively) neurofilament; NGFR, p75 nerve growth factor receptor, PERIPH, peripherin; SYN, synaptophysin; VF, vimentin.

Olfactory Circuitry in Neurological Disorders

the sensory epithelium, conceivably reflecting influences of aging and cumulative environmental insults (Getchell et al., 1995; Rama Krishna et al., 1995; Smutzer et al., 1998). In general, the number of sensory neurons decreases across the human life span, with the most notable decrease occurring after the age of 65 years (Doty, 2001). The OE has been of considerable interest to students of AD (Brouillard et al., 1994). The major reasons are that smell dysfunction is an early clinical sign of this disorder and that the OE can be readily biopsied. Thus, a relatively large number of studies have searched for molecular markers within the OE that may detect AD pathology (Table 1). If AD-related pathology is present in this epithelium, then a potential means is available for establishing a histological diagnosis of AD during the life of the patient, perhaps even before the onset of clinical systems. Biopsy samples are excellent mechanisms for collecting biological tissue for the potential development of stable cell lines that could be used to examine the etiology of diseases such as AD. Thus, an olfactory biopsy, if reliable, could theoretically replace the neuropathological examination in establishing the presence or absence of the disease (Kaakkola et al., 1994). Early detection of AD is critical for subsequent administration of medications that may mitigate, delay, or even reverse the disease process. Although the accessibility of the OE for biopsy makes this tissue a promising system for clinical analysis, no presently identified marker proteins or messenger RNAs expressed in the OE of AD patients are specific to AD pathology (see Table 1). In addition, studies have not correlated the olfactory function of AD patients with the presence of these markers for AD. It is conceivable that damage to the OE alone, for whatever reasons, is the factor involved in inducing such pathology, rather than the disease process under consideration. In a recent review, Arnold et al. (1998) described the numerous non–ADrelated proteins found within the OE such as keratin 8 and microtubule-associated proteins (MAPs). Therefore, a primary focus of this chapter is on the detection of protein markers (and the expression levels of these markers) believed to be associated in some manner with neurodegenerative diseases and schizophrenia. 1. Dystrophic Neurites and Neurofibrillary Tangles Interest in employing olfactory system biopsies in the diagnosis of AD came to the forefront in 1989, when Talamo et al. (1989) reported that autopsied olfactory neuroepithelia from 8 patients with AD not only had a decreased number of olfactory receptor neurons relative to controls, but exhibited selective neuropathology similar to that seen in other regions of the brains of AD patients

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(i.e., the presence of dystrophic neurites). However, neuritic plaques and neurofibrillary tangles were not observed in the epithelia, a finding confirmed by others (Lee et al., 1993). In contrast to control tissue, the epithelia from AD patients had increased reactivity to antibodies raised against NF proteins as well as abnormal neural structures. Thus, olfactory nerve axon bundles in postmortem AD tissue reacted with antibodies to human neuron-specific enolase (NSE) and to the dephosphorylated form of the midsized NF subunit, NFM (P-), phosphate-independent forms of NFM, and to the low molecular weight subunit of neurofilament protein (NFL). The control subjects of this study, however, were not age-matched to AD patients, being significantly younger (respective medians
64 and 84 years), with over half falling below the age of the youngest AD patient. Hence, the reported differences could reflect age, rather than AD, per se. Nevertheless, this work received support from a study by Tabaton et al. (1991) that examined the immunoreactivity of biopsied olfactory mucosa from 8 AD patients and 6 age-matched controls to tau, a microtubuleassociated protein that is an integral component of paired helical filaments that make up neurofibrillary tangles, and to ubiquitin, a proteolytic stress protein that is essential for cell viability. Both of these proteins may contribute to aggregation events associated with the pathogensis of AD. Tau-reactive and partially ubiquitin-reactive dystrophic neurites were detected in the lamina propria of all AD patients, but in no control subjects. No plaques or tangles were observed within the olfactory mucosa of AD patients. Subsequent, and more extensive, studies have been more equivocal on the uniqueness of such pathology within the olfactory epithelia of AD patients. Trojanowski et al. (1991) found dystrophic neurites in the olfactory mucosa of all 11 AD cases examined (where they were not particularly abundant). However, these neurites were also found in 6 of 8 neurologically normal adult controls, 4 of 4 parkinsonism cases associated with AD, 2 of 2 cases of progressive supranuclear palsy (PSP), a case of ShyDrager syndrome without dementia, a case of diffuse cortical Lewy body disease with dementia, a case of schizophrenia, and a case of idiopathic degeneration of the substantia nigra with parkinsonism. Interestingly, only 1 of 4 elderly individuals with Down syndrome (DS) and AD exhibited such neurites. In contrast, dystrophic neurites were lacking in all 9 fetal and neonatal cases examined. These dystrophic neurites expressed growth-associated protein 43 (GAP-43), neural cell adhesion molecules (NCAMs), MAP 1B, MAP 2, tau, peripherin, and synaptophysin, as well as the high (NF-H), middle (NF-M), and low (NF-L) molecular weight NF triplet proteins, and were most dense at the interface of the OE and lamina propria,

Authors

Talamo et al.

Tabaton et al.

Trojanowski et al.

Lee et al.

Kaakkola et al.

Year

1989

1991

1991

1993

1994

11 AD

12 AD 8C 4 AD/PD 4 AD/DS 6 Fetuses 2 Term 2 PSP 1 DLDB 1 Schiz. 1 ND 1 S-D

11 AD 8C 4 AD/PD 4 AD/DS 6 Fetuses 2 Term 2 PSP 1 DLDB 1 Schiz. 1 ND 1 S-D

8 AD 6 AMC

9 AD 14 C

Number of patients and controls

58 (52–76) 46 (17-28)

73.9 (8.55) 60.3 (23.4) 67.8 (6.8) 57.8 (3.3) 5.2 M (1.0) 5 D, 3 M 78, 82 58 75 84 55

67.0 (6.67) 60.3 (23.4) 67.8 (6.8) 57.8 (3.3) 5.2 M (1.0) 5 D, 3 M 78, 82 58 75 84 55

68 (6.91) 63 (4.00)

83.3 (7.0) 58.7 (21.0)

Mean or median age (range, SD or SE)

6:5 2:6

5:7 6:2 2:2 2:2 3:3 0:2 1:1 1:0 0:1 0:1 1:0

5:6 6:2 2:2 2:2 4:2 0:2 1:1 1:0 0:1 0:1 1:0

4:4 NR

3:6 7:7

M:F sex ratio

B

A

A

B

A

Autopsy or biopsy

-and ubiquitin-immunopositive dystrophic neurites in lamina propria of all AD, but no AMC, mucosal specimens.

NF 68, NF 150, NF 200, NF 150 (p), NF 200 (p), MAP 2,

No differential expression of NF proteins in AD; NF neurons coincided with distribution of SYN, a ubiquitous neuronal marker; no MAP 2 or  found in AD mucosa.

The following antiNeuritic pathology seen in 10 of 12  antibodies: AD, 4 of 4 AD/PD, 3 of ALZ50, 133, 134, 135, 4 AD/DS, 6 of 6 non-AD E1, Z7, 189, 304, T1, degenerative cases, and in 4 of T14, T3p, T46, PHF1 8 controls. All  epitopes examined were expressed in dystrophic neurites within the OE of AD, but certain ones seemed preferentially expressed.

LCB, GAP 43 MAP 2, Dystrophic neurites seen in 11 MAP 5, NF-H, NF-M, AD, 11 of 14 cases of other NF-L, Peripherin, neurodegenerative diseases, PKC,  ChA, SYN, and 6 of 8 neurologically normal GFAP, Desmin, Keratin controls, but zero of 9 fetal and 8, N-CAM, NGFR, neonatal cases. Vimentin

NSE, ubiquitin

RM024, DP1TA51, AD mucosa shows unique HO14, RM0254, NFL, pathological changes in ALZ50, Anti-NF, OMP, morphology, distribution, and NSE, GFAP, P57 immunoreactivity of neuronal Vimentin, SE2 structures relative to C mucosa.

Antibodies used

Major conclusions relative to the olfactory mucosa of AD and C

Table 1 Details of Studies Examining the Olfactory Mucosa from Alzheimer’s Disease (AD) Patients, Some Other Neurodegenerative Diseases, and Controls (C)

Kishikawa et al.

Yamagishi et al.

Crino et al.

Getchell et al.

Kulkarni-Narla et al.

1990

1994a

1995

1995

1996

5 AD 4 NDC 4 C† 6C

3 AD 3 AMC 2 Fetuses 1 Infant 13 Others

12 AD

6 AD including 1 familial AD 7C

61 “of various diseases”

76.8 (15.9) 83.5 (10.0) 72.0 (7.20) 37.8 (14.5)

72, 74, 85 76, 78, 82 16–26 wks 10 wks 55.61 (17.8)

NR; most same as Lee et al., 1993 10 C 3 AD/PD 4 AD/DS 6 Fetuses 2 PSP 1 PD 1 S-D

73.5 66.4

“Of various ages”

4:1 2:2 2:2 3:3

2:1 1:2 1:1 1:0 7:6

NR

1:5 6:1

NR

A

A

A

B

A

Mn-SOD, Cu, Zn-SOD

HSP-70

(continued)

Pronounced increase in Mn- and Cu, Zn-SOD immunoreactivity in AD relative to C.

HSP-70 expressed in olfactory, supporting, and acinar cells of Bowman’s glands at all ages, being greatest in prenatal specimens; expression decreased with age, and was ~4 less in AD than in AMC ORCs.

The majority of each class of patients evaluated showed immunoreactivity to these antibodies. 20–30% of controls had such immunoreactivity.

A senile plaque-like extracellular mass found that reacted strongly to an anti-Tau antibody, and weakly to an anti-amyloid-beta protein antibody. Ubiquitin immunoreactivity was also observed in dendrites of sensory neurons.

-antibody, amyloid- protein, ubiquitin

Up107, 4G8, LN39, TFS

Positive reaction to anti--antibody in 65.5% of non-demented subjects, including young persons; no reaction to anti--amyloid antibody, but pseudo-like straining present mainly after age of 60 years.

-amyloid antibody, -antibody

Yamagishi et al.

Hock et al.

Yamagishi et al.

Duda et al.

1996

1998

1998

1999

7 AD 5 PD 2 MSA 1 AD/PD 1 PD/DLB 1 DLB 1 ND 11 C

9 AD 10 C

For biopsy, 2 mild and 3 moderate AD For autopsy, 2 severe AD and 5 C

9 AD 6 C* 8C

Number of patients and controls

80.9 (8.2) 65.4 (24.6) 55, 60 71 63 58 86 73.6 (15.0)

3:4 5:0 0:2 1:0 0:1 1:0 0:1 5:6

NR

0:2

AD autopsy ages: 86, 88 76.2 (11.4)

5:0

7:2

M:F sex ratio

AD biopsy ages ` 46–82:

75.0 (3.9) 80.4 (4.0) 34.5 (16 wks–90 y)

Mean or median age (range, SD or SE)

A

A

B The 7 A cases served as positive and negative controls

A

Autopsy or biopsy

No AD: C differences in olfactory receptor neuron density in contiguous OE strips; density of apoE staining in PGP-staining ONs 3.5  greater in AD than C. Although antibodies to - and -synucleins detected abnormal dystrophic neurites in OE of patients with neurodegenerative disorders, similar pathology also was observed in the OE of con trols.

-, -, -synucleins

Cytoskeletal changes and  pathology within the OE are not primary (or specific) features of AD, and may occur predominantly in late stages of the disease.

 antibody AT8, -amyloid -A4, NSE

ApoE, PGP 9.5

ON from all patients contained OMP, Spot 35, and NSE. Number of Spot 35 immunoreactive neurons decreased in AD relative to C; Mean number of OMP- and NSE-reactive neurons were no different.

Calbindin-D28 (Spot 35), NSE, OMP

Antibodies used

Major conclusions relative to the olfactory mucosa of AD and C

A
autopsy; AD/PD: AD with parkinsonism; AD/DS
AD with Down syndrome; AMC
age-matched controls; apoE
apolipoprotein E; B
biopsy; D
days; DLBD
diffuse Lewy body disease; M
months; MSA
multiple system atrophy; ND
idiopathic nigral degeneration; NDC
nondemented controls; NR
not reported; OE
olfactory epithelium; ON
olfactory receptor neurons; ORC
nonneuronal olfactory cell; PD
Parkinson’s disease; PSP
progressive supranuclear palsy; S-D
Shy-Drager syndrome; and Schiz.
schizophrenia. Antibodies designated as follows: HSP-70 = heat shock protein 70; MAP
microtubule-associated protein; LN39
an antibody to -amyloid precursor protein; NF
neurofilament; NFM (P-)
antibody to dephosphorylated from of mid-sized neurofilament subunit; NSE
neuron-specific enolase; OMP
olfactory marker protein; p
phosphorylated epitope; PGP
protein gene product 9.5; SYN
synaptophysin; TFS
thioflavin-S; Up107
an antibody to β-amyloid peptide. *Six of these controls were age-matched to the AD patients; the remaining 8 were not and included specimens ranging from a 16-week-old fetus to a 90 year-old man with hypertension. †These controls were not age-matched, but were confirmed to have, as were the other controls, brains with no significant AD pathology.

Authors

Year

Table 1 (continued)

Olfactory Circuitry in Neurological Disorders

as well as the lower portion of the sensory epithelium. These data suggested that (1) the mere presence of dystrophic neurites within the olfactory mucosa is not specific to AD, (2) dystrophic neurites are a common feature of the olfactory neuroepithelium of neurologically normal adults, and (3) the relevance, if any, of such neurites to aging or specific disease processes was not clear at that time. Later studies by this same group further evaluated these specimens or large subsets thereof. Lee et al. (1993) employed a panel of antibodies to epitopes present in subdomains, which span much of the lengths of all six known isoforms of human central nervous system (CNS) tau. All six epitopes were expressed in dystrophic OE neurites of AD patients. While the general findings noted above were observed, some antibodies (e.g., antibody 304) did not label dystrophic neurites in non-AD normal cases. These results suggest that within the OE, alterations occur in tau protein that are specific to AD. Another important observation of this work was that dystrophic neurites did not contain paired helical filaments characteristic of AD pathology in the CNS (Lee et al., 1991), but instead were composed of numerous bundles of 10–15 nm long filaments. These anatomical studies are significant because human olfactory receptor neurons from normal individuals do not typically express NF proteins, the neuron-specific intermediate filament protein peripherin, or the synaptic protein synaptophysin except within dystrophic neurites (Trojanowski et al., 1991). The cause of dystrophic neurite formation in the OE is unknown, but could be induced by respiratory disease or response to injuries. Dystrophic neurites could also result from neuronal populations that sustain damage from external stimuli such as pathogens, toxic chemicals, or their metabolites (Trojanowski et al., 1991). Additionally, some dystrophic neurite populations could arise from aberrant basal cell development in patches of epithelium that have undergone replacement of damaged sensory neurons. Since a small number of NF-positive olfactory neurons give rise to NF-positive dystrophic neurites, these dystrophic neurites could reflect abnormally induced proliferation of specific neuron populations (Carrell, 1988, Talamo et al., 1989). Alternatively, these OE neurites could represent sensory neurons that fail to form specific axonal connections with their glomerular targets in the olfactory bulb (Smutzer et al., 1998). 2.

Amyloid Plaques

A second histological marker of AD pathology is the formation of extracellular senile plaques composed of -amyloid peptide. Amyloid plaques contain two major amyloidogenic peptides that are formed by enzymatic cleavage

509

of amyloid precursor protein (APP) by an aspartyl protease. Recent evidence suggests that presenilin 1 may be the aspartyl protease that cleaves APP to -amyloid protein A 1–40 and -amyloid protein A 1–42 (Li et al., 2000). Nasal mucosal biopsy tissue from AD patients has also been examined by Yamagishi et al. (1994a; 1994b). These researchers detected tangle-like abnormal tau protein immunoreactivity in dendrites and perikarya of olfactory receptor neurons and in nerve bundles. They also detected ubiquitin immunoreactivity in the OE. These results suggested that ubiquitin conjugated with other polypeptides within dendrites, which marked these proteins for selective degradation. These authors concluded that in AD patients, similar pathologic changes occur in both the CNS and the olfactory mucosa. In a postmortem study, Crino et al. (1995) examined APP and -amyloid protein within the olfactory mucosa of patients diagnosed with AD. They utilized antibodies directed at -amyloid and against flanking sequences of APP. -Amyloid immunolabeling was observed in 10 of 12 AD cases, and 2 of 3 cases with combined AD and PD. -Amyloid staining was detected primarily in the basal third of the OE, in axons projecting through the lamina propria, and in metaplastic respiratory epithelium dispersed throughout the sensory OE. These authors concluded that their data “suggest that deposition of A occurs in a variety of circumstances and is not restricted to patients with AD, PD, or DS.” Recently, Hock et al. (1998) used a panel of specific antibodies to examine whether staining for tau or -amyloid differed between (1) OE tissue obtained by biopsy from five clinically mild to moderate AD patients and (2) OE tissue obtained at autopsy from advanced AD cases. No positive staining was found in any of the mild to moderate AD cases, although tau immunoreactivity was noted in the fine nerve fibers of the lamina propria and in a few OE cells of advanced AD cases. The authors concluded, “these results are consistent with other reports showing that cytoskeletal changes and tau pathology in the OE are not primary (or specific) features of AD and may occur predominantly in late stages of the disease.” 3.

Synuclein

The synucleins are a family of neuronal proteins that have been implicated in the pathogenesis of both AD and PD (Kruger et al., 1998; Lippa et al., 1998). Of the synucleins, -synuclein is a major component of cytoplasmic inclusions, and this protein was originally isolated from plaques obtained from the CNS tissue of AD patients (Ueda et al., 1993). -Synuclein is a soluble protein of 140 amino acids, and localized primarily to presynaptic axon terminals in

510

Smutzer et al.

the nervous system (Chase, 1997). When insoluble, this protein forms aggregates within plaques of AD tissue. Synuclein is also the major fibrillary component of Lewy bodies and Lewy neurites (Spillantini et al., 1998). Lewy bodies are the primary histopathological marker for PD. The -, -, and -synucleins are differentially expressed in olfactory and respiratory epithelia, with synuclein being the most abundant synuclein in the OE. In addition to its expression within dystrophic neurites, synuclein is expressed within the perikarya and cilia of olfactory receptor neurons. -, -, and -Synuclein proteins are also expressed in Bowman’s glands and in basal cells of the OE (Duda et al., 1999). These observations are consistent with a role for synucleins in the development, regeneration, and plasticity of olfactory receptor neurons in the normal adult human OE (Duda et al., 1999). These synucleins have also been identified in the olfactory mucosa of AD, PD, dementia with Lewy body disease, multiple system atrophy, and control patients.

the olfactory neuroepithelium to damage from a variety of agents. An increase in hsp 70 expression has been reported in frontal cortex white matter from postmortem AD tissue, implying a stress response in other AD tissue (Harrison et al., 1993). 6.

Apolipoprotein E

Another protein, superoxide dismutase (SOD), is reportedly increased in the OE of AD patients relative to controls. Manganese-SOD and Cu-Zn SOD are two ubiquitous enzymes that protect cells against destructive superoxide anion free radicals and their reaction products during cellular processes that include mitochondrial oxidative phosphorylation. SODs scavenge free radicals and protect cells from oxidation. Immunocytochemical analysis of human OE for Mn- and Cu-Zn SOD protein expression in postmortem tissue identified both enzymes in olfactory receptor cells (including axons), sustentacular cells, basal cells, Bowman’s glands, and vascular endothelium (KulkarniNarla et al., 1996). Significantly, immunoreactivity for both SOD enzymes heavily labeled cells near the surface of the OE and in basal region of the OE from AD patients. This pronounced increase in SOD enzyme immunoreactivity in the OE supports the hypothesis that oxidative stress in the sensory epithelium could be partially responsible for olfactory deficits observed in these patients.

In addition to inclusions within OE cells, several genes and their gene products show unusual expression patterns within the human olfactory mucosa of AD patients. In a postmortem study, Yamagishi et al. (1998) examined apolipoprotein E (apoE) protein levels in human olfactory mucosa. ApoE is a 34 kDa glycoprotein component of most serum lipoproteins, whose function is to mobilize cholesterol for membrane formation primarily during cellular differentiation and cell repair. ApoE is encoded by a polymorphic gene with three common alleles. A genotype containing a fourth allele has been associated with an increased risk for familial AD. The 4 isoform of apoE is thought to associate with tau or MAP 2 proteins in neurofibrillary tangles and with -amyloid protein within senile plaques (Rebeck et al., 1993). This association may partly explain why the 4 allele is a risk factor for familial AD. In the OE of elderly individuals, apoE has been detected throughout the cytoplasm of sensory neurons and Schwann cells associated with olfactory nerve bundles, in macrophages, and in blood vessels along the olfactory basement membrane. Its origin is presumably glial cells, including Schwann cells, and possibly intraepithelial macrophages (more prevalent in the OE of older than of younger persons), since no evidence of its expression within olfactory neurons has surfaced. Indirect support for the hypothesis that olfactory receptor neurons may endocytose apoE from surrounding cells comes from the demonstration of receptors for its uptake on CNS neurons (Rebeck et al., 1993). While it does not appear to be a specific marker for AD, it is noteworthy that apoE levels are more than threefold greater in AD than in control olfactory receptor cells (Yamagishi et al., 1998).

5.

7.

4.

Superoxide Dismutaste

Heat Shock Protein

Heat shock proteins are highly conserved proteins that are upregulated in cells after exposure to various stressors that include temperature shock, ischemia, injury, and toxic substances (Welch, 1992). Such upregulation presumably aids in the protection of cells from injury. It has been demonstrated that heat shock protein (hsp) 70 is markedly lower (~fourfold decrease in immunoreactivity) in the OE of AD patients relative to controls (Getchell et al., 1995). This downregulation may result in increased susceptibility of

Calbindin D28k (Spot 35 protein)

Intracellular levels of Ca2 are maintained at submicromolar concentrations to prevent Ca2 toxicity and to inhibit the formation of insoluble phosphorylated and carboxylated Ca2 salts (Hubbard, 2000). Calbindin D28k is a soluble protein that binds cellular Ca2 and buffers cytosolic Ca2 concentration within cells. Calbindin D28k localizes to cells of the OE, olfactory bulb, and AON of the human adult (Ohm et al., 1991; Yagamishi et al., 1996). In the OE, all known cell types express this Ca2-binding protein.

Olfactory Circuitry in Neurological Disorders

Intracellular Ca2 flux within olfactory tissue is critical for both odorant signal transduction (Kleene 1993; Zufall et al., 2000) and perireceptor events that include secretion (Smutzer et al., 1997). For odorant signal transduction, the cyclic nucleotide-gated channel (CNG) is a nonspecific cation channel that regulates cation influx after odorant stimulation and activation with cyclic nucleotides. An increase in divalent cations reduces the sensitivity of the CNG channel to ligand (Kleene, 1999). Also, intracellular Ca2 activates a Ca2-dependent chloride channel during odorant signal transduction (Kleene, 1993). Calcium is also required for type III adenylate cyclase activity, and this cation may amplify the response of this enzyme. In addition, Ca2 may be involved in the migration of neural progenitor cells from the mitotically active subventricular zone of the forebrain to the bulb and cerebral cortex (Baker et al., 2001; Haughey et al., 2002). In cells, Ca2 regulates its own release from endoplasmic reticulum (ER) stores through activated IP3 receptors (Bezprozvanny et al., 1991) and/or activated ryanodine receptors (Li and Chen, 2001). The emptying of ER Ca2 stores by either or both of these Ca2 channels could trigger capacitative-like Ca2 entry into the dendrites and soma of olfactory receptor neurons via Ca2release–activated currents similar to those identified in nonexcitable cells (Zufall et al., 2000). This intracellular Ca2 pool could modulate secretory activity, or possibly amplify the magnitude and duration of Ca2 transients that spread the odor-induced Ca2 wave in receptor neurons (Zufall et al., 2000). It is possible that a decrease in calbindin D28k expression within neurons could affect the ability of these cells to buffer Ca2, which in turn could adversely affect intracellular signaling, neuroblast migration and differentiation, or perireceptor processes. Furthermore, an increase in cytosolic Ca2 could cause these cells to be more susceptible to damage or necrosis. For example, the expression of calbindin D28k in a pancreatic islet beta-cell line protects these cells against cytokine-induced apoptosis and necrosis (Rabinovitch, 2001). An age-related decrease in calbindin D28k protein expression in olfactory receptor cells has been reported. The number of immunoreactive olfactory receptor cells were greatest in tissues from prenatal fetuses and noticeably less in tissue from a 10-week-old postnatal infant, three “young” (24-, 45-, and 47-year-old) and two “old” (65- and 90-year-old) specimens (Yamagishi et al., 1996). Although the number of calbindin D28k immunoreactive olfactory receptor neurons was similar among the postnatal groups examined in this study, statistically fewer immunoreactive neurons were found in the elderly group relative to the fetal group, and an age-related decrease in

511

epithelial thickness was observed. Importantly, a significantly lower number of calbindin D28k reactive receptor cells was noted in AD epithelia than in age-matched control epithelia. 8.

Metallothionein

Metallothioneins (MTs) are a family of proteins, present in a number of mammalian tissues, that are characterized by atypical cysteine abundance and high heavy metal [Cu(I), Zn(II)] content. All MT isoforms have numerous physiological functions, such as aiding in zinc and copper metabolism and in protecting against stress and oxygen free radical species (e.g., superoxide radicals). In mammals, four major isoforms have been described in numerous tissues, including the brain. One of these isoforms, MT-3 (also termed growth inhibitory factor), may play a role in neuromodulatory events and in the pathogenesis of AD (Richarz and Bratter, 2002). MT expression is increased in cortical glial cells of confirmed and possible AD cases (Adlard et al., 1998). Chuah and Getchell (1999) reported that immunostaining for MT in OE obtained from the septum of AD patients was increased relative to that of age-matched controls; indeed, four of six control specimens showed no MT staining at all. More intense and more frequent immunostaining was observed in the epithelium, Bowman’s glands, and underlying lamina propria. In a mouse study described in the same publication, such staining did not differ in the OE or olfactory bulbs between apo-E–deficient and wild-type mice. The authors concluded that “the induction of MT in a peripheral organ such as the olfactory mucosa is possibly indicative of the widespread increased level of ROS reactive oxygen species associated with AD.” In summary, the data from these and other studies are mixed as to whether specific neuropathology discernible by various antibodies may be present in the OE of AD patients that would meaningfully distinguish them from normal elderly persons. Clearly, more research is needed in this area, particularly longitudinal biopsy studies to examine if quantifiable changes occur over time in persons who develop AD. Such studies could determine whether such changes are distinct enough to provide a sensitive and specific diagnosis of AD. As noted earlier, no study has correlated the occurrence of such markers with actual psychophysical measurements of olfactory function, which could readily be performed in conjunction with biopsy studies. This approach would seem to be important if one is to identify complex AD-related factors in the OE.

512

B.

Smutzer et al.

Olfactory Bulb Pathology in Alzheimer’s Disease

As described in other chapters of this volume (e.g., Chapter 7), the olfactory bulb is the first olfactory system relay station in the brain. After forming synapses with incoming olfactory receptor cell axons within the glomeruli, the major bulbar projection neurons — the mitral and tufted cells — send their axons via the olfactory tract to cortical regions collectively termed the olfactory cortex (see next section). Included among such regions is the anterior olfactory nucleus (AON), which is a relatively well-defined structure largely comprised of large multipolar neurons located deep within the bulb’s granular cell layer (Esiri and Wilcock, 1984; ter Laak et al., 1994). The AON, in turn, sends projections to a number of contralateral structures via the anterior commissure, including the contralateral AON and olfactory bulb. Marked age-related changes occur in the olfactory bulb, and even nondemented elderly persons exhibit neurofibrillary tangles within the bulb and AON (Kishikawa et al., 1990; Meisami et al., 1998; Okamoto et al., 1990; Smith, 1942; Yousem et al., 1998). The massive loss of olfactory bulb glomeruli in older persons has been known for over 60 years (Smith, 1942). Kishikawa et al. (1990) reported the following frequencies of intrabulbar tangles as a function of decade of life in nondemented individuals: 5th decade, 17.9% (n
28); 6th decade, 25.6% (n
43); 7th decade, 57.1% (n
28); 8th decade, 86.7% (n
15); and 9th decade, 100% (n
2). Similar findings were reported by Okamoto et al. (1990). Such age-related changes are accelerated and exacerbated in AD, where decrements in some neurotransmitter receptors (e.g., dopamine) have also been reported (Loopuijt and Sebens, 1990). The formation of neurofibrillary tangles and cell loss occurs in all layers of the bulb and within the AON early in the disease process of AD (Averback, 1983; Kovacs et al., 2001; Ohm and Braak, 1987; Struble and Clark, 1992; ter Laak et al., 1994). For example, Esiri and Wilcock (1984) reported finding 62% fewer cells in the AON of AD patients relative to age-matched controls. Interestingly, while neurofibrillary tangles are frequently present within the neurons of the AON, they are relatively rare within the mitral, tufted, or granule cells of the bulb. However, severe and specific losses of olfactory bulb cells occur even in the earliest stages of AD (Davies et al., 1993; Struble and Clark, 1992). Such losses do not require the prior formation of tangles or plaques and appear to be among the major pathological changes of AD (Struble and Clark, 1992). A loss of ~50% of myelinated axons, associated with a reduction in cross-sectional area of the olfactory tract, has been reported in AD patients (Davies et al., 1993). Recent data suggest that the olfactory bulb is

among the first olfactory structures to exhibit neurofibrillary tangle formation and neuronal damage in AD (Kovacs et al., 2001). This neurodegeneration in the bulb would likely affect olfactory information processing in other regions of the CNS. C.

Olfactory Cortex Pathology of Alzheimer’s Patients

As described in detail in Chapters 8 and 9, the olfactory tract projects to central structures collectively termed the primary olfactory cortex. These structures are found largely within the medial temporal and basal frontal areas of the brain and include elements of the AON, the piriform and prepiriform cortices, the corticomedial amygdala, the olfactory tubercle, and the entorhinal cortex. Most mitral cell axons terminate within the prepiriform or piriform cortices, which have extensive connections with CNS structures involved in both cognition and behavior (Reyes et al., 1987). The latter structures include insular, orbitofrontal, and dorsolateral cortices, which collectively are considered the secondary olfactory cortex. Of these structures, the entorhinal cortex serves as a link between the neocortex and the limbic system and contains a large number of connections with association cortices in all four lobes of the brain. Perhaps the most important projection of the entorhinal cortex is to the hippocampus by way of the perforant path, as this is the main conduit by which information regarding sensory experience reaches the hippocampus (van Hoesen et al., 2000). It is well established that in AD, central nervous system regions closely associated with the olfactory system exhibit major pathological changes (Arnold et al., 1998). Neurofilament proteins and -amyloid plaques occur most prominently in the association areas of the cerebral cortex, which are interconnected with elements of the olfactory cortex. This finding is in contrast to the primary motor, somatosensory, auditory, and visual areas, which exhibit relatively little AD pathology (Pearson, 1996). Thus, pronounced loss of cholinergic neurons in the basal forebrain nuclei, entorhinal area, and hippocampus have all been reported in AD (Hyman et al., 1984; Kasa et al., 1997). Histopathological examination of the prepiriform cortex has shown increased numbers of neurofibrillary tangles and neuritic plaques in this region. Arnold et al. (1991a) mapped the densities of neurofibrillary tangles and senile plaques in 39 cortical areas of brains of AD patients, and observed a distinct distribution of NF tangles. The brain regions with the highest densities of neurofibrillary tangles were the entorhinal cortex, subiculum, temporal pole, perirhinal cortex, amygdala, and prepiriform cortex. Other areas that were less affected included limbic proisocortical

Olfactory Circuitry in Neurological Disorders

regions and the neocortical association areas. Senile plaques were more evenly distributed than were neurofibrillary tangles in Alzheimer’s brains. Recent studies further showed that antibodies to presenilin proteins stained neurofibrillary tangles in AD tissue (Murphy et al., 1996). These antibodies reacted with a subset of neurofibrillary tangles, but not with dystrophic neurites in the entorhinal cortex and hippocampus. In a large series of cases from across the adult life span, Braak and Braak (1991) staged the presence of neurofibrillary pathology throughout the brain. They described six stages of neuropathological severity, with dementia presenting in the third or fourth stage. Neurofibrillary tangles were first noted in the “transentorhinal” region (comprising the perirhinal and lateral portion of entorhinal cortex) before being found more extensively in the entorhinal cortex, hippocampus and other limbic regions, and ultimately the neocortex. Other studies have examined olfactionrelated cortices from AD brains in considerable detail, and these studies have demonstrated particularly severe pathology in the entorhinal cortex. Severe pathology has also been reported in the corticomedial nuclei of the amygdala (Kromer Vogt et al., 1990), prepiriform cortex (Reyes et al., 1987), temporal pole (Arnold et al., 1994), insular and orbitofrontal cortices (Chu et al., 1997), and basal forebrain (Whitehouse et al., 1982). Recently, McShane et al. (2001) reported that anosmia is more closely associated in demented patients with Lewy bodies than with typical measures of AD-related pathology. However, olfactory testing in this study was rudimentary since patients were simply asked whether or not they perceived an odor. A large number of studies have established that relatively few patients with either AD or PD have anosmia, making this all-or-none single-stimulus classification questionable. Moreover, the brain regions sampled for Lewy bodies were apparently not the same as those sampled for AD-related pathology, making the comparisons somewhat enigmatic. Nonetheless, this is perhaps the first study to attempt a direct correlation between pathology and function. Clearly, more refined studies are needed to determine whether this relatively startling conclusion is valid. Price et al. (2001) determined, in a postmortem study, the volume and number of neurons in the entorhinal cortex and hippocampus of preclinical AD cases using a stereological procedure (Price et al., 2001). These subjects had no measurable cognitive decline, but postmortem analysis demonstrated neuropathological evidence of AD (Price et al., 2001). No significant decrease in neuron number or cell volume was found in the control group, and little or no decrease was detected between the control and preclinical AD groups. In contrast, substantial decreases in neuron number and volume were observed in very mild AD cases.

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Larger decreases were observed in the severe AD group, suggesting that clinical deficits occur only when significant neural loss is present (Price et al., 2001). Overall, the olfactory losses observed in AD patients may reflect, in whole or in part, the neuropathology occurring in the olfactory bulb and primary and secondary olfactory cortices. Clearly, increased numbers of neurofibrillary tangles and senile plaques are present in regions of the olfactory system believed important for odor discrimination, identification, and even odor detection. While such inclusions may correlate with the well-characterized losses of olfactory function in AD, more studies are required to link these inclusions with quantifiable olfactory dysfunction in humans. Moreover, the classical pathology of AD may not entirely explain the olfactory loss, since patients with vascular dementia exhibit a similar degree of olfactory dysfunction (Gray et al., 2001). While vascular dementia is frequently accompanied with an admixture of ADlike pathology, such pathology is generally believed to be more variable than in AD (Kalaria and Ballard, 1999).

III.

PARKINSON’S DISEASE

Parkinson’s disease is the second most common neurodegenerative disorder in humans. Several forms of parkinsonism exist, and these forms may be idiopathic, familial, or induced by respiratory chain inhibitors such as MPP. Parkinsonism also occurs in many patients diagnosed with AD. PD is a movement disorder that is characterized by tremor, rigidity, and bradykinesia. PD can be identified in postmortem tissue by a loss of pigmented dopaminergic neurons of the substantia nigra pars compacta of the brainstem and gliosis. PD is further characterized by a distinct pathology that includes the formation of Lewy bodies and Lewy neurites (Forno, 1996; Lewy, 1912; Trojanowski and Lee, 1998). Lewy bodies are composed of a dense core of filamentous and granular material surrounded by radially oriented filaments (Roy and Wolman, 1969). A number of proteins have been identified in Lewy bodies and Lewy neurites of PD patients. These proteins include NF proteins (Schmidt et al., 1991), -synuclein (Spillantini et al., 1998), and ubiquitin (Forno, 1996). The transformation of soluble synuclein proteins into insoluble aggregates (or interactions with other neural proteins or molecules to produce aggregates) may lead to Lewy body formation. After their formation, these Lewy bodies could compromise the survival of affected neurons (Trojanowski and Lee, 1998), including those of the olfactory pathway. Olfactory dysfunction is one measurable feature of individuals who suffer from PD (Doty et al., 1988). Clinical evidence indicates that olfactory deficits may occur even

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before the onset of movement disorders that are characteristic of PD (Berendse, 2001). Thus, olfactory testing is one promising tool to identify olfactory deficits that may occur prior to clinical symptoms (Berendse, 2001; Tissingh et al., 2001; Wolters et al., 2000). A.

Olfactory Epithelium Pathology in PD

Relatively few studies have characterized the olfactory system of patients with PD at the cellular level. The few published studies have focused on the OE and olfactory bulb. Crino et al. (1995) were the first to identify -amyloid protein in the OE of postmortem tissue from PD patients. As in AD, immunostaining was observed in the basal third of the OE, in axons that projected through the lamina propria, and in metaplastic respiratory epithelium within the OE. This group also examined postmortem tissue from PD patients with dementia due to AD. As in AD, dystropic neurites were detected in all four cases examined. The dystropic neurites stained with antibodies against GAP-43, and GAP-43 protein is a marker for postmitotic immature neurons. Dystrophic neurites also expressed proteins that were not detected in normal olfactory receptor neurons. These proteins included synaptophysin, tau, and NF proteins. More recent studies have utilized synuclein immunoreactivity for positive identification of dystrophic neurites in the OE of PD patients. -Synuclein has received intense scrutiny because two missense mutations in the -synuclein gene (an A53T and an A30P nucleotide substitution) have been associated with a rare autosomal dominant form of familial PD (Kruger et al., 1998; Papadimitriou et al., 1999; Polymeropoulos et al., 1997). Also, -synuclein aggregates of wild-type protein are a major protein component of filaments of Lewy bodies and Lewy neurites in idiopathic PD (Spillantini et al., 1998; Trojanowski and Lee, 1998). As in AD, -synuclein was the most abundant synuclein species found in dystrophic neurites of the OE of PD patients. Again, no synuclein pathology was specific to the OE of PD patients (Duda et al., 1999). -Synuclein can associate with the cytoplasmic protein synphilin-1. Synphilin-1 forms intracellular inclusions that resemble Lewy bodies in cells that are co-transfected with the central domain of synuclein (Engelender et al., 1999). Furthermore, synphilin-1 has been identified as a protein component of Lewy bodies in PD (Wakabayashi et al., 2000). This 90 kDa protein is thought to allow -synuclein to interact with intracellular proteins involved in vesicle transport or in cytoskeletal functions (Engelender et al., 1999). These immunocytochemical studies within the OE are significant because the expression of -synuclein protein in cortical Lewy bodies correlates well with dementia

in PD (Hurtig et al., 2000). Thus, the pathology of the OE could have potential use in antemortem analysis of PD. B.

Olfactory Bulb Pathology in PD

Recent evidence also suggests that disease-specific pathology may localize to the olfactory bulb and olfactory tract of PD patients. A number of investigators (Daniel and Hawkes, 1992; Hawkes et al., 1997; Pearce et al., 1995) have detected Lewy body pathology by classic ubiquitin immunostaining in the AON of PD cases. Lewy bodies in the olfactory bulb and tract were similar in morphology to those found in the cerebral cortex. Surprisingly, Lewy bodies were not detected in control olfactory bulb tissue. In addition, the hippocampus (which is a secondary olfactory center) also contained high densities of synuclein-positive lesions in presynaptic axon terminals. These results are consistent with synaptic dysfunction in the hippocampus (Galvin et al., 1999). Pearce et al. (1995) detected a dramatic cell loss in the AON of patients with PD, a phenomenon also observed in the AON of patients with PD-dementia complex of Guam (Doty, 1997). The cell loss correlated well with the number of Lewy bodies, a noteworthy observation because Lewy body formation in PD is almost exclusively confined to the brainstem, cerebral cortex, and hippocampus. As in AD, these results suggest a susceptibility of certain neuronal populations to PD pathology. If Lewy bodies localize to the olfactory bulb and tract tissue in PD but not in control tissue, then it is possible that PD could be diagnosed in postmortem tissue by immunostaining this tissue for Lewy body proteins. Whether dopaminergic pathology is associated with the olfactory dysfunction of patients with PD is not known. The adult human olfactory bulb is thought to contain tyrosine hydroxylase activity in superficial tufted cells and in small periglomerular neurons (Halasz et al., 1981; Hoogland and Huisman, 1999; Liberini et al., 2000; Smith et al., 1991), although the ability of these neurons to synthesize dopamine and the role of this neurotransmitter in olfactory function is unclear. The olfactory dysfunction of PD patients is not restored with dopamine repletion, suggesting that the deficit, if dopamine dependent, reflects a lack of (or dysfunctional) receptors rather than the availability of dopamine (Doty et al., 1992). Nevertheless, animal studies suggest that dopaminergic neurons may modulate olfactory function to some degree. For example, dopamine receptor activation in rat olfactory bulbs causes a significant depression of synaptic transmission at the first synapse between olfactory receptor neurons and mitral cells (Hsia et al., 1999). Systemic injection of the dopamine D2 receptor agonist quinpirole impairs operantly

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determined odor detection performance of rats. In contrast, injection of the dopamine D1 receptor agonist SKF 38393 enhances such performance (Doty and Risser 1989; Doty et al., 1998), effects that could be modulated at the level of the bulb and/or in more central regions (see next section). Ligands that directly or indirectly enhance dopaminergic neurotransmission stimulate sniffing behavior, and this effect is blocked by selective dopamine D2 antagonists, including 1-sulperide (Le Moal, 1995). Dopaminergic neurons may also play a role in olfactory bulb cell differentiation. In rodents and nonhuman primates, the constant differentiation of dopaminergic neurons from precursor cells takes place in the olfactory bulb (Kornack and Rakic, 2001), where these cells differentiate into periglomerular cells. The latter cells originate as neuroblasts in the subventricular zone of the forebrain (Haughey et al., 2002). The mitotically active neuronal progenitor cell population undergoes restricted chain migration to the bulb and cerebral cortex via the rostral migratory stream. A subset of these neuroblasts then migrates to the bulb and differentiates into dopaminergic neurons (Baker et al., 2001; Pencea et al., 2001). In the absence of disease, these neuroblasts may provide a reservoir for replacement of neurons lost during cell turnover in the olfactory bulb or after brain injury (Haughey et al., 2002). In the presence of disease, however, neurogenesis may be deficient.* In addition to a possible role in olfaction, bulbar dopaminergic neurons may be important for the future treatment of PD. When human neural progenitor (embryonic) cells were transplanted into the subventricular zone of neonatal rat brains, these embryonic cells underwent migration to, and differentiation within, the olfactory bulb (Englund et al., 2002). However, it is not known if the continuous differentiation of dopaminergic neurons occurs in the human olfactory bulb. If this does occur, one promising source of transplantable neurons in PD patients could be adult olfactory bulb dopamine progenitor cells that originate in the anterior subventricular zone (Baker et al., 2001). * Interestingly, amyloid -peptide, a marker for AD that is also present in some patients with PD, may contribute to this process. This protein specifically impairs neurogenesis in the subventricular zone and cerebral cortex of adult mice and in human embryonic cortical neural progenitor cells grown in culture (Haughey et al., 2002). The adverse effects of amyloid -peptide on neurogenesis parallels a disruption of Ca2  regulation in neural progenitor cells. If this situation occurs in vivo, then a systematic examination of the inhibitory role of amyloid -peptide on neurogenesis could allow insights into the olfactory loss of PD and AD, and perhaps even into the progressive elements of these disorders.

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C.

Olfactory Cortex Pathology of Parkinson’s Patients

PD is characterized by a decrease in CNS dopamine production. If dopamine affects CNS olfactory function in humans, then a loss of dopaminergic neurons could negatively modulate olfactory processing in the brain. In rodents, recent studies have identified dopaminergic fibers in the piriform cortex (Datiche and Cattarelli, 1996). In addition, biochemical studies and binding studies have shown that the rodent piriform cortex contains high concentrations of dopamine and dopamine receptors (Descarries et al., 1987). For example, the olfactory tubercle is the main source of innervation to the piriform cortex, and this region has markedly decreased dopamine levels in PD (Bogerts et al., 1983). Histopathological studies of postmortem PD tissue have shown that neurofibrillary tangles and Lewy bodies were found within lamina II of the entorhinal cortex (Braak and Braak, 1990). In one study, Lewy bodies were detected in this region in 48% of the cases examined (20/42), whereas neurofibrillary tangles were positively identified in 98% of the cases (41/42) (Mattila et al., 1999). In a related immunohistochemical study, -synuclein-immunoreactive cortical Lewy bodies were identified in the amygdala (primary olfactory cortex), hippocampus (secondary olfactory processing center), or cortical gyrus of 43 of 45 PD patients (Mattila et al., 2000). This study further demonstrated that -synuclein–positive cortical Lewy bodies were associated with cognitive impairment in PD that was independent of any AD-type pathology.

IV.

SCHIZOPHRENIA

Schizophrenia, a severe psychiatric illness that affects approximately 1% of the world’s population, is characterized by thought disorder, hallucinations, delusions, and impaired cognition, particularly in memory, attention, and executive functioning. Numerous neuropsychological and clinical neurobiological studies have implicated abnormalities of fronto-temporal-limbic circuitry in the disorder (Arnold, 1999; Moberg et al., 1999). However, in contrast to AD and PD, schizophrenia is not defined by distinct pathological lesions (Arnold and Trojanowski, 1996; Damadzic et al., 2002; Harrison, 1999), and no good animal models are presently available to examine its etiology. While research has highlighted both neurodevelopmental and neurodeteriorative aspects of the disorder, neurodevelopmental factors seem to play the predominant determining roles. This disorder is associated with such factors as (1) maternal starvation during the first trimester of

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pregnancy (Susser et al., 1996), (2) influenza infection during the second trimester (Mednick et al., 1988), (3) rhesus and ABO blood type incompatibility (Hollister et al., 1996), and (4) perinatal anoxic birth injuries (Cannon et al., 2000). Monozygotic twin studies suggest that schizophrenia has both epigenetic and genetic components, since concordance for schizophrenia is between 30 and 50%. Antipsychotic drugs used to treat the positive symptoms of schizophrenia specifically block dopamine receptors, and this observation has implicated aberrant dopamine transmission in the pathogenesis of schizophrenia (Goldstein and Deutch, 1992). As noted in Chapter 23, olfactory deficits in schizophrenia are generally less severe relative to those observed in AD or PD.* A.

The Olfactory Epithelium in Schizophrenia

The question as to whether the OE of patients with schizophrenia differs from that of controls has been addressed in several studies. Smutzer et al. (1998), in an early qualitative study, found that olfactory neurons and basal cell populations of both schizophrenic and control groups equally expressed MAP 1B and N-CAM, and all receptor neurons were immunopositive for protein gene product 9.5. Basal cells from both groups also expressed nerve growth factor receptor. Schizophrenic and control subjects exhibited dystrophic neurites immunoreactive for either synaptophysin, MAP 1B, tau, or NF proteins, and evidenced seemingly equivalent immunostaining patterns,

* One of the most consistent neurobiological findings in schizophrenia has been ventriculomegaly seen in CT and magnetic resonance imaging (MRI) studies (Frith et al., 1995). The degree of ventricular enlargement correlates with poor premorbid adjustment and has been found at the first onset of symptoms. Notably, many of the postmortem brain findings suggestive of aberrant neurodevelopment in schizophrenia have been observed in olfactory cortices and closely related areas. Cytoarchitectural and neuronal morphometric studies have described abnormalities in neuron arrangement, size, shape, orientation, and packing density in various limbic and prefrontal regions (Arnold and Trojanowski, 1996). Several groups have reported abnormalities in interstitial white matter neuron density and placement, which suggests aberrant neuronal migration or subplate neuron pruning during cerebrogenesis (Akbarian et al., 1996; Arnold et al., 1991a). Other findings have been interpreted as subtle “miswiring” in cortical connections that may be neurodevelopmental or neuroplastic in nature (Benes, 1993). Strengthening the neurodevelopmental theory has been the failure to find any evidence for postmaturational neural injury or neurodegeneration (Arnold, 1999; Arnold and Trojanowski, 1996; Arnold et al., 1991b, 1998; Bloom, 1993; Honer et al., 1995).

suggesting that the olfactory deficits associated with schizophrenia could be due to abnormalities in more central olfactory structures. Two more recent studies, however, have found evidence for abnormalities in the OE of patients with schizophrenia. In the first study, neuronal cultures derived from biopsy tissue of living patients were evaluated (Feron et al., 1999). Tissue from the schizophrenics exhibited, relative to control tissue, a reduced ability to attach to culture slides and contained a greater proportion of cells undergoing mitosis. Interestingly, the addition of dopamine to the cultures from schizophrenia patients reduced the level of apoptotic cell death. In a subsequent histopathologic study, the OE of 13 elderly subjects with schizophrenia and 10 controls were removed at autopsy (Arnold et al., 2001a). Sections were immunolabeled with antibodies that distinguish OE neurons in different stages of development, including basal cells (low-affinity nerve growth factor receptor, p75NGFR), postmitotic immature neurons (GAP-43), and mature olfactory receptor neurons (olfactory marker protein). These results are shown in Figure 2. The density of p75NGFR basal cells in specimens from schizophrenic patients was decreased to 37% of that in controls, whereas the density of GAP-43–positive postmitotic immature neurons was increased 316% relative to controls. Marked increases were noted in (1) the ratio of GAP-43–positive postmitotic immature neurons to p75NGFR positive cells (665%) and (2) the ratio of olfactory marker protein–positive mature neurons to p75NGFR-positive basal cells (328%). These findings led these authors to conclude that “abnormal densities and ratios of OE neurons at different stages of development indicate dysregulation of OE neuronal lineage in schizophrenia.” This dysregulation could be due to intrinsic factors controlling differentiation or an inability to gain trophic support from axonal targets in the olfactory bulb. While caution is necessary in extrapolating developmental findings in mature OE to early brain development, similarities in molecular events suggest that such studies may be instructive. B.

The Olfactory Bulb in Schizophrenia

Recently it was shown that patients with schizophrenia exhibit a 20% decrease in bilateral olfactory bulb volume relative to controls, as measured by quantitative MRI (Turetsky et al., 2000). Such a decrease is of greater magnitude than the decreases noted in other brain structures in this disorder. For example, bilateral reduction of hippocampus volumes in patients with schizophrenia is 4% (Gur et al., 1987). In the bulb this phenomenon could reflect a decrease in cell numbers, a decrease in cell volumes, or both. One possible explanation for a decrease in bulb size could be

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Figure 2 The olfactory epithelium (OE) in schizophrenia. (a) Coronal section of nasal cavities stained with toluidine blue. The OE lines the walls of the left and right nasal cavities along turbinates (lateral walls) and septum (medial walls). Segments for analysis were from the OE adjacent to the apices of the nasal cavities. (Bar indicates 1 mm.) Nonpsychiatric control (b) and schizophrenia case (c) OE was double-labeled for p75 nerve growth factor receptor (p75NGFR) plus basal cells (brown), and immature olfactory receptor neurons (ORN) expressing growth-associated protein-43 (GAP-43) plus immature neurons (black or dark gray). Nonpsychiatric control (d) and schizophrenia case (e). OE was double-labeled for p75NFGR and basal cells (brown), and olfactory marker protein (black). BC indicates basal cell layer; and LP, lamina propia. Bar indicates 10 mm for b to e. (See color insert.)

apoptosis of mitral cells possibly induced by noninnervation (Naruse and Keino, 1995), incorrect innervation of regenerating olfactory neurons, or interruption of neuroblast migration via the rostral migratory stream (Chazal et al, 2000). Whatever its basis, this finding suggests that the olfactory bulb may be especially vulnerable to the disease processes that produce structural brain changes in this disease. To date, only two preliminary studies of OB immunohistopathology have been reported. Arnold et al. (2001b) used a panel of antibodies and quantitative microscopy to characterize olfactory bulb glomeruli in 17 elderly schizophrenics and 17 matched controls. They found significant decreases in glomerular expression of the presynaptic SNARE protein SNAP-25, decreases in receptor tyrosine kinase B expression (this protein binds brain-derived neurotrophic factor), and a decrease in expression of the postsynaptic dendritic protein MAP 2. In a related study, a significant reduction in the expression of phosphorylationindependent MAP 2 protein was observed in olfactory bulbs from an elderly, highly chronic, sample of subjects with schizophrenia (Rioux et al., 2001). If aberrant synaptic reinnervation is an underlying feature of schizophrenia, then olfactory receptor cell regeneration and synapse formation in the bulb could be an excellent model to examine aberrant neurodevelopment in this disorder.

C.

The Olfactory Cortex in Schizophrenia

A number of abnormalities in cytoarchitecture, neuronal morphology, innervation, and neuronal protein expression have been reported in schizophrenia that suggests a disturbance in nascent neuronal migration during fetal development (Arnold et al., 1998). One example is in the entorhinal cortex, a component of the primary olfactory cortex (Arnold et al., 1991c; Falkai et al., 2000; Jakob and Beckmann, 1986, 1994). Several reports have described (1) poorly formed neuron clusters in layer II, (2) apparent heterotopic displacement of layer II-type neurons deep into layer III, (3) developmental anomalies in pre-alpha cell clusters, and (4) poor laminar differentiation and attenuation of deeper layers of the cortex (Arnold et al., 1991b; Jakob and Beckmann, 1986). Other findings include smaller neuron size (Arnold et al., 1995), increased density of glutamatergic vertical axons (Longson et al., 1996), and decreased MAP 2 protein expression (Arnold et al., 1991c). In contrast, several studies have reported no significant differences between schizophrenics and controls in entorhinal cytoarchitecture (Akil and Lewis, 1997; Heinsen et al., 1996; Krimer et al., 1997a). Krimer and colleagues (1997a) qualitatively studied all sectors of the

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entorhinal cortex and found no major differences between schizophrenics and controls. They examined neuronal numbers, density, and layer volumes in the entorhinal cortex. However, their quantitative analyses were limited to a small group of seven schizophrenics and eight controls. They did report a trend toward lower neuronal numbers and density (by 12–18%) in almost all layers of the most rostral entorhinal sectors (Krimer et al., 1997b). Due to the role of the entorhinal cortex in olfactory processing, anatomical or physiological disturbances within this tissue could mediate the observed aberrant behavior and/or olfactory dysfunction in schizophrenic patients. The parahippocampal region has been implicated in olfactory memory processes. This region consists of the presubiculum, parasubiculum, entorhinal cortex, perirhinal cortex, and the parahippocampal cortex proper (Arnold, 2000). Global changes in the parahippocampal region of schizophrenics have been suggested based on volumetric changes in the parahippocampal gyrus. These changes are manifested as a decrease in gyral volume in the left hemisphere of the brain. The first report was by Bogerts et al. (1985), who conducted a systematic, but nonstereological, analysis of serial brain sections of unmedicated schizophrenics and normal controls that underwent autopsy from 1928 to 1953. These authors reported a 44% mean reduction in parahippocampal gyrus volume. This decrease in volume was greater than that observed in any other brain structure they studied in these autopsy cases. These other brain structures included the amygdala, caudate, hippocampal formation, nucleus accumbens, putamen, and external pallidum.

V.

SUMMARY

At present, the physiological mechanisms that underlie olfactory dysfunction in neuropsychiatric disorders remain largely unknown. In both AD and PD, olfactory deficits occur early in the disease process, and considerable data suggest that CNS neural networks involved in processing olfactory information may be especially vulnerable to neurodegeneration (Liberini et al., 2000). Whether or to what degree environmental agents play a role in the etiology of the olfactory dysfunction in these disorders is not known. In AD, PD, and schizophrenia, alterations in all major stages of the neural circuitry responsible for identifying and processing olfactory information appear to be present. Which of these degenerative processes or neurodevelopmental anomalies are responsible for the olfactory dysfunction remains a mystery. On a positive note, the olfactory system has generated intense interest among scientists within the last decade,

and significant advances have been made in our basic understanding of odorant processing and related phenomena, including neuronal regeneration within the OE and bulb. The next decade should allow scientists and clinicians to utilize this and future knowledge as a tool to examine and dissect the pathology and etiology of neurological disorders that include AD, PD, and schizophrenia. In particular, a better understanding of the mechanisms behind the continuously regenerating neurons of the OE, their inherent ability to form synapses with specific glomeruli within the bulb, the nature of higher-order olfactory processing, and the roles of various neurotransmitters, including dopamine, in determining, modulating, and coordinating olfactory information will likely provide key information. This information is necessary for elucidating the pathology responsible for the olfactory anomalies of a number of neurological diseases and, in some cases, the diseases themselves. Such an understanding will be aided not only by the painstaking examination of specific lesions or genetic polymorphisms in neuropsychiatric disorders, but by the application of advanced molecular approaches to this problem. Such approaches will most likely include the use of single-cell mRNA expression profiling in conjunction with cDNA microchip array technology, thereby allowing for the examination of differences in expression profiles of key genes that may modulate olfactory function in neurological disorders (Mirnics et al., 2000). VI.

ACKNOWLEDGMENTS

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Olfactory Circuitry in Neurological Disorders Smutzer, G., Zimmerman, J. E., Hahn, C. G., Ruscheinsky, D. D., Rodriguez, A., Han, L. Y., and Arnold, S. E. (1997). Inositol 1,4,5-trisphosphate receptor expression in mammalian olfactory tissue. Brain Res. 44:347–354. Smutzer, G., Lee, V. M., Trojanowski, J. Q., and Arnold, S. E. (1998). Human olfactory mucosa in schizophrenia. Ann. Otol. Rhinol. Laryngol. 107:349–355. Spillantini, M. G., Crowther, R. A., Jakes, R., Hasegawa, M., and Goedert, M. (1998). Alpha-synuclein in filamentous inclusions of Lewy bodies from Parkinson’s disease and dementia with Lewy bodies. Proc. Natl. Acad. Sci. USA 95:6469–6473. Struble, R. G., and Clark, H. B. (1992). Olfactory bulb lesions in Alzheimer’s disease. Neurobiol. Aging 13:469–473. Susser, E., Neugebauer, R., Hoek, H. W., Brown, A. S., Lin, S., Labovitz, D., and Gorman, J. M. (1996). Schizophrenia after prenatal famine. Further evidence. Arch. Gen. Psychiatry 53:25–31. Tabaton, M., Cammarata, S., Mancardi, G. L., Cordone, G., Perry, G., and Loeb, C. (1991). Abnormal tau-reactive filaments in olfactory mucosa in biopsy specimens of patients with probable Alzheimer’s disease. Neurology 41:391–394. Talamo, B. R., Rudel, R., Kosik, K. S., Lee, V. M., Neff, S., Adelman, L., and Kauer, J. S. (1989). Pathological changes in olfactory neurons in patients with Alzheimer’s disease. Nature 337:736–739. Talamo, B. R., Feng, W. H., and Stockmayer, M. (1994). Human olfactory epithelium: normal patterns and types of lesions found in the general population. Inhal. Toxicol. 6:249–275. ter Laak, H. J., Renkawek, K., and van Workum, F. P. (1994). The olfactory bulb in Alzheimer disease: a morphologic study of neuron loss, tangles, and senile plaques in relation to olfaction. Alz. Dis. Assoc. Dis. 8:38–48. Tissingh, G., Berendse, H. W., Bergmans, P., DeWaard, R., Drukarch, B., Stoof, J. C., and Wolters, E. C. (2001). Loss of olfaction in de novo and treated Parkinson’s disease: Possible implications for early diagnosis. Mov. Disord. 16:41–46. Trojanowski, J. Q., and Lee, V. M. (1998). Aggregation of neurofilament and alpha-synuclein proteins in Lewy bodies: implications for the pathogenesis of Parkinson disease and Lewy body dementia. Arch. Neurol. 55:151–152. Trojanowski, J. Q., Newman, P. D., Hill, W. D., and Lee, V. M. (1991). Human olfactory epithelium in normal aging, Alzheimer’s disease, and other neurodegenerative disorders. J. Comp. Neurol. 310:365–376. Turetsky, B. I., Moberg, P. J., Yousem, D. M., Doty, R. L., Arnold, S. E., and Gur, R. I. (2000). Olfactory bulb volume is reduced in patients with schizophrenia. Am. J. Psychiatry 157:828–830.

523 Ueda, K., Fukushima, H., Masliah, E., Xia, Y., Iwai, A., Yoshimoto, M., Otero, D. A., Kondo, J., Ihara, Y., and Saitoh, T. (1993). Molecular cloning of cDNA encoding an unrecognized component of amyloid in Alzheimer disease. Proc. Natl. Acad. Sci. USA 90:11282–11286. van Hoesen, G. W., Augustinack, J. C., Dierking, J., Redman, S. J., and Thangavel, R. (2000). The parahippocampal gyrus in Alzheimer’s disease. Clinical and preclinical neuroanatomical correlates. Ann. NY. Acad. Sci. 911:254–274. Wakabayashi, K., Engelender, S., Yoshimoto, M., Tsuji, S., Ross, C. A., and Takahashi, H. (2000). Synphilin-1 is present in Lewy bodies in Parkinson’s disease. Ann. Neurol. 47:521–523. Welch, W. J. (1992). Mammalian stress response: cell physiology, structure/function of stress proteins, and implications for medicine and disease. Physiol. Rev. 72:1063–1081. Whitehouse, P. J., Price, D. L., Struble, R. G., Clark, A. W., Coyle, J. T., and Delon, M. R. (1982). Alzheimer’s disease and senile dementia: loss of neurons in the basal forebrain. Science 215:1237–1239. Wolters, E. C., Francot, C., Bergmans, P., Winogrodzka, A., Booij, J., Berendse, H. W., and Stoof, J. C. (2000). Preclinical (premotor) Parkinson’s disease. J. Neurol. 247 (suppl 2):II103–II109. Yamagishi, M., Ishizuka, Y., and Seki, K. (1994a). Pathology of olfactory mucosa in patients with Alzheimer’s disease. Ann. Otol. Rhinol. Laryngol. 103:421–427. Yamagishi, M., and Ishizuka, Y. (1994b). (Abnormal tau protein expression in biopsied human olfactory mucosa) (in Japanese). Nippon Jibiinkoka Gakkai Kaiho J. Oto-RhinoLaryngol. Soc. Jpn. 97:466–472. Yamagishi, M., Takami, S., and Getchell, T. V. (1996). Ontogenetic expression of spot 35 protein (calbindin-D28k) in human olfactory receptor neurons and its decrease in Alzheimer’s disease patients. Ann. Otol. Rhinol. Laryngol. 105:132–139. Yamagishi, M., Getchell, M. L., Takami, S., and Getchell, T. V. (1998). Increased density of olfactory receptor neurons immunoreactive for apolipoprotein E in patients with Alzheimer’s disease. Ann. Otol. Rhinol. Laryngol. 107: 421–426. Yousem, D. M., Geckle, R. J., Bilker, W. B., and Doty, R. L. (1998). Olfactory bulb and tract and temporal lobe volumes. Normative data across decades. Ann. NY Acad. Sci. 855:546–555. Zufall, F., Leinders-Zufall, T., and Greer, C. A. (2000). Amplification of odor-induced Ca2release and its role in olfactory signal transduction J. Neurophysiol. 83:501–512

25 Multiple Chemical Intolerance Claudia S. Miller University of Texas Health Science Center, San Antonio, Texas, U.S.A.

I.

substances these patients implicate are structurally unrelated, an observation unsettling for toxicologists and immunologists, who tend to think of receptors, biochemical pathways, and target organs as being substance- or at least class-specific. Third, these patients report a baffling array of symptoms involving any and every organ system, and often several systems simultaneously. Fourth, the vast majority of patients attest that chemical odors and even nonodorous chemical exposures trigger cognitive difficulties and mood disturbances, symptoms physicians tend to see as psychogenic. The fact that these patients’ symptoms overlap with those of chronic fatigue syndrome, somatoform disorder, fibromyalgia, panic disorder, and posttraumatic stress disorder adds to the confusion. Despite the phenomenon’s seeming implausibility, in recent years similar patterns of multisystem symptoms and multiple chemical, food, and drug intolerances have surfaced in more than a dozen countries (nine European nations, the United States, Canada, Japan, Australia, New Zealand) among demographically diverse groups— groups having little in common, save some initial chemical exposure event (Ashford and Miller, 1998). Among these groups are radiology workers in New Zealand exposed to x-ray developer solutions containing glutaraldehyde (Genton, 1998), EPA employees in the agency’s Washington, D.C., headquarters exposed to airborne organic chemicals arising from construction, painting, and new carpeting (Hirzy and Morison, 1989), families in Germany exposed to pentachlorophenol used to preserve log homes (Ashford et al., 1995), sheep dip-

BACKGROUND

From time to time, physicians encounter patients with a chief complaint of hyperosmia accompanied by multisystem symptoms and intolerances for a wide variety of chemicals, foods, and/or drugs (Ashford and Miller, 1998; Berglund et al., 1992; Doty et al., 1998). Often these patients say they become ill when exposed to various odors, often in response to cacosmia (Ryan et al., 1988) or dysosmia (Miller, 1996). For example, they may describe everyday exposures to fragrances, diesel exhaust, new plastic car interiors, household cleaners, etc. (Table 1), as being overpowering, “stronger than ever before,” or “extremely irritating” and triggering symptoms such as headaches, fatigue, memory difficulties, mental confusion, anxiety, irritability, depression, myalgias, arrhythmias, dyspnea, and every sort of gastrointestinal problem (Table 2). This clinical presentation has come to be known as multiple chemical sensitivity (MCS) or multiple chemical intolerance. Patients presenting with this peculiar combination of subjective hyperosmia, multisystem symptoms, and multiple intolerances appear to be on the increase (AOEC, 1992; NIEHS, 1997; NRC, 1992). Later in this chapter, epidemiological and clinical studies of this phenomenon will be reviewed. Several things are puzzling about these patients. First, the levels of chemicals they say trigger their symptoms are orders of magnitude below established safety limits, leading some physicians to dismiss the illness on the basis that it violates a fundamental tenet of toxicology—evidence of a dose-response relationship (Waddell, 1993). Second, the 525

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Table 1 Triggering Exposures Reported by 80% or More of Persons with Chemical Intolerances That Developed Following an Exposure to Pesticides (n
37) or Indoor Air Contaminants (n
75) New carpeting New automobile interior Poorly ventilated meeting rooms Perfume Detergent aisle in grocery Newspaper/printed materials Fresh asphalt/tar Diesel exhaust Felt-tip markers Nail polish/remover Restroom deodorizers Fabric stores Heavy traffic New plastic shower curtain Hairspray

Enclosed mall Oil-based paint Particle board Gas engine exhaust Hotel rooms Phenolic disinfectants Dry-cleaned clothes Insecticides Gasoline Potpourri New tires Cigar smoke Cigarette smoke Incense Insect repellent

Source: Miller and Mitzel, 1995.

pers in Great Britain exposed to organophosphate pesticides (Ashford and Miller, 1998; Monk, 1996; Stephens et al., 1995), hospital workers in Nova Scotia exposed to building air contaminants (Ashford and Miller, 1998), casino card dealers in Lake Tahoe, California, exposed to solvents and pesticides (Cone and Sult, 1992), breast and temporomandibular joint implant recipients (Miller and Prihoda, 1999a,b), and Gulf War veterans exposed to solvents, smoke, fuels, pesticides, and various drugs (Bell et al., 1998, Fiedler et al., 1996b; Miller and Prihoda, 1999a,b). What has attracted scientific attention to this problem is the fact that such diverse groups—people from different occupations, socioeconomic classes, countries and cultures, people who do not see the same doctors, watch the same television shows, or read the same books—are presenting with such similar patterns of multisystem symptoms and new-onset intolerances preceded by an initial chemical exposure event. The fact that the newonset intolerances these patients report include medications, foods, alcohol, and caffeine—not just chemical inhalants—makes these worldwide observations compelling. Some scientists regard this repeating pattern as early evidence of an emerging new paradigm or theory of disease, one with the potential to explain a broad spectrum of illnesses including certain cases of asthma, migraine headaches, and depression, as well as chronic fatigue syndrome, fibromyalgia, and Gulf War syndrome.

Table 2 Symptoms Commonly Reported by Chemically Intolerant Individualsa Neuromuscular Loss of consciousness Stumbling/dragging foot Seizures Print moving/vibrating on page Feeling off balance Tingling in fingers/toes Double vision Muscle jerking Fainting Numbness in fingers/toes Clumsiness Problems focusing eyes Cold or blue nails/fingers Uncontrollable sleepiness Head-related Head fullness/pressure Tender face/sinuses Sinus infections Tightness in face/scalp Brain feels swollen Ringing in ears Headache Feeling groggy Musculoskeletal Joint pain Muscle aches Weak legs Weak arms General stiffness Cramps in toes/legs Painful trigger points Gastrointestinal Abdominal gas Foul gas Problems digesting food Abdominal swelling/ bloating Foul burping Diarrhea Abdominal pain/cramping Constipation a

Cardiac Heart pounding Rapid heart rate Irregular heart rate Chest discomfort Affective Feeling tense/nervous Uncontrollable crying Feeling irritable/edgy Depressed feelings Thoughts of suicide Nerves feel like vibrating Sudden rage Loss of motivation Trembling hands Insomnia Airway Cough Bronchitis Asthma or wheezing Postnasal drainage Excessive mucus production Shortness of breath Eye burning/irritation Susceptible to infections Dry eyes Enlarged/tender lymph nodes Hoarseness Cognitive Memory difficulties Problems with spelling Slowed responses Problems with arithmetic Problems with handwriting Difficult concentration Difficulty making decisions Speech difficulty Feelings of unreality/spacey Other Feeling tired/lethargic Dizziness/lightheadedness

Categories were derived via factor analysis of symptoms reported by 112 individuals who said they became ill following exposure to indoor air contaminants (n
75) or cholinesterase-inhibiting pesticides (n
37). Source: Miller and Mitzel, 1995.

Multiple Chemical Intolerance

A.

Historical Background

In the 1950s an allergist named Theron Randolph described a cosmetic saleswoman who experienced dyspnea, asthma, fatigue, irritability, depression, and intermittent loss of consciousness whenever she smelled “man-made combustion products and derivatives of gas, oil, and coal” (Randolph, 1962; Randolph and Moss, 1980). Randolph coined the term “chemical susceptibility” to describe her condition. Subsequently, other physicians, seeing similar problems in their patients, allied with Randolph to found the Society for Clinical Ecology (renamed in 1984 the American Academy of Environmental Medicine). These clinicians adopted Randolph’s principal diagnostic and therapeutic approach— trial avoidance of common chemicals and foods and, if patients’ symptoms cleared, re-introduction of single substances one at a time to determine which, if any, triggered symptoms. Over time, some clinical ecologists adopted unorthodox diagnostic and treatment approaches, such as the administration of “neutralizing” chemical and food extracts (via injection or sublingually) and sauna “detoxification” to “sweat out” chemical contaminants, practices that drew criticism from professional medical societies (AAAAI, 1981, 1986, 1999; ACP, 1989; AMA, 1992). Professional concerns over these and other “alternative” treatments continue. Recently, there has been a softening of positions taken against the illness as a new group of doctors—board-certified occupational and environmental medicine physicians in universities—have begun to study this phenomenon (ACOEM, 1999; Ashford and Miller, 1998). The American College of Occupational and Environmental Medicine now “supports scientific research into the phenomenon of MCS to help explain and better describe its pathophysiologic features and define appropriate clinical interventions” (ACOEM, 1999). There is widespread agreement that these patients report certain distinctive features—multisystem symptoms and multiple intolerances—whether they have seen a clinical ecologist or not (Davidoff and Keyl, 1996). In 1987, Mark Cullen at Yale University edited a compendium of papers entitled Workers with Multiple Chemical Sensitivities: An Overview, introducing occupational/environmental medicine practitioners to the problem (Cullen, 1987). He defined “multiple chemical sensitivity” as “an acquired disorder characterized by recurrent symptoms, referable to multiple organ systems, occurring in response to demonstrable exposure to many chemically unrelated compounds at doses far below those established in the general population to cause harmful effects. No single widely accepted test of physiologic function can be shown to correlate with symptoms.”

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B.

Recent Developments

Over the past decade there has been an outpouring of technical reports, concept papers, and hypotheses concerning chemical intolerance. The terms “multiple chemical sensitivity” and “environmental illness” appear on the National Library of Medicine’s bibliographical database, Medline. While it is generally agreed that a problem exists and that patients are suffering, medical opinion remains polarized as to whether the condition is a unitary one or a potpourri, and whether it arises from chemical exposures, psychological factors, or a blend of these. There is mounting concern that if low-level chemical exposures were found to cause this problem, the implications for environmental policy, product liability, workers’ compensation, and medical treatment would be staggering. Several nations have examined the issue, with Canada leading the way through its 1985 Thomson Report (Thomson, 1985) and sponsorship of clinical studies and scientific meetings. In the United States, the phenomenon has been explored by New Jersey, Maryland, and California (Ashford and Miller, 1989; Bascom, 1989; Kreutzer et al., 1999), various federal environmental agencies (ATSDR, 1994; Fiedler and Kipen, 1997a), the National Academy of Sciences (NRC, 1992), and professional organizations (ACS, 1999; AOEC, 1992). While promising research strategies have emerged from these meetings (summarized in Ashford and Miller, 1998), few comprehensive or illuminating studies have been funded. At this time, underlying mechanisms remain unknown, treatments are empirical, and no environmentally controlled hospital facility is available in the U.S. for clinical research, diagnosis, or treatment. Patients continue to suffer, while funding for serious scientific study is mired by the very medical debate such studies are needed to settle. Amid the confusion of opinion swirling around the illness, affected individuals and their caregivers are in need of rational, low-risk interim interventions with the potential to alleviate suffering and foster recovery. Of equal importance, there is a need to prevent exposures (e.g., to pesticides, chemicals associated with new construction) that could disable currently healthy, but potentially susceptible, people.

II.

DEFINING THE PROBLEM

At the present time, physicians and researchers cannot agree upon a name for this condition, much less whether it is a single illness or a group of related or unrelated conditions that simply share the symptom of chemical intolerance. One thing is clear. The problems these patients report do not con-

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stitute a syndrome: By definition, a syndrome is “a group of symptoms or signs typical of a disease” (Webster’s, 1986). The symptoms these patients report are simply too heterogeneous to be collapsed into a single syndrome—perhaps a collection of syndromes, but not a single one. A.

in this chapter. The latter presumes no particular etiology; instead, it nourishes fresh ideas and encourages new discoveries. Tolerance, as used here, is the ability to withstand an insult. Chemically intolerant individuals appear to have lost their prior natural or innate tolerance for a wide variety of chemicals, foods, and drugs.

Sensitivity or Intolerance?

The various meanings of the term “sensitivity” contribute to the confusion surrounding the condition(s). The word sensitivity is used in three relatively distinct ways (Ashford et al., 1995): 1.

The heightened responses of certain individuals to known toxicants or allergens, i.e., the responses of people who are especially susceptible to toxic substances like mercury or carbon monoxide or to allergens like housedust mites or bee venom. 2. The responses of certain individuals to identifiable exposures which cannot be explained by disease mechanisms generally understood by doctors. This category includes: (a) sick building syndrome, involving individuals who respond adversely to one or several air contaminants which may or may not be identifiable. Evidence for sick building syndrome’s existence rests on the fact that affected individuals’ symptoms resolve when they leave the problem building; (b) sensitivity, such as that induced by toluene diisocyanate (TDI), which starts out as hypersensitivity to a specific chemical, or a single chemical class, but evolves into nonspecific hyperresponsiveness (further described in category 3 below). 3. The heightened, extraordinary, or unusual responses of certain individuals to structurally unrelated chemicals at exposure levels orders of magnitude below those affecting most people. Multiple chemical sensitivity fits in this third category. Synonyms and related terms for MCS include environmental illness (EI), chemical intolerance, ecological illness, idiopathic environmental intolerance (IEI), universal allergy, and toxicant-induced loss of tolerance (TILT). A bright line needs to be drawn between MCS and antibody-mediated sensitivities or allergies. Allergists use the term “chemical intolerance” (not “chemical sensitivity”) to distinguish this third category from classical allergies. The word “sensitivity” poses a problem: it implies that sensitization has occurred, when, in fact, the loss of tolerance these individuals experience might arise from something entirely different, e.g., cell membrane disruption or gene activation. So instead of “sensitization,” the term “chemical intolerance” is used

B.

Proposed Case Definitions

Despite the differing opinions and semantic difficulties in this area, several case definitions for this phenomenon have been proposed (summarized in Ashford and Miller, 1998), some of which may prove useful, e.g., for research, medical evaluation purposes, or compensation (AOEC, 1992; Bartha et al., 1999; Nethercott et al., 1993; NRC, 1992). The original Cullen case definition, used in some early studies, unfortunately excludes “diagnosable” conditions such as asthma or depression (Cullen, 1987), when in fact chemical intolerance might underlie certain cases of asthma or depression. One operational case definition calls for sick individuals to be removed from background chemical, food, and drug exposures to determine whether their symptoms clear, and, if they do, administering single, double-blinded, placebo-controlled chemical challenges to see which, if any, trigger symptoms. The latter, patient-focused approach to “defining” multiple chemical intolerance is considered the “gold standard” for the field and has emerged as a principal research recommendation from several scientific meetings (AOEC, 1992; Miller et al., 1997; NRC, 1992). Bartha et al. (1999) offer six “consensus criteria” for multiple chemical sensitivity culled from a survey of 89 clinicians and researchers familiar with the illness but whose opinions concerning its origins differed (Nethercott et al., 1993): (1) a chronic condition (2) with symptoms that recur reproducibly (3) in response to low levels of exposure (4) to multiple unrelated chemicals and (5) improve or resolve when incitants are removed (6) with symptoms that occur in multiple organ systems. The authors urge that multiple chemical intolerance be formally diagnosed “in addition to any other diagnosable disorders (e.g., migraine, asthma, depression) in all patients in whom the above six criteria are met and for whom no single other organic disorder can account for all the signs and symptoms. . . .” Patients who experience short-lived symptoms associated with a particular odor or exposure, e.g., tobacco smoke, mothballs, or paint vapors, but whose symptoms stop when the exposure ends with no recurrence or spreading to other substances should not be labeled as having multiple chemical intolerance.

Multiple Chemical Intolerance

III.

PHENOMENOLOGY

About 50–60% of chemically intolerant individuals say their illness began following a specific chemical exposure (or a series of exposures), referred to as an initiating event, e.g., a chemical spill, repeated exposure to solvents, a pesticide application, indoor air contaminants associated with new construction, combustion products (Fiedler et al., 1996a; Miller, 1994). Only a subset of those exposed appear to develop chronic symptoms and intolerances. What makes some individuals more susceptible remains a mystery. Initially, patients may describe “flu-like” symptoms that fail to resolve, or feeling as though they are in a “perpetual fog.” Next to develop are multisystem symptoms that seem to wax and wane unpredictably, followed by a dawning awareness of specific intolerances, frequently involving alcoholic beverages or medications at first. Over time, these intolerances spread to include a wide variety of everyday exposures—chemical odors (low levels of volatile compounds), foods, drugs, caffeine, alcoholic beverages, and skin contactants. Food intolerances may appear but not be recognized as such. Instead, patients may complain of digestive difficulties, feeling ill after meals, or becoming irritable if a meal is missed or delayed. These intolerances may begin within weeks of an acute, high-level exposure or, as in the case of a sick office building, emerge insidiously over months or years. Symptoms may be triggered via any exposure route—inhalation, ingestion, injection (e.g., drugs), or skin or mucosal contact. Some patients report that breathing through their mouths instead of their noses, e.g., around traffic exhaust or people wearing fragrances, mitigates their symptoms somewhat. Particular odors or exposures (see Table 1)—whether fragrances, chemicals outgassing from new furnishings or carpeting, traffic exhaust, cleaning agents, etc.—may trigger different constellations of symptoms in different individuals. An individual patient often reports different responses with different exposures. Symptom intensity may range from mild (e.g., nasal congestion, nausea, or slight headache) to severe (e.g., mental confusion, depression or seizures) (Table 2). There is consistency, however: a particular exposure, e.g., diesel exhaust or a certain fragrance, in a particular person tends to elicit a characteristic constellation of symptoms, a so-called “signature response.” Responses may occur at below-olfactory-threshold concentrations, with symptoms developing within seconds to hours after a triggering exposure and persisting minutes to days. Hyperresponsiveness to physical stimuli, including light, noise, and touch, is commonly reported (Miller and Prihoda, 1999 a,b). Patients may wear sunglasses indoors or dim the room to keep bright light from reaching their eyes. Although some patients report hypersensitivity to

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odorants, there are anecdotal reports of anosmic individuals suffering from multiple chemical intolerance. Various studies and surveys suggest that patients who systematically avoid problem exposures find some relief (Johnson, 1996; Lax and Henneberger, 1995), but comprehensive avoidance is challenging, as well as socially isolating. Common odors involving low-level volatile organic chemicals (VOC) in the parts per billion or parts per trillion range are near-ubiquitous. Making matters worse, physicians, and even the patients themselves, may fail to discern symptom-exposure relationships (Ashford and Miller, 1998). Several factors may contribute to this. For example, habituation can occur with chronic or repeated exposure to the same substance(s), e.g., volatile organic chemicals in a sick office building. Second, apposition, i.e., overlapping symptoms resulting from various exposures (chemicals, foods, drugs), may hide or “mask” the effects of particular exposures. According to this scenario, the intolerant individual who applies hairspray and fragrances in the morning, cooks breakfast on a gas stove, and drives through heavy traffic to a sick office building may experience near-continuous symptoms (highly masked) and fail to recognize any single exposure as causal (Miller 1996, 1997). In effect, background symptom “noise” might hide any particular “signal.” “Withdrawal” symptoms reportedly develop when patients avoid problem exposures for several days, e.g., over a weekend or while on vacation. Such avoidance can be inadvertent or deliberate, e.g., a physician-recommended trial avoidance period (see Sec. V. E). Later, with reexposure, as on a Monday morning after a weekend away from work, symptoms may return “with a vengeance.” Some chemically intolerant individuals quit their jobs so as to avoid coworkers’ fragrances, carbonless copy paper, cleaning agents, etc. Others switch employers, occupations, and residences in search of safer surroundings. In science, anomalies expose the limitations of existing paradigms and drive the search for new ones. In the late 1800s, physicians observed that certain illnesses seemed to spread from sick, feverish individuals to their families and neighbors. These anomalous observations paved the way for the germ theory of disease. This germ theory, so obvious to us today, enabled scientists and the public to grasp for the first time the origins of dozens of seemingly unrelated illnesses affecting every organ system. Today, we are witnessing another anomaly—a repeating pattern of illness appearing in groups of people, including Gulf War veterans, from more than a dozen countries following chemical exposures. What unites the Gulf War veterans and these civilian groups is their common experience of an initiating chemical exposure event followed by newly acquired intolerances and

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multisystem symptoms. These observations provide compelling scientific evidence for a shared, underlying disease mechanism—one involving a fundamental breakdown in natural tolerance. This two-stage process—an initial chemical exposure (initiation) leading to newly acquired intolerances, with symptoms subsequently triggered by multiple common exposures (triggering)—has been referred to as toxicant-induced loss of tolerance, or “TILT” (Fig. 1). It does not appear to matter which exposure causes the breakdown in tolerance—be it pesticides, solvents, indoor air contaminants, smoke from oil well fires, or medications. It is the aftermath of these exposures, the new-onset intolerances to various substances, that appears to perpetuate the symptoms. Four observations suggest that toxicantinduced loss of tolerance might be a new theory of chemically induced disease: 1. The appearance of the same pattern of symptoms and new-onset odor and other intolerances in demographically diverse groups worldwide following well-defined exposures to pesticides, solvents, indoor air contaminants, etc. 2. The fact that these groups’ new-onset intolerances involve not only chemical inhalants, but also various foods, medications, caffeine, alcoholic beverages, and skin contactants. These observations in particular constitute what Kuhn (1970) called a “compelling” or “critical” anomaly. Just as fever is the hallmark symptom for infection, a signal that sends doctors down certain diagnostic pathways, new chemical, food, and drug intolerances are the hallmark symptom for TILT. 3. Recent animal models replicating key features of TILT (see Sec. VI). 4. The striking parallels between this phenomenon and addiction, suggesting shared neural mechanisms likely involving multiple neurotransmitter pathways (see Sec. VI). TILT has the potential to explain certain cases of asthma, migraine headaches, and depression, as well as chronic fatigue, fibromyalgia, and Gulf War syndrome (Fig. 2). But both stages of TILT—initiation and triggering—are in need of testing. Some argue that TILT’s second stage, triggering, should be studied first and that to accomplish this, a special scientific “apparatus” needs to be built—an environmentally controlled in-patient hospital unit (environmental medical unit) in which patients can reside for a week or longer, allowing them reach a “clean” exposure baseline (Miller et al, 1997; NRC, 1992). Assuming this occurs and patients’ exposure-related symptoms resolve, subjects could then be exposed to various potential triggers, including caffeine, gasoline, perfume, foods, medications, and tobacco smoke,

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one at a time, to determine the source of their symptoms. One limitation of the TILT theory is that it may not explain every case of chemical intolerance: not every patient is able to identify an initiating event or events. An initiating event, e.g., a pesticide application, may go unnoticed. Alternatively, genetic, psychological, nutritional and other factors may underlie their intolerances. IV.

PREVALENCE

Several large surveys suggest that 15–30% of the U.S. population consider themselves “especially” or “unusually” sensitive to certain chemical odors, while approximately 2–6% claim a physician’s diagnosis of “multiple chemical sensitivity,” “environmental illness,” or significant daily impairment from chemical exposures (Table 3) (Kreutzer et al., 1999; Meggs et al., 1996; Voorhees, 1998). In the largest of these studies, a statewide randomized telephone interview survey conducted by the California Department of Health Services, 15.9% of participants said they were “allergic or unusually sensitive to everyday chemicals,” 11.9% identified two or more chemicals that made them sick, and 6.3% reported doctor-diagnosed “environmental illness” or “multiple chemical sensitivity” (Kreutzer et al., 1999). Female gender and Hispanic ethnicity were associated with greater self-reporting of sensitivity (adjusted odds ratios of 1.63 and 1.82, respectively). Neither self-reported chemical sensitivity nor doctor-diagnosed multiple chemical sensitivity was associated with employment, educational level, marital status, geographic location, or income. The similar rates seen in California, New Mexico, and North Carolina suggest that multiple chemical intolerance could be among the most prevalent, if not the most prevalent, chemically caused medical condition(s) in the United States. The California study concluded that “surprising numbers” of people believe that common chemical exposures make them sick and that “the homogeneity of responses across race-ethnicity, geography, education, and marital status is compatible with a physiological response or with widespread societal apprehensions in regard to chemical exposure” (Kreutzer et al., 1999). While the media have fanned public fears of environmental exposures, this by no means proves the problem is psychogenic. Only careful studies can settle questions concerning etiology. More women than men have participated in clinical studies of chemical intolerance to date (4:1 female: male ratio), with an average age in the fourth decade and educational level of at least 2 years of college (Fiedler and Kipen, 1997b). In contrast, the California statewide survey cited above found a 5:3 female:male ratio in a random general population sample. Among military and industrial populations, more males report the problem, likely reflect-

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Figure 1 Phenomenology of toxicant-induced loss of tolerance (TILT). Illness appears to develop in two stages: (1) initiation, i.e., loss of prior, natural tolerance resulting from an acute or chronic exposure (pesticides, solvents, indoor air contaminants, etc.), followed by (2) triggering of symptoms by small quantities of previously tolerated chemicals (traffic exhaust, fragrances), foods, drugs, and food/drug combinations (alcohol, caffeine). The physician sees only the tip of the iceberg—the patient’s symptoms—and formulates a diagnosis based on them (e.g., asthma, chronic fatigue, migraine headaches). Masking hides the relationship between symptoms and triggers. The initial exposure event causing breakdown in tolerance may also go unnoticed. (©UTHSCSA 1996.)

Figure 2 Conditions that may result from toxicant-induced loss of tolerance. Illnesses like depression, migraine, arthritis, and chronic fatigue may have various underlying mechanisms, one of which might be TILT.

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Table 3 Frequency of Self-Reported Chemical Intolerance from Several Large Surveys

Population (Ref.) EPA office workers (Wallace et al., 1993) Rural North Caroliniansa (Meggs et al., 1996) California residentsa (Kruetzer et al., 1999) New Mexico residentsa (Voorhees, 1998) a Randomly

Number of people studied

Those considering themselves especially or unusually sensitive to certain chemicals (%)

Those reporting physiciandiagnosed multiple chemical intolerance or daily symptoms triggered by chemicals(%)

3948

31

Not evaluated

1027

33

3.9

4046

15.9

6.3

1814

17

1.9

sampled.

ing underlying gender ratios (Miller and Prihoda, 1999a,b; Simon et al., 1990;). In sick buildings, the condition is commonly reported by college-educated white females in the 30- to 50-year age range and of middle- to upper-middle-class socioeconomic status (Ashford and Miller, 1998). Why more women then men report the problem and why more chemically intolerant patients work in office buildings and service industries than in heavy industry remains unknown (Black et al., 1990; Lax and Henneberger, 1995; Miller and Mitzel, 1995;). The gender disparity may reflect women’s greater willingness to report symptoms, something unique about indoor air pollutants that women may be less able to escape, e.g., as secretaries or realtors, or gender-based biological response differences. The paradox that more multiple chemical intolerance cases arise from the service sector than heavy industry may be due to (1) “the healthy worker” selection effect, i.e., individuals bothered by chemical exposures would tend to choose nonchemical jobs, (2) the fact that women, who may be biologically more vulnerable, are less apt to work in heavy industry, mining, construction, etc., or (3) some unknown but insidious effect of indoor air pollution mixtures. Over the past decade, occupational medicine physicians have witnessed a surge in the numbers of these patients in their practices (Ashford and Miller, 1998)—another puzzle. While increased media coverage of environmental exposures has fueled our awareness, at the same time major increases have occurred in the variety and quantity of chemicals people encounter each day, all within a few generations. For example, U.S. production and use of synthetic organic chemicals have risen exponentially since World War II, rising from a billion pounds produced annually in the early 1940s to 400 billion pounds in the 1980s (U.S. International Trade Commission). And the nature of the chemicals has changed. For example, cholinesteraseinhibiting pesticides (organophosphates and carbamates) have supplanted chlorinated pesticides like DDT, on eco-

logical grounds. The former currently account for nearly half of all pesticides used in the United States. These relatively new neurotoxic compounds, developed for agricultural use, also worked well in homes, schools, and office buildings, controlling pests with few “callbacks” for exterminators. Today, thousands of different synthetic organic chemicals are released into the air from fuels, paints, clothing, and consumer products of every description. Ninety percent of the U.S. population spends more than 90% of the day indoors, exposed to complex mixtures of volatile organic chemicals (VOCs) emitted by new carpet, pesticides, cleaners, fragrances, particle board, and furnishings. These airborne chemicals tend to accumulate in modern, sealed indoor environments. Even the air inside vehicles, especially new ones, contains myriad VOCs emitted by plastic interiors, rubber floor mats, and other components. VOCs are entrained from traffic exhaust and freshly tarred roads. Thus, the vast majority of the population is exposed near-continuously to thousands of biologically “foreign” VOCs, albeit at low levels (parts per billion or trillion). Add to this the oil embargo of the 1970s, when U.S. home owners received tax credits for installing insulation, caulking cracks, etc., thus sealing in indoor air contaminants. Responding to the energy crunch, the American Society of Heating, Refrigeration and Air Conditioning Engineers (ASHRAE) further reduced U.S. recommendations for fresh outside air entering commercial buildings, schools, public spaces, etc., which were 30 cubic feet per minute (cfm) per occupant in 1900, down to 5 cfm per occupant—a sixfold decrease in fresh air in commercial and public buildings.* Home ventilation systems generally

* In recent years, ASHRAE fresh air requirements for public and commercial spaces have been raised to a minimum of 15 cfm per occupant, 20 cfm in offices, because of health complaints associated with the 5 cfm recommendation (ASHRAE 1999).

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bring in no fresh outside air; occupants breathe only what little leaks through cracks, crevices, open doors, or windows. With more tightly sealed, “energy-efficient” structures and less fresh air brought in from outside, VOC contaminants inside homes and workplaces have crept to unprecedented levels, often orders of magnitude greater than outdoor levels. As a consequence, sick building syndrome has spread across the United States, most notoriously affecting the Environmental Protection Agency’s (EPA) Washington, D.C., headquarters, where several hundred individuals fell ill following remodeling and new carpet installation (Hirzy and Morison, 1989). Several dozen EPA employees subsequently developed multiple chemical intolerances. More than a decade later, many of these individuals remain disabled (Ashford and Miller, 1998).

V. CLINICAL RECOGNITION AND EVALUATION Physicians may be reluctant to diagnose multiple chemical intolerance even when it provides the most parsimonious description for a patient’s problems. The diagnosis of multiple chemical sensitivity is frequently challenged by workers’ compensation boards, employers, and others.* Some doctors opt to apply “piecemeal” but recognized and compensable diagnostic labels such as asthma, toxic encephalopathy, or migraine headache. There are no symptoms, clinical signs, or laboratory tests that are pathognomonic for multiple chemical intolerance. As for other medical conditions for which no objective diagnostic tests are available, e.g., depression, schizophrenia, and migraine headaches, health care providers must rely on careful history taking and observation over time to make the diagnosis. Circumstances permitting, patients’ intolerances should be assessed through trial avoidance of suspected substances and, if improvement occurs, judicious reintroduction of single exposures under the supervision of a knowledgeable practitioner. Where symptoms and circumstances warrant, doctors should discuss with patients the possibility of multiple chemical intolerance. Commonly reported complaints include hyperosmia, fatigue, memory and concentration difficulties, mood changes, and multisystem health problems (Table 2). Although only one organ system may

*“Toxicant-induced loss of tolerance,” which describes the break-

down in tolerance resulting from exposure—a phenomenon that has been widely witnessed by reputable scientists and reported on in numerous, peer-reviewed medical articles—has not been similarly scrutinized or challenged.

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be affected (especially early in the illness), multisystem involvement is the norm. The probability that the practitioner is dealing with multiple chemical intolerance rises if (1) an identifiable chemical exposure, e.g., to solvents, pesticides, combustion products, or volatile organic chemicals from a sick building, remodeling, or new construction, clearly preceded onset of the symptoms; (2) a major change in the patient’s health occurred, ideally documented by increased health care utilization and/or absenteeism; (3) clinical signs or abnormal laboratory tests appear postexposure, e.g., increased liver function tests, a depressed white blood cell count, or decreased cholinesterase level; (4) new-onset depression, asthma, severe headaches, etc., appear in the absence of other clear causes; (5) others who shared the same exposure event became ill, especially but not necessarily with similar symptoms; or (6) previously tolerated chemical exposures now provoke symptoms. (7) If the patient reports newly acquired intolerances for medications, alcohol, caffeine, or foods (or feeling ill after meals), and (8) if clinical laboratory abnormalities (e.g., pulmonary function tests) improve with trial avoidance and/or worsen with reexposure, then the likelihood increases further. No one of these features “proves” the diagnosis, but the more the patient manifests, the more the practitioner should suspect toxicant-induced loss of tolerance. Physicians need to discuss with these patients the polar opinions surrounding the illness’s origins, steering them to sympathetic specialists and helping them explore treatment options, including psychological therapies and environmental interventions, while warning that all therapies currently are uproven, the underlying mechanism remains a mystery, and no test(s) are diagnostic. Much research will be needed before these patients’ most pressing questions can be answered with any degree of scientific certainty. Patience on the parts of both patient and practitioner is essential. “The evaluation of a patient presenting with MCS may take several hours and it is necessary to allot sufficient time, even if inadequately reimbursed” (Sparks et al., 1994b). It is not unusual for these individuals to consult 10 or more practitioners before the problem is recognized (Miller and Mitzel, 1995). In one study, chemically intolerant patients averaged 23 health care provider visits per year (Buchwald and Garrity, 1994). Physicians can easily underestimate the illness’s multisystem impact. Chemically intolerant patients tend to migrate from specialist to specialist, accumulating diagnostic labels like toxic encephalopathy, chronic fatigue syndrome, psychosomatic illness, migraines, and fibromyalgia while remaining oblivious to the underlying dynamic. Hyperosmia, a hallmark symptom, may be overlooked in patients reporting a profusion of symptoms. Physicians’

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professional opinions, even when science to support them is lacking, greatly influence insurors’ determinations, compensation boards, and disability reviewers, as well as employers, friends, and family. Consequently, patients tend to avoid doctors who seem skeptical or ill-informed.

reluctant to deal with chemical exposures. Chemically intolerant patients “fall in the crack” between these two specialties.

A.

Patients typically report multisystem symptoms, with fatigue being most common (Table 2). Symptoms often mimic chronic fatigue syndrome or fibromyalgia, diagnoses many patients eventually acquire (Ashford and Miller, 1998; Buchwald and Garrity, 1994; Chester and Levine, 1994; Miller and Mitzel, 1995). Mood changes, including irritability, anxiety, and depression, are commonly reported. Exposure-triggered memory and concentration difficulties have led some patients to abandon cognitively demanding careers. Affected groups report strikingly similar symptoms, whether the “initiating” event involved pesticides or construction-related indoor air contaminants (Miller and Mitzel, 1995). Central nervous system symptoms predominate. Gastrointestinal (e.g., problems digesting food), respiratory (e.g., shortness of breath or being unable to get enough air), and musculoskeletal (e.g., muscle/joint pain) problems are commonly reported (Miller and Mitzel, 1995). Besides chemical intolerances, almost invariably these patients report responding adversely to various foods, drugs, alcoholic beverages, or caffeine. Ninety-seven percent of 112 self-reported chemically intolerant individuals in one study reported significant food intolerances, causing them to avoid their problem foods or follow elimination diets (Miller and Mitzel, 1995). Food intolerances often involve frequently eaten items and those commonly craved, e.g., bread (wheat), corn chips, chocolate, caffeinated beverages, milk, etc. Patients who experience an immediate, sharp reaction after eating a particular food tend to avoid that food, particularly if the food is unusual (e.g., avocado or cashews) and eaten only occasionally (Randolph, 1956). On the other hand, foods eaten frequently (twice weekly or more) may precipitate fatigue, headaches, mood problems, digestive difficulties, etc., but go unrecognized, due to masking. Because patients may experience a slight “pickup” shortly after these foods are eaten, they may actually crave these foods (e.g., “chocoholics”). Food-intolerant patients may insist upon having their meals on time. If they miss a meal or eat late, they may report feeling tired, jittery, achy, etc. Some snack between meals, eat just before bedtime (to avoid nighttime withdrawal symptoms or awakening), stash food by the bedside or in the car, or carry large cups of coffee or tea wherever they go, “titrating” themselves throughout the day—unconscious ploys to ward off food-withdrawal symptoms. Randolph called this phenomenon “food addiction.”

Exposure History

Busy doctors seldom take occupational or environmental histories even when circumstances warrant (IOM, 1995). The process is time-consuming, and physicians often feel ill-equipped to interpret the information gathered. But here the value of a careful exposure history cannot be overemphasized. Standard forms for collecting basic occupational and environmental exposure information are available and should be part of every patient’s chart (IOM, 1995). However, these alone are not sufficient for evaluating chemically intolerant individuals, since many common exposures (e.g., home remodeling, pesticide use) that bother these patients may be omitted from these forms. One approach is to have patients construct their own symptom/exposure time lines (one line per year), with symptoms and medical problems recorded along the top of the line and life events (e.g., changes in jobs, residences, military service, surgeries, pregnancies, remodeling, pesticide use, etc.) along the bottom. A clear, concise chronology, preferably in this format, may ferret out contributory exposures. A standardized and validated screening questionnaire, the Quick Environmental Exposure and Sensitivity Inventory (QEESI), has been described in the medical literature and is available for evaluating these patients’ symptoms and chemical/odor intolerances, monitoring their clinical course, and measuring their treatment response (Miller and Prihoda, 1999a). This 50-item inventory permits patients to rate their symptoms and intolerances before and after an exposure event. It can also help affected individuals identify potential home and workplace triggers and determine whether they may be more susceptible to various alcoholic beverages, caffeine, foods, and drugs. If intolerances to any of these preceded the alleged initiating exposure event, it may help clarify why this person, rather than others who were similarly exposed, fell ill. Whenever possible, material safety data sheets (MSDSs) should be obtained to clarify the nature of the patients’ exposures. Occupational medicine physicians, toxicologists, and industrial hygienists may help with collecting and evaluating exposure data. Occupational medicine doctors routinely take detailed exposure histories, but most do not delve into patients’ food intolerances. On the other hand, allergists who are trained to evaluate food intolerances using elimination diets may be

B.

Symptoms and Intolerances

Multiple Chemical Intolerance

Chemically intolerant individuals may report acute symptoms with minimal caffeine intake and/or caffeine withdrawal symptoms lasting a week or longer, including headaches, irritability, abdominal cramps, heart pounding, insomnia, or fatigue. Blinded, crossover studies have demonstrated that certain people who consume as little as one cup of regular coffee per day (about 100 mg of caffeine) reliably develop caffeine withdrawal symptoms when they stop (Silverman et al., 1992). Even a few sips of decaffeinated coffee (about 10 mg of caffeine per cup) are said to precipitate symptoms in some chemically intolerant patients. Initially, these individuals may consume copious quantities of caffeine in an effort to stave off withdrawal symptoms. Caffeine intolerance can be confirmed by stopping all xanthines (coffee, tea, cola, chocolate, caffeinated soft drinks) for 1–2 weeks to determine whether symptoms improve and, if improvement occurs, reintroducing caffeine while observing whether symptoms return. Withdrawal symptoms may persist 7–10 days after all xanthines are stopped. Because sudden xanthine cessation can precipitate severe withdrawal symptoms in patients prone to debilitating headaches, depression, etc., gradual tapering may be preferable in such cases, albeit withdrawal may be protracted. Alcohol intolerance may be the first intolerance a patient notices (Miller and Prihoda, 1999a,b), perhaps because of ethanol’s rapid absorption rate and the fact that most people tend to use it intermittently, often on an empty stomach. As little as one drink may precipitate acute symptoms or may be followed by several days’ withdrawal symptoms (hangover). Patients rarely tell physicians they are alcohol intolerant unless the practitioner asks. Smokers may report, if queried, that smoking one more cigarette than usual or borrowing someone else’s stronger brand precipitates acute symptoms such as headache, lightheadedness, shortness of breath, gagging, coughing, nervousness, or nausea. Some individuals quit or switch to lighter brands. Patients commonly report adverse reactions to medications, for example, flu-like symptoms persisting days after methacholine challenge; becoming extremely irritable and eating ravenously after a steroid injection; chest tightness and chills following radiographic contrast dye injection; or panic attacks or floating feelings with antidepressants. Elevated liver function tests with some drugs, e.g., piroxicam, may occur. Previously well-tolerated drugs may no longer be tolerated, e.g., over-thecounter decongestants. Skin and/or systemic symptoms may occur with skin or mucosal contactants, e.g., adhesive tape, topical creams or ointments, jewelry, soaps, shampoos, plastic mouth appliances, toothpaste, contact lenses, various fabrics (wool, polyester), condoms, spermicides, cosmetics, deodorants, laundry soaps, fabric softeners, and chlorinated pool water.

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C.

Past Medical History and Associated Conditions

Whenever possible, past medical records should be examined, even if voluminous and difficult to obtain. A past history of unexplained illness(es) is common in this patient population. Aerospace workers who developed multiple chemical intolerances when a new composite plastic was introduced into their workplace averaged 6.2 unexplained physical symptoms preceding the change in process versus only 2.9 unexplained symptoms in unaffected coworker controls (Simon et al., 1990). Fifty-four percent of the ill workers had histories of anxiety or depression that preceded their exposure, compared with 4% of controls. Other investigators have found that past psychiatric history does not explain the illness (Fiedler et al., 1992). Even if some chemically intolerant individuals have histories of depression that predate their “initial exposure event,” the question remains whether depression causes chemical intolerance, whether depressed individuals are more susceptible to chemical exposures (i.e., have vulnerable neurochemistry), or whether the earlier depression may have been due to prior, unidentified intolerances (Davidoff and Fogarty, 1994). Medical and psychiatric diagnoses reported more frequently by chemically intolerant college students include nasal allergies, hives, breast cysts, premenstrual disorder, and childhood hyperactivity (Bell et al., 1996), and among community-based chemically intolerant middle-aged individuals, rhinitis, migraines, irritable bowel, ovarian cysts, menstrual dysfunction, depression, anxiety, and panic disorder (Bell et al., 1995). Another community sample found chemically intolerant individuals more likely to seek medical help for heart problems, bronchitis, asthma, and pneumonia, and to report parental heart disease, asthma, and diabetes (Baldwin and Bell, 1998). Black et al. (1999) reported that chemically intolerant patients versus controls reported more first-degree relatives with major depression, alcoholism, panic disorder, obsessive-compulsive disorder, and antisocial personality disorder and who had made more suicide attempts and received more psychiatric treatment. D.

Physical Examination and Laboratory Evaluation

Abundant anecdotal evidence suggests that chemically intolerant individuals improve when they identify and learn to avoid exposures that trigger their symptoms. Various federally sponsored consensus groups have recommended research/diagnostic studies using an environmentally controlled, inpatient hospital unit analogous to a drug detoxification unit, in which patients could be taken to a “clean” baseline, allowing exposure-related symptoms to resolve (Fig. 3) (AOEC, 1992; Miller et al., 1997; NRC,

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1992). Subjects could then be exposed to various potential triggers, including caffeine, gasoline, perfume, individual foods, medications, and tobacco smoke, one at a time, to determine what is causing their symptoms, but at this time no such facility is available in the United States. Although a comprehensive physical examination is essential, findings frequently are unremarkable. Baseline laboratory tests such as a complete blood count and chemistry profile can be helpful, as well as tests suggested by history or physical findings, such as thyroid function tests, pulmonary function tests, peak flow monitoring over time, tests for collagen-vascular disease, and neuropsychological evaluations in some patients (Weaver, 1996). Blood tests for environmental chemicals should be used only if there is reason to suspect specific exposures, and these substances can reasonably be expected to persist in tissues (e.g., a chlorinated pesticide or recent organophosphate pesticide exposure, but not most solvent or indoor air volatile organic chemical exposures). Referrals to specialists are often necessary, given the multisystem nature of the illness and the need to rule out contributing or coexisting conditions, such as an autoimmune disease, endocrine disorder, demyelinating disease, brain tumor, etc. (Moorhead and Suruda, 2000). While specialists’ evaluations can be reassuring for patients and referring physicians, unnecessary invasive testing and polypharmacy are potential pitfalls. One physician, preferably a primary care

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physician, needs to oversee the entire evaluation and treatment, integrating specialists’ advice as appropriate. To date, no consistently abnormal laboratory findings have been demonstrated in these patients. Various studies have reported abnormalities in T- and B-lymphocyte counts; helper/suppresser T-cell ratios; immunoglobulin levels; autoimmune antibodies (e.g., anti-nuclear, antismooth muscle, anti-thyroid, anti-parietal cell, etc.); activated T lymphocytes (TA1 or CD26); quantitative EEGs; evoked potentials; SPECT and other brain scans (Heuser and Mena, 1997; Hu et al., 1999; Mayberg, 1994; Rossi et al., 1999; Waxman, 2000); vitamin, mineral, amino acid, and detoxification enzyme levels; and blood or tissue levels of pesticides, solvents, and other chemicals. Common flaws in studies conducted to date include failures to (1) define the study population (no case definition used), (2) compare cases with age- and sex-matched controls, (3) blind specimens, and (4) document the test method’s accuracy and reproducibility. Some illness proponents claim that different immunological abnormalities occur in different patients. However, if enough tests are done, statistically a certain number can be expected to be abnormal (e.g., 1 in 20), a fact frequently forgotten. Mitchell et al. (2000) recommend that “[p]atients should be informed that subtle differences in individual immunological parameters, especially if observed in only one laboratory, are common and do not necessarily indicate the presence of a systemic

Figure 3 Use of an environmental medical unit (EMU) in the evaluation of health effects from low level chemical exposures. The figure illustrates stages in the evaluation of a patient in an EMU. At left, prior to entering the EMU, a patient is experiencing overlapping symptoms in response to everyday exposures and is unable to discern the effects of any particular exposure. Background symptom “noise” is high, and, to the patient, symptoms seem to wax and wane unpredictably over time. (1) With entry into the EMU and the avoidance of all chemical, food, and drug triggers simultaneously, remission of symptoms should begin. During the first few days of “withdrawal,” irritability, headaches, and depression are expected complaints. Within a week, the individual should be at a clean baseline and ready for challenge testing. (2) Following a chemical, food, or drug challenge (arrows), the patient should report a specific constellation of symptoms. (3) When the challenge ends, patients should gradually return to baseline and be free of symptoms. If the individual is re-challenged with the same substance 4 to 7 days after the first challenge, the same constellation of symptoms should occur. Challenges involving unrelated chemicals or foods may take place in the interim.

Multiple Chemical Intolerance

disorder, unless there are additional clinical correlates and the results are verified.” E.

Trial of Avoidance

There are anecdotal reports of patients diagnosed with this condition early in its course who avoided further exposure and apparently recovered (Hileman, 1991), suggesting that timely recognition and avoidance might reverse the illness and prevent disability. Our current inability to treat the condition once entrenched underscores the importance of early intervention. Physicians need to recognize patients who may be in the initiation phase of the illness and who may have ongoing exposures to pesticides, remodeling, solvents, etc. These individuals may benefit from a period of trial avoidance to determine whether they improve, followed by judicious, medically supervised reexposure to determine whether their symptoms recur. Unfortunately, at this time most patients are diagnosed long after their illness begins. Almost uniformly, patients report that identifying and avoiding chemical and food triggers benefit them most (Lax and Henneberger, 1995). While conceptually simple, avoiding potential incitants is no simple task, particularly for patients with cognitive difficulties. As a rule, patients need help identifying and minimizing exposures to volatile organic chemicals released by fragrances, cleaners, and other products. Simply telling them to avoid exposures that trigger their symptoms is not sufficient. Initially, they may be loathe to relinquish their favorite fragrances, soaps, candles, gas stoves, foods, etc., even on a trial basis. Because avoidance of alcoholic beverages, nicotine, caffeine, medications, and problem foods can precipitate severe withdrawal symptoms in susceptible individuals, e.g., severe headaches, disorientation, depression, and malaise, such regimens should not be attempted on an outpatient basis unless supervised by a knowledgeable medical practitioner. Patients with histories of severe asthma, headaches, depression, or other medical conditions (especially if emergency management was ever required) should be hospitalized in an environmentally controlled medical unit during the initial stages of avoidance. The vast majority of patients find that avoiding problem foods makes them feel better (Johnson, 1996; LeRoy et al., 1996; Miller and Mitzel, 1995). Various elimination diets have been used with these patients, most often the rotary diversified elimination diet, in which no food or food group is consumed more than once every 4 days (Rinkel, 1944; Rinkel et al., 1951). Popular versions of this diet have appeared (Mandell and Scanlon, 1979). Adhering to restrictive diets is difficult, and research is needed to determine their efficacy. However, modifying patients’ timing of food intake in order to uncover possible food intolerances is relatively innocuous,

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provided nutrition is maintained. If multiple food intolerances are found, achieving adequate nutrition may be difficult. Long-standing digestive difficulties and/or use of restrictive diets can result in nutrient deficiencies. It may be helpful to have patients maintain a daily log or diary (one page per day), recording each symptom (scored on a 0–10 intensity scale) and listing all inhalant and ingestant exposures and the times at which exposures and symptoms occurred. Review of this log may help reveal problem exposures. It is important to recognize that patients who smoke, use alcohol or caffeine on a regular basis, or have ongoing workplace or other chemical exposures may be unable to link their symptoms with specific exposures due to masking.

VI.

PROPOSED MECHANISMS

Under the assumption that most of the symptoms described in this chapter stem from a unitary disease process, then the unifying dynamic remains a mystery. Even if one assumes these symptoms arise from multiple causes or different disease processes, their underlying physiological bases are difficult to imagine. Some physicians and researchers view these patients’ symptoms as psychogenic phenomena, resembling depression, somatoform disorder, or posttraumatic stress disorder. Others see them as chemically induced and offer various physiological explanations (Ashford and Miller, 1998; summarized in Sorg, 2000). To date, surprisingly little research has been done in this area when one considers the fact that 2–6% of the U.S. population think they suffer from it (see Sec. IV). Early mechanistic studies focused on immune dysregulation as a potential explanation for these patients’ problems. These studies suffered from major methodological limitations, and no consistent immunological abnormality was found (reviewed in Mitchell et al., 2000). To assess inter- and intra-laboratory reliability, Mitchell et al. (2000) sent split blood specimens to six laboratories. Good agreement was found for T-cell subsets, but not for more difficult tests, e.g., immune activation markers. Subsequently, patient and control samples were sent to two validated laboratories for T-cell subset and antibody analyses. Despite these validated laboratories and tests, most of the statistically significant differences between the chemically intolerant subjects and healthy controls were observed in only one of the two laboratories used, the only exception being that CD4 lymphocyte percentages were higher and CD8 percentages lower in the chemically intolerant group. Data analysis is ongoing. A key question is why these people are bothered by low-level exposures that the vast majority tolerate. Doty

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et al. (1988) asked whether chemically intolerant individuals might have lower olfactory thresholds. Eighteen patients and 18 controls received graded, low-level concentrations of rose oil (phenyl ethyl alcohol) and methyl ethyl ketone via olfactometer. There were no significant differences in olfactory thresholds; however, during stimulus presentations, the patients had significantly higher nasal resistance and respiration rates. Fiedler et al. (1996a) found no difference in odor identification ability between chemically intolerant patients and controls. The heightened nasal resistance seen by Doty et al. suggests an inflammatory process, a mechanism proposed by others (Bascom, 1991; Meggs, 1994; Meggs and Cleveland, 1993). Fiberoptic rhinolaryngoscopic examination of 10 chemically intolerant patients whose illness began after a chemical exposure revealed “cobblestoning” of the pharyngeal mucosa, resembling lymphoid hyperplasia, in 6 patients and pale mucosal foci in 8 patients (Meggs and Cleveland, 1993). Nasal biopsies of 13 chlorine dioxide–exposed workers who developed chemical intolerances likewise showed inflammation and nerve fiber proliferation (Meggs et al., 1996). In some specimens, mucosal epithelial cells were detached from the basement membrane and intraepithelial cell junctions were disrupted. Eosinophils did not appear elevated. Further studies incorporating control subjects and blinded assessments are needed. German investigators have been examining the chemosensory event–related potentials of chemically intolerant patients in response to olfactory (hydrogen sulfide) and trigeminal (carbon dioxide) cues (Hummel et al., 1996), both before and after exposure to a common solvent (2-propanol). While solvent exposure did not affect subjects’ odor thresholds, their ability to discriminate odors appeared heightened after solvent versus clean air exposure. No normal subjects have yet been tested. Other investigators propose that odor conditioning may play a role in multiple chemical intolerance (Bolla-Wilson et al., 1989; Guglielmi et al., 1994; Schottenfeld and Cullen, 1986; Schusterman and Dager, 1991; Siegel and Kreutzer, 1997). Bell et al. (1999) and Sorg (2000) propose that multiple chemical intolerance results from central neural sensitization processes. Neural sensitization is the increased neuronal responsiveness to a stimulus following repeated exposures to the same stimulus or a different stimulus, a form of learning being explored in laboratory animals repeatedly exposed to drugs of abuse, especially cocaine and amphetamines. The limbic and mesolimbic brain regions are particularly susceptible to sensitization, a process that appears to involve excitatory amino acids, which may alter pain perception, olfaction, learning, and memory. In this rodent model, physical stressors, e.g., foot shock and tail pinching, augment stimulant drug responses.

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Features of this animal model that fit the clinical picture (Sorg, 2000) include the progressive increase in drug/chemical response over time and with additional exposures, the apparent permanence of the sensitivity, the absence of symptoms in the absence of chemical/drug exposure, the greater sensitivity of females versus males, and the spreading of sensitivity to chemically dissimilar substances. Sorg (1996) hypothesized that sensitization to formaldehyde and other chemicals might affect the same brain pathways involved in cocaine sensitization, e.g., the mesolimbic dopamine system. Rats inhaling 1ppm of formaldehyde 1 hour daily for 20 days, as compared to sham-exposed rats, later showed heightened responses to cocaine. This sensitivity persisted 4–6 weeks post–formaldehyde exposure (when the experiment ended) (Sorg, 1996). Kay (1996) showed that rats exposed to toluene vapors, but not most food odors (except mint), exhibited narrow-band, high-amplitude 15–30 Hz oscillations in the olfactory-limbic tract. Repeated low-level exposure to toluene (below OSHA legal exposure limits) in rats adversely affected their performance of complex learning and spatial memory tasks (Rogers et al., 1999; Von Euler et al., 1993). Consistent with these limbic changes in animals, chemically intolerant individuals score higher on the McLean limbic system checklist (Teicher et al., 1993), a questionnaire probing symptoms associated with temporal lobe seizures which frequently originate in the limbic system (amygdala) (Bell, 1996). Notably, women with temporal lobe epilepsy have increased rates of self-reported polycystic ovary disease, possibly due to amygdala-regulated release of hypothalamic reproductive hormones. Chemically intolerant women also self-report more menstrual problems than do controls (Bell et al., 1995). In a series of inhalation challenge studies using various low level exposures, Bell et al. (1996, 1997a,b,c) found evidence for sensitization of heart rate, blood pressure, plasma beta-endorphins, and EEG activity in chemically intolerant individuals, but not in controls. Cholinesterase-inhibiting pesticide exposure has been implicated in the initiation of multiple chemical intolerance and the Gulf War veterans’ unexplained illnesses (Cone and Sult, 1992; Haley et al., 1999; Miller and Mitzel, 1995). Rats bred for organophosphate sensitivity show greater susceptibility to various cholinergic agonists but, interestingly, are also less tolerant of nicotine, serotonin agonists, dopamine antagonists, diazepam, ethanol, and sucrose—structurally unrelated substances (Djuric et al, 1995; Overstreet, 1996). These specially bred rats have about 20% more cholinergic receptors in certain limbic regions, including the hippocampus and striatum. When sensitized to egg protein (ovalbumin) via

Multiple Chemical Intolerance

intraperitoneal injection and subsequently fed ovalbumin, these cholinergically sensitive rats showed greater gut permeability than did control rats (Djuric et al., 1995). Increased gut permeability in humans is thought to underlie various food intolerances. Likewise, inhaled methacholine and inhaled ovalbumin following ovalbumin presensitization via intraperitomal injection resulted in greater airway reactivity in cholinergically sensitive rats than controls (Djuric et al., 1998). Differences in the ability to absorb, metabolize, and excrete various chemicals might explain why some people are more vulnerable to developing multiple chemical intolerance, for example, decreased sulfation capacity (McFadden, 1996), abnormal porphyrin metabolism (Morton, 1995), or paraoxonase (organophosphate-detoxifying enzyme) deficiency (Costa et al., 1999; Haley et al., 1999). To date, no adequately controlled studies exploring these possibilities have been published. Other proposed explanations include: (1) neurogenic inflammation—e.g., increased c-fiber neuron density in affected tissues; greater neuropeptide and prostanoid production by susceptible subjects; increased and protracted response to c-fiber activators like capsaicin; increased central autonomic response following c-fiber stimulation; and decreased mucosal neutral endopeptidase; (2) nonneurogenic inflamation—e.g., increased inflammation in affected tissues resulting in augmented neurosensory response; increased inflammatory response to chemical exposure; and (3) altered perceptual and central integration—e.g., differences in adaptation, habituation, cortical representation, perception, cognition, and/or hedonics; different qualitative and quantitative interactions between trigeminal and olfactory systems; different higher integration of sensory inputs (Bascom et al., 1997). Randolph first noted striking parallels between chemical intolerance and drug addiction (Randolph, 1962), including the presence of stimulatory and withdrawal symptoms, cravings, and cross-addiction/intolerances to structurally diverse substances (summarized in Table 4). In this model, both addiction and chemical intolerance involve a fundamental breakdown in tolerance that occurs only in certain susceptible individuals following repeated exposures, whether to drugs of abuse or to chemicals. It is hypothesized that repeated exposures to drugs or chemicals result in amplified stimulatory and withdrawal symptoms (Miller 1997, 1999). Subsequently, in order to prevent unpleasant withdrawal symptoms, drug abusers take another “hit,” and then another, leading to addiction. In contrast, chemically intolerant individuals who link their withdrawal symptoms to alcohol, caffeine, etc., shun these substances, but perhaps for the same reason addicts

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remain addicted—to avoid withdrawal symptoms. The theory is that toxicant-induced loss of tolerance leads to amplified stimulatory and withdrawal symptoms and that the outwardly polar behaviors of the drug addict and the chemically intolerant patient may both be strategies for avoiding withdrawal symptoms (Miller, 1999, 2001a; Newlin, 1997). Proposed psychological mechanisms for multiple chemical intolerance include odor conditioning, physicianinduced (iatrogenic) beliefs, panic disorder, toxic agoraphobia, posttraumatic stress disorder (e.g., illness resulting from a traumatic chemical spill or childhood sexual abuse), somatoform disorder, and depression (Binkley and Kutcher, 1997; Göthe et al., 1995; Gots, 1995; Guglielmi et al., 1994; Kurt, 1995; Pennebaker, 1994; Simon, 1994; Sparks et al., 1994a, b; Spyker, 1995; Staudenmayer, 1999; Staudenmayer and Selner, 1987; Staudenmayer et al., 1993). Davidoff and Fogarty (1994) examined 10 published studies that explored possible psychogenic theories for chemical intolerance. All drew scientifically unsupportable conclusions concerning cause and effect, and erroneously assumed that psychological symptoms were psychogenic when chemical exposures might also explain them. Some symptoms these patients report mimic panic disorder, including hyperventilation, lightheadedness, chest discomfort, palpitations, paresthesias, and impaired mentation, leading researchers to wonder whether anxiety underlies chemical intolerance. Eleven of 15 chemically intolerant patients exposed to their own problem triggers, e.g., hairspray or the yellow pages, reported that these reproduced their characteristic responses, including tachycardia, tremor, and pallor (Leznoff, 1997). Exhaled CO2 in all 11 responders decreased, consistent with hyperventilation due to an anxiety reaction. A few responded to odorless stimuli, e.g., sugar or water, as well. In a singleblind study, Binkley and Kutcher (1997) administered saline (placebo) intravenous sodium lactate (known to elicit attacks in persons with panic disorder) to 5 chemically intolerant patients, all of whom responded with panic-like symptoms. In another single-blind case-control study, 31 chemically intolerant patients and 31 healthy controls inhaled a single breath of air alone or air spiked with CO2 (35%) (Poonai et al., 2000). Inhaled CO2 elicits panic symptoms in the majority of patients with panic disorder, but only 5% of healthy controls. Significantly more chemically intolerant patients (71%) than controls (26%) fulfilled panic criteria after CO2 inhalation. Both groups showed significant changes in heart and breathing rates after CO2 versus air, with no significant difference between patients and controls. The high percentage of controls responding in this study and the fact that the CO2

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Table 4 Parallel and Opposing Features of Addiction and Abdiction (Chemical Intolerance) Feature Multisystem symptoms, especially central nervous system symptoms Multiple, chemically unrelated substances affecting same individual Caffeine, alcohol, nicotine, drugs implicated Size of doses “tolerated” vs. those tolerated by general population Inhalation, ingestion, injection or transmucosal routes Stimulatory and withdrawal symptoms Heightened sensitivity to physical stimuli (noise, light, heat, cold, touch, vibration) during withdrawal phase Cravings, bingeing Habituation Heightened sensitivity following period of avoidance Genetic predisposition Demographics

Gender ratio (M:F) Age of onset Ill-defined physiological mechanisms Lack of biological markers Lack of effective drugs for treating condition Primary therapeutic strategy Detox/withdrawal requiring 4–7 days Societal views concerning nature of problem

Addiction

Abdiction





 (cross-tolerance)  large

 (cross-tolerance or “spreading”)  small





 

 

   (e.g., tobacco)  Poorly educated males, lower socioeconomic status

 (caffeine, foods)  

2:1 Teens, 20–30 years    Abstinence  Disease vs. lack of willpower to avoid substances (underavoidance)

Patients viewed as difficult, demanding Linked to violence, physical/sexual abuse, suicide Disruption of work, family and social relationships

  

 College educated females, middle to upper socioeconomic status 3:5 30–50 years    Avoidance  Disease vs. belief system leading to avoidance of substances (overavoidance)  a 

a

If chemical intolerance and addiction are interrelated and tend to cluster in families, then the higher rates of childhood abuse described by some investigators (Staudenmayer et al., 1993) among chemically intolerant women conceivably could be the consequence of alcoholic or drug-addicted elders who abused them.

mixture “had a marked taste” suggests study design problems. The other two panic studies cited above did not use controls. Many symptoms of chemical intolerance deviate from the panic profile. Hyperventilation during chemical (or even placebo) exposure in a setting where patients are being scrutinized by researchers and administered chemical challenges does not prove that the illness is psychogenic. A sizable percentage of healthy controls appear to respond similarly. Results of the lactate infusion study do not rule out possible metabolic problems in these patients, e.g., abnormal red blood cell oxygen-carrying

capacity. Carefully conducted studies are needed to untangle the current confusion of competing hypotheses. Funding for such studies has been scant.

VII.

MEDICAL MANAGEMENT

Forty percent of chemically intolerant patients in one study had consulted 10 or more medical practitioners (Miller and Mitzel, 1995). Some see clinical ecologists, while others remain with their family doctors. Still others seek out aca-

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demic occupational and environmental medicine doctors, particularly when workplace exposures are involved. Even physicians familiar with the phenomenon have difficulty managing these cases. Patients are apt to ask about various unorthodox therapies. At this time, controlled studies for any treatments are lacking. Ideally, physicians should not chastise patients who attempt alternative treatments, but display their desire to see the patients improve, help them avoid potentially dangerous interventions, and serve as their advocates during a perplexing illness. “Increasingly, in difficult circumstances, the reasonable trend in medicine is to explain the options and allow the patient to decide” (Vasey, 1995). Ample time should be allotted for visits and/or telephone consultations if patients cannot travel. Often, other patients and support groups can offer assistance. A multidisciplinary approach, paralleling that used for chronic pain, has been suggested (Weaver, 1996). Weaver advises: “Regardless of the treatment chosen, it is important to emphasize that functional improvement and increased patient control, not cure, are the goals. Complete resolution of odor sensitivity may not be possible.” Patients can exhaust their financial resources, their families, and themselves pursuing alternative treatments, when time and money might be better spent modifying their home environment. Consistently, the single most helpful intervention reported by these patients has been avoidance of problem exposures. In a survey of 206 MCS patients with an average educational level of nearly 4 years of college, 71% rated avoidance of problem chemicals, and 54% avoidance of problem foods, as “very helpful.” Although 52% had tried psychological or psychiatric therapies, only 17% of those who had tried them rated them as “very helpful” (Miller, 1995). DePaul University researchers found that at least three fourths of 305 chemically intolerant subjects who had tried the following interventions found them to be of “enormous” or “major” help: avoiding chemicals that cause reactions (93%), creating an environmentally safe (odorfree) living space (86%), moving to a less polluted area (76%), and avoiding foods that provoke reactions (75%) (LeRoy et al., 1996). In a grass-roots survey of 243 chemically intolerant patients, nearly half of whom were on disability, at least three fourths reported these interventions to be of “enormous help” or “major help”: avoidance of chemical exposures (95%), relocation to avoid pollution (79%), and avoidance of problem foods (76%) (Johnson, 1996). Given the patients’ clear consensus that chemical and food avoidance benefit them most, increasing numbers of physicians have begun to recommend avoidance strategies for these patients.

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While the vast majority of patients view avoidance of odor/chemical and food triggers as paramount (Johnson, 1996; LeRoy et al., 1996; Miller, 1995), psychological support (to be distinguished from traditional psychotherapy) can be helpful in any illness, whether the condition is psychogenic or physical in origin. Some authors have advocated psychological interventions as the preferred or only acceptable treatment modality for multiple chemical intolerance (Sparks et al., 1994b), expressing concern that the illness may be iatrogenic. At this time, placing sole reliance on psychological therapies, to the exclusion of trial avoidance, is at best premature and, at worst, potentially harmful (Miller, 1995). A multifaceted approach is called for, one that includes the identification and avoidance of odor/chemical and food triggers, low- or no-cost alterations of patients’ workplace and home environments, and appropriate psychological support. In the United States, affected individuals must chart their own course to recovery. Balanced medical information and knowledgable, sympathetic physicians are difficult to find. Fatigue and concentration difficulties may undermine care seeking, the ability to identify triggers, implementing lifestyle changes, obtaining social support services, etc. Chemically intolerant patients frequently report adverse reactions to medications and poor tolerance for standard drug doses. They appear to be at increased risk of developing both known drug side effects (even at low doses) and idiosyncratic reactions (McLellan, 1987). When new symptoms surface, it can be difficult to determine whether they are due to a medication (the drug itself, a dye, excipient, or diluent), an environmental exposure, or some new medical problem, e.g., cancer or coronary artery disease. Exhaustively evaluating each new symptom quickly becomes costly and exposes patients to other risks, including anesthetic agents or x-ray contrast dye, potentially adding more layers of “unexplained” symptoms. Patients may improve as medications are removed from their regimens. Some patients benefit from lower-than-normal drug doses (e.g., analgesics or antidepressants, but not antibiotics), a noteworthy clinical observation consistent with their amplified responses to environmental pollutants. Some find they can tolerate certain drugs better than others and make special requests, e.g., dental anesthetics without epinephrine. Any reasonable request should be honored. Environmental testing rarely contributes to understanding these patients’ health problems. However, professional industrial hygienists or indoor air consultants sensitive to these concerns can help detect contaminant sources and make recommendations for minimizing exposures following a walk-through workplace or home evaluation. These consultants need to be nonsmokers with an excellent sense

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of smell. Smokers and passive smokers (as well as workers routinely exposed to petroleum products) are less able to perceive various low-level odors, perhaps as a consequence of sensory deficit or habituation (Ahlstrom et al., 1986, 1987). Indoor air contaminant concentrations of importance for chemically intolerant patients appear to be orders of magnitude below those prescribed by law (OSHA) for occupational environments. Unless exceeded, legally adequate occupational exposure levels have little relevance and should not be invoked. As for any chronic illness, psychological support may be helpful, depending upon the patient’s needs and willingness. Tremendous disruption of work, family, and social lives can occur with this illness, e.g., divorces from spouses who smoke. Suicides have been reported. Support can be provided by psychologists, psychiatrists, social workers, or primary care doctors, although patients rarely find that psychological interventions alter their intolerances (Johnson, 1996; Miller 1995). Actively psychotic or suicidal patients need to be taken seriously and treated and protected accordingly. Various investigators advocate psychological or psychiatric interventions, but only anecdotal data suggest that these approaches may be helpful (Amundsen et al., 1996; Bolla-Wilson et al., 1989; Guglielmi et al., 1994; Schottenfeld and Cullen, 1985; Spyker, 1995). Several authors claim their patients improved as a result of psychotherapeutic interventions, but adequate follow-up is not provided (Amundsen et al., 1996; Bolla-Wilson et al., 1989; Guglielmi et al., 1994; Schottenfeld and Cullen, 1985; Spyker, 1995). Staudenmeyer et al. (1993) similarly attest, without providing follow-up or appropriate study design, that among their patients who agreed to undergo psychotherapy, 75% had a successful outcome. Among 243 chemically intolerant individuals who had taken an antidepressant, 10–20% found them of “major” or “enormous” help; 50–65% reported harmful effects; and 10–30% reported they were not helpful or their effect was unclear (Johnson, 1996). Two single-case reports have appeared in the literature involving individuals whose odor intolerances apparently resolved while they were taking selective serotonin-reuptake inhibitor antidepressants, one case involving psychological desensitization as an adjunct (Stenn and Binkley, 1998) and the other using drug alone (Andiné et al., 1997). Accommodating these individuals in the workplace can be challenging but vital to their self-esteem and livelihoods. Some patients are able to continue working provided that they avoid problem exposures, e.g., coworkers’ fragrances or copier machines. Workplace accommodations can include increasing fresh air supply and air circulation, removing business machines (fax machines, copiers, laser

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printers) from the immediate work environment, providing an alternative work space, removing carpeting, selecting odorless and less toxic cleaning agents, adopting integrated pest management, and allowing employees to work from home (Miller 2001; Miller et al., 1999). Physicians can facilitate the accommodation process by sending reasonable written requests to employers or schools on their patient’s behalf, but only with the patient’s full knowledge and written consent, so as to protect the physician should the patient’s job be eliminated. Multiple chemical intolerance is increasingly being viewed as a disability (Winterbauer, 1997). Internal memoranda of the Social Security Administration and Department of Housing and Urban Development recognize the illness for purposes of compensation and housing accommodation, respectively. Recent Equal Employment Opportunity Commission (EEOC) statistics show that from November 1, 1993, through March 31, 2000, 564 multiple chemical intolerance discrimination–related complaints were filed against employers, 60% alleging failure to provide reasonable accommodation and nearly 50% wrongful discharge (EEOC, 2000). The courts have struggled over whether the illness should be viewed as a disability, issuing conflicting opinions. Current law would make it difficult for an employer to claim that a condition that so greatly restricts daily activity is not a disability (Winterbauer, 1997). The Americans with Disabilities Act (ADA) obligates employers to seek inexpensive, practical solutions that will reduce troublesome odorous and odorless (e.g., pesticide) exposures (Winterbauer, 1997). It does not require that a chemical-free workplace be provided.

VIII. ILLNESS COURSE, PROGNOSIS, AND PREVENTION Few patients report full recoveries, even decades after they become sick. There are those rare individuals whose illness was recognized early in its course, who avoided further exposure, and who appear to have recovered (Hileman, 1991). At this time, early recognition and avoidance of further exposure offer the greatest potential to prevent disability. Difficulties in treating the illness, once entrenched, underscore the importance of prompt action. In one clinical series, chemically intolerant individuals who succeeded in avoiding at least half of their self-reported incitants reported feeling better than did nonavoiders at follow-up 6 months to 21/2 years after their initial visit (Lax and Henneberger, 1995). In the future, understanding multiple chemical intolerance will be essential for establishing sound national

Multiple Chemical Intolerance

and corporate environmental policy. If there is a subset of the population that is especially susceptible to various “odors,” i.e., common low-level chemical exposures, strategies to protect these people must be found. If only certain chemical exposures initiate the illness process, then the focus should be on avoiding or minimizing those exposures, for example, preventing chemical spills, prohibiting building occupancy during finish-out, and using integrated pest management. Besides regulating exposures that may initiate the illness, notifying people in advance of anticipated exposures, e.g., reroofing or painting, should be standard practice. Such strategies may protect more vulnerable individuals from becoming sick in the first place and thereby prevent unnecessary and costly overregulation of environmental exposures in the future. REFERENCES AAAAI (American Academy of Allergy, Asthma and Immunology) (1981). Position statements: controversial techniques. J. Allergy Clin. Immunol. 67(5):333–338. AAAAI (American Academy of Allergy, Asthma and Immunology) (1986). Position statements: clinical ecology. J. Allergy Clin. Immunol. 72(8):269–271. AAAAI (American Academy of Allergy, Asthma and Immunology) (1999). Idiopathic environmental intolerances. J. Allergy Clin. Immunol. 103:36–40. ACOEM (American College of Occupational and Environmental Medicine) (1999). Multiple chemical sensitivities: idiopathic environmental intolerance. J. Occ. Environ. Med. 41(11):940–942. ACP (American College of Physicians) (1989). Clinical ecology: position statement. Ann. Intern. Med. 111:168–178. ACS (American Chemical Society) (1999). Special issue on multiple chemical sensitivity. Toxicol. Ind. Health 15(3–4):283–437. Ahlstrom, R., Berglund, B., Berglund, U., Lindvall, T., and Wennberg, A. (1986). Impaired odor perception in tank cleaners. Scand. J. Work Environ. Health 12(6): 574–581. Ahlstrom, R., Berglund, B., Berglund, U., Engen, T., and Lindvall, T. (1987). A comparison of odor perception in smokers, nonsmokers, and passive smokers. Am. J. Otolaryngol. 8(1):1–6. AMA (American Medical Association, Council of Scientific Affairs) (1992). Clinical ecology. JAMA 268:3465–3467. Amundsen, M., Hanson, N., Bruce, B., Lantz, T., Schwartz, M., and Lukach, B. (1996). Odor aversion or multiple chemical sensitivities: recommendations for a name change and description of successful behavioral medicine treatment. Reg. Toxicol. Pharmacol. 24:S116–S118. Andiné, R., Rönnbäck, L., and Järyholm, B. (1997). Successful use of a selective serotonin reuptake inhibitor in a patient with multiple chemical sensitivities. Acta Psychiatr. Scand. 96:82–83.

543 AOEC (Association of Occupational and Environmental Clinics) (1992). Advancing the understanding of multiple chemical sensitivity. Toxicol. Ind. Health 8(4):1–257. Ashford, N., and Miller, C. (1989). Chemical sensitivity: a report to the New Jersey State Department of Health. Trenton, NJ. Ashford, N., and Miller, C. (1998). Chemical Exposures: Low Levels and High Stakes. John Wiley and Sons, Inc., New York. Ashford, N., Heinzow, B., Lütjen, K., Marouli, C., Mølhave, L., Mönch, B., Papadopoulos, S., Rest, K., Rosdahl, D., Siskos, P., and Velonakis, E. (1995). Chemical sensitivity in selected European countries: an exploratory study. A Report to the European Commission. Ergonomia, Athens, Greece. ASHRAE (American Society of Heating, Refrigeration and Air Conditioning Engineers). (1999). Ventilation for acceptable indoor air quality, ASHRAE Standard 62–99, Atlanta, GA. ATSDR (Agency for Toxic Substances and Disease Registry) (1994). Proceedings of the Conference on Low-Level Exposure to Chemicals and Neurobiologic Sensitivity. Toxicol. Ind. Health 10(4/5):253–669. Baldwin, C., and Bell, I. (1998). Increased cardiopulmonary disease risk in a community-based sample with chemical odor intolerance: implications for women’s health and health-care utilization. Arch. Environ. Health 53:347–353. Bartha, L., Baumzweiger, W., Buscher, D., Callender, M., Dahl, K., Davidoff, A., et al. (1999). Multiple chemical sensitivity: a 1999 consensus. Arch. Environ. Health 54(3):147–149. Bascom, R. (1989). Chemical hypersensitivity syndrome study: options for action. A literature review and a needs assessment. Prepared for the State of Maryland Department of Health. February 7. Bascom, R. (1991). Multiple chemical sensitivity: a respiratory disorder. Toxicol. Ind. Health 8(4):221–228. Bascom, R., Meggs, W., Frampton, M., Hudnell, K., Killburn, K., Kobal, G., Medinsky, M., and Rea, W. (1997). Neurogenic inflammation: with additional discussion of central and perceptual integration of nonneurogenic inflammation. Environ. Health Perspect. 105(suppl. 2): 531–537. Bell, I. R. (1996). Clinically relevant EEG studies and psychophysiological findings: possible neural mechanisms for multiple chemical sensitivity. Toxicology 111:101–117. Bell, I., Peterson, J., and Schwartz, G. (1995). Medical histories and psychological profiles of middle-aged women with and without self-reported illness from environmental chemicals. J. Clin. Psychiatry 56(4): 151–160. Bell, I., Miller, C., Schwartz, G., Peterson, J., and Amend, D. (1996). Nueropsychiatric and somatic characteristics of young adults with and without self-reported chemical odor intolerance and chemical sensitivity. Arch. Environ. Health 51(1):9–21. Bell, I., Rossi, J., Gilbert, M., Kobal, G., Morrow, L., Newlin, D., Sorg, B., and Wood, R. (1997a). Testing the neural sensitization and kindling hypothesis for illness from low levels of environmental chemicals. Environ. Health Perspect. 105(suppl. 2):539–47. Bell, I., Schwartz, G., Baldwin, C., Hardin, E., Klimas, N., Kline, J., Patarca, R., and Song, Z. (1997b). Individual differences in neural sensitization and the role of context in illness from

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Miller Djuric´, V., Overstreet, D., Bienenstock, J., and Perdue, M. (1995). Immediate hypersensitivity in the Flinders rat: further evidence for a possible link between susceptibility to allergies and depression. Brain Behav. Immun. 9(3): 196–206. Djuric´, V., Cox, G., Overstreet, D., Smith, L., Dragomir, A., and Steiner M. (1998). Genetically transmitted cholinergic hyperresponsiveness predisposes to experimental asthma. Brain Behav. Immun. 12:272–284. Doty, R.L., Deems, D.A., Frye, R.E., Pelberg, R., and Shapiro, A. (1988). Olfactory sensitivity, nasal resistance, and autonomic function in patients with multiple chemical sensitivities. Arch. Otolaryngol. Head Neck Surg. 114:1422–1427. EEOC (2000). Statistics on MCS-related complaints. May 31, 2000. Personal communication from the Director of the EEOC Office of Research, Information and Planning, Deidre Flippen, Washington, DC. Fiedler, N., and Kipen, H. (1997a). Experimental approaches to chemical sensitivity. Environ. Health Perspect. 105(suppl. 2):405–547. Fiedler, N., and Kipen, H. (1997b). Chemical sensitivity: the scientific literature. Environ. Health Perspect. 105(suppl. 2):409–415. Fiedler, N., Maccia, C., and Kipen, H. (1992). Evaluation of chemically-sensitive patients. J. Occup. Med. 34:529–538. Fiedler, N., Kipen, H., DeLucia, J., Kelly-McNeil, K., and Natelson, B. (1996a). A controlled comparison of multiple chemical sensitivities and chronic fatigue syndrome. Psychosom. Med. 58:38–49. Fiedler, N., Kipen, H., Natelson, B., and Ottenweller, J. (1996b). Chemical sensitivities and the Gulf War: Department of Veterans Affairs Research Center in basic and clinical science studies of environmental hazards. Reg. Toxicol. Pharmacol 24(1 Pt 2):S129–S138. Genton, M. (1998). Shedding light on darkroom disease: progress and challenges in understanding radiology workers’ occupational illness. Can. J. Med. Radiation Tech. 2(2):60–66. Göthe, C., Molin, C., and Nilsson, C. (1995). The environmental somatization syndrome. Psychosomatics 36(1):1–11. Gots, R. (1995). Multiple chemical sensitivities—public policy (editorial). J. Toxicol. Clin. Toxicol 33(2):111–113. Guglielmi, R., Cox, D., Spyker, D. (1994). Behavioral treatment of phobic avoidance in multiple chemical sensitivity. J. Behav. Ther. Exp. Psychiatry 25(3):197–209. Haley, R., Billecke, S., and La Du, B. (1999). Association of low PON1 type Q (type A) arylesterase activity with neurologic symptom complexes in Gulf War veterans. Toxicol. Appl. Pharmacol. 157(3):227–233. Heuser, G., and Mena, L. (1998). Neurospect in neurotoxic chemical exposure demonstration of long-term functional abnormalities. Toxicol. Ind. Health 14(6):813–827. Hileman, B. (1991). Multiple chemical sensitivity. Chem. Eng. News 69(29):26–42. Hirzy, J., and Morrison, R. (1989). Carpet/4-phenylcyclohexene toxicity: the EPA headquarters case. Presented at the Annual Meeting of the Society for Risk Analysis, San Francisco. Hu, H., Johnson, K., Heldman, R., Jones, K., Komaroff, A. L., Schacterle, R., Barsky, A., Becker, A., and Holman, L. (1999).

Multiple Chemical Intolerance A comparison of single photon emission computed tomography in normal controls, in subjects with multiple chemical sensitivity syndrome, and in subjects with chronic fatigue syndrome. Department of Labor and Industries, State of Washington. Hummel, T., Roscher, S., Jaumann, M., and Kobal, G. (1996). Intranasal chemoreception in patients with multiple chemical sensitivities: a double-blind investigation. Reg. Toxicol. Pharm. 24:S79–S86. Institute of Medicine (IOM) (1995). Environmental Medicine: Integrating Missing Elements into Medical Education, A. Pope and D. Rall (Eds.). National Academy Press, Washington, DC. Johnson, A. (1996). MCS Information Exchange. Brunswick, ME. Kay, L. (1996). Support for the Kindling Hypothesis in multiple chemical sensitivity syndrome (MCSS) induction. Presentation at the 26th Annual meeting of the Society for Neuroscience, Washington, DC, November 16–21. Kruetzer, R., Neutra, R., and Lashuay, N. (1999). Prevalence of people reporting sensitivities to chemicals in a populationbased survey. Am. J. Epidemiol. 150(1):1–12. Kuhn, T. 1970. The Structure of Scientific Revolutions. The University of Chicago Press, Chicago, IL. Kurt, T. (1995). Multiple chemical sensitivities—a syndrome of pseudotoxicity manifest as exposure perceived symptoms. J. Toxicol. Clin. Toxicol. 33(2):101–105. Lax, M., and Henneberger, P. (1995). Patients with multiple chemical sensitivities in an occupational health clinic: presentation and follow-up. Arch. Environ. Health 50(6):425–431. LeRoy, J., Davis, T., and Jason, L. (1996). Treatment efficacy: a survey of 305 MCS patients. CFIDS Chronicle. Winter:52–53. Leznoff, A. (1997). Provocative challenges in patients with multiple chemical sensitivity. J. Allergy Clin. Immunol. 99: 438–442. Mandell, M., and Scanlon, L. (1979). Dr. Mandell’s 5-Day Allergy Relief System. Thomas Y. Crowell, New York. Mayberg, H. (1994). SPECT studies of multiple chemical sensitivity. Toxicol. Ind. Health 10(4–5):661–666. McFadden, S. (1996). Phenotype variation in xenobiotic metabolism and adverse environmental response: focus on sulfurdependant detoxification pathways. Toxicology 111:43–65. McLellan, R. (1987). Biological interventions in the treatment of patients with multiple chemical sensitivities. In Workers with Multiple Chemical Sensitivities, Occupational Medicine State of the Art Reviews. Cullen M (Ed.). Hanley & Belfus Philadelphia, pp. 755–777. Meggs, W. (1994). RADS and RUDS—the toxic induction of asthma and rhinitis. Clin. Toxicol. 32(5):487–501. Meggs, W., and Cleveland, C. (1993). Rhinolaryngoscopic examination of patients with the multiple chemical sensitivity syndrome. Arch. Environ. Health 41(1):14–18. Meggs, W., Dunn, K., Bloch, R., Goodman, P., and Davidoff, L. (1996). Prevalence and nature of allergy and chemical sensitivity in a general population. Arch. Environ. Health 51(4):275–282. Miller, C. (1994). Multiple chemical sensitivity and the Gulf War veterans. NIH Workshop on the Persian Gulf Experience and

545 Health, April 27–29, 1994. Bethesda, MD: National Institutes of Health. Miller, C. (1995). Letter to the editor. J. Occup. Med. 37(12):1323. Miller, C. (1996). Chemical sensitivity: symptom, syndrome or mechanism for disease? Toxicology 111:69–86. Miller, C. (1997). Toxicant-induced loss of tolerance: an emerging theory of disease? Environ. Health Perspect. 105(suppl 2): 445–453. Miller, C. (1999). Are we on the threshold of a new theory of disease? Toxicant-induced loss of tolerance and its relationship to addiction and abdiction. Toxicol. Ind. Health 15: 284–294. Miller, C. (2000). Toxicant-induced loss of tolerance: addiction, abdiction, and the chemical environment. Addiction 96(1):115–139. Miller, C. (2001). Multiple chemical intolerance and indoor air quality. In Handbook of Indoor Air Quality, Samet, J., and Spengler, J. (Eds.). McGraw-Hill, NY, pp. 27.1–27.32 Miller, C., and Mitzel, H. (1995). Chemical sensitivity attributed to pesticide exposure versus remodeling. Arch. Environ. Health 50(2):119–129. Miller, C., and Prihoda, T. (1999a). The Environmental Exposure and Sensitivity Inventory (EESI): a standardized approach for measuring chemical intolerances for research and clinical applications. Toxicol. Ind. Health 15:370–385. Miller, C., and Prihoda, T. (1999b). A controlled comparison of symptoms and chemical intolerances reported by Gulf War veterans, implant recipients and persons with multiple chemical sensitivity. Toxicol. Ind. Health 15:386–397. Miller, C., Ashford, N., Doty, R., Lamielle, M., Otto, D., Rahill, A., and Wallace, L., (1997). Empirical approaches for the investigation of toxicant-induced loss of tolerance. Environ. Health Perspect. 105(suppl. 2):515–519. Miller, C., Gammage, R., and Jankovic, J. (1999). Exacerbation of chemical sensitivity: a case study. Toxicol. Ind. Health 15:398–402. Mitchell, C., Donnay, A., Hoover, D., and Margolick, J. (2000). Immunologic parameters of multiple chemical sensitivity. In Multiple Chemical Sensitivity/Idiopathic Environmental Intolerance, Occupational Medicine State of the Art Reviews, Sparks, P., (Ed.). Hanley and Belfus, Inc., Philadelphia, pp. 647–665. Monk, J. (1996). Farmers fight chemical war. Chem. Ind. February 5:108. Moorhead, J., and Suruda, A. (2000). Occipital lobe meningioma in a patient with multiple chemical sensitivities. Am. J. Ind. Med. 37:443–446. Morton, W. E. (1995). Redefinition of abnormal susceptibility to environmental chemicals. Presented at the Second International Congress on Hazardous Waste: Impact on Human Ecological Health, Atlanta, Georgia, June 6. Nethercott, J., Davidoff, L., Curbow, B., and Abbey, H. (1993). Multiple chemical sensitivities syndrome: toward a working case definition. Arch. Environ. Health 48:19–26. Newlin, D. (1997). A behavior-genetic approach to multiple chemical sensitivity. Environ. Health Perspect 105(suppl. 2):505–508.

546 NIEHS (National Institute of Environmental Health Sciences) (1997). Chemical sensitivity. Environ. Health Perspect. 105(suppl. 2):405–547. NRC (National Research Council) (1992). Multiple Chemical Sensitivities: Addendum to Biologic Markers in Immunotoxicology, National Research Council, National Academy of Sciences. National Academy Press, Washington, DC. Overstreet, D. H. (1996). Alternatives to naltrexone in animal models. Alcohol Clin. Exp. Res. 20:231A–235A. Pennebaker, J. (1994). Psychological bases of symptom reporting: perceptual and emotional aspects of chemical sensitivity. Toxicol. Ind. Health 10(4/5):497–511. Poonai, N., Antony, M., Binkley, K., Stenn, P., Swinson, R., Corey, P., Silverman, F., and Tarlo, S. (2000). Carbon dioxide inhalation challenges in idiopathic environmental intolerance. J. Allergy Clin. Immunol. 106(2 part 1):358–363. Randolph, T. (1956). The descriptive features of food addiction. Q. J. Stud. Alcohol 17:198–224. Randolph, T. G. (1962). Human Ecology and Susceptibility to the Chemical Environment. Charles C. Thomas, Springfield, IL. Randolph, T. G., and Moss, R. W. (1980). An Alternative Approach to Allergies. Lippincott and Crowell, New York. Rinkel, H. (1944). Food allergy: the role of food allergy in internal medicine. Ann. Allergy 2:115–124. Rinkel, H., Randolph, T., and Zeller, M. (1951). Food Allergy. Charles C. Thomas, Springfield, IL. Rogers, W., Miller, C., and Bunegin, L. (1999). A rat model of neurobehavioral sensitization to toluene. Environ. Health Perspect. 152:356–369. Rossi, G., Rea, W., Johnson, A., Hickey, D., and Simon, T. (1999). Neurotoxicity in single photon emission computed tomography brain scans of patients reporting chemical sensitivities. Toxicol Ind. Health 15(3–4):415–420. Ryan, C., Morrow, L., and Hodgson, M. (1988). Cacosmia and neurobehavioral dysfunction associated with occupational exposure to mixtures of organic solvents. Am. J. Psychiatry 145(11):1442–1445. Schottenfeld, R., and Cullen, M. (1985). Occupational-induced posttraumatic stress disorders. Am. J. Psychiatry 142(2):198–202. Schottenfeld, R., and Cullen, M. (1986). Recognition of occupational induced posttraumatic stress disorders. J. Occ. Med. 28:365–369. Schusterman, D., and Dager, S. (1991). Prevention of psychological disability after occupational respiratory exposures. Occup. Med. 6:11–27. Siegel, S., and Kreutzer, R. (1997). Pavlovian conditioning and multiple chemical sensitivity. Environ. Health Perspect. Suppl. 105:521–526. Silverman, K., Evans, S., Stain, E., and Griffiths, R. (1992). Withdrawal syndrome after the double-blind cessation of caffeine consumption. N. Engl. J. Med. 327(16):1109–1114. Simon, G., Katon, W., and Sparks, P. (1990). Allergic to life: psychological factors in environmental illness. Am. J. Psychiatry 147:901–906.

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547 Webster’s Third New International Dictionary of the English Language (Unabridged) (1986). Merriam-Webster, Springfield, MA. Winterbauer, S. (1997). Multiple chemical sensitivity and the ADA: taking a clear picture of a blurry object. Employee Relations Law J. 23(2):69–104.

26 The Olfactory System and the Nasal Mucosa as Portals of Entry of Viruses, Drugs, and Other Exogenous Agents into the Brain Harriet Baker Cornell University, White Plains, New York, U.S.A.

Mary Beth Genter University of Cincinnati, Cincinnati, Ohio, U.S.A.

I.

circulation from the nasal cavity. The first route occurs via internalization of xenobiotics into the receptor neurons of the olfactory epithelium. Substances are transported into the brain through the axons of olfactory receptor neurons in a process also called the olfactory nerve pathway by Mathison et al. (1998). In an older publication, McMartin et al. (1987) referred to an analogous mechanism in their reference to both transcytosis (uptake of the agent into vesicles with subsequent discharge into interstitial space) and transcellular transport (passage of agents via pores or carriers across the cell membrane, diffusion across the cytoplasm, and transport out of the cell to the glomerular region of the olfactory bulb). Within the glomerulus, axodendritic contacts between receptor cells and mitral cells serve as one transport pathway. For some xenobiotics, transneuronal transport has been observed in widespread areas of the brain. Both anterograde and retrograde transport have been reported, the latter perhaps a result of the large centrifugal afferent innervation of the olfactory glomerular region where receptor afferent fibers terminate (see Chapter 7). Anterograde labeling can then be observed in mitral cell terminal fields such as the piriform cortex. Retrograde transport is also observed in brain regions with projections to the olfactory bulb including the horizontal limb of the diagonal band, substantia nigra, locus ceruleus, and dorsal raphe nucleus.

INTRODUCTION

The important role the olfactory system plays in food acquisition and mating behaviors is reflected in the substantial development of this sensory modality in the majority of vertebrate species with, perhaps, the exception of primates. In view of these life-sustaining roles, the common dogma posits that a system as important to survival as olfaction must have mechanisms to protect the sensory cells within the olfactory receptor epithelium from the materials in inspired air, both toxicants and odorants. Also, a means should exist to prevent access of extrinsic substances to the central nervous system (CNS). As discussed below (see also Chapter 3), several mechanisms have been proposed that would protect the epithelium from toxic exposure. Included among these mechanisms are intracellular detoxification of molecules, removal of substances by ligand-specific binding proteins contained in mucosal secretions, surveillance by immune system cells, as well as degeneration and replacement of damaged receptor neurons with new cells derived from basal stem cells. Recent data indicate, however, that these protective mechanisms can be overwhelmed, resulting in access of xenobiotics from the nares to the CNS and to the systemic circulation. Figure 1 (Fig. 1; see color plate) indicates three major routes of entry for agents to the CNS and systemic 549

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Figure 1 (A) Sagittal view of the rat brain showing olfactory epithelium (OE) receptor cells and their axonal projections to the olfactory glomeruli (G). In the glomerulus, receptor cell axons contact the dendrites of periglomerular (PG) and mitral (M) cells. Mitral cells project to the piriform cortex (PC). Centrifugal afferent innervation comes from the horizontal limb of the diagonal band (HLDB), the substantia nigra (SN), the dorsal raphe (DR) and the locus ceruleus (LC). (B) The olfactory mucosa includes an epithelial cell layer (OE) and the lamina propria (LP) separated by the basal lamina (BL). The OE contains sustentacular (S), basal (B), and receptor (R) cells. Receptor cell axons fasciculate to form the olfactory nerve (ON) that crosses the cribriform plate (CP) to enter the CNS. The axon and nerve are surrounded by a perineural sheath that forms the perineural space (PN). The lamina propria contains mucus-secreting Bowman’s glands (BG), axons of receptor cells, and numerous blood vessels (BV). Red and green dots depict possible entry pathways through neurons, glands, and blood vessels. (C) Respiratory epithelium consists of columnar ciliated (C), goblet (G), and basal (B) cells and is highly vascular (BV). (D) H & E stained section illustrating the layers of the OE and LP showing the numerous blood vessels in the lamina propria. Olfactory marker protein (OMP) immunostained section shows that only mature receptor neurons and their axons, not basal or sustentacular cells, contain OMP. (Panels B and C adapted from Lewis and Dahl, 1995.) (See color insert.)

Xenobiotics also may gain access to the CNS by two other routes, as indicated in Figure 1. A number of drugs (see below) and metals, including gold, are hypothesized to reach the subarachnoid space by movement through perineural spaces, a process also called the olfactory epithelial pathway or paracellular transport (Jackson et al., 1979; Mathison et al., 1998; McMartin et al., 1987). In fact, many drugs that otherwise would not have access to the brain can be applied intranasally, including antiviral drugs for the treatment of human immunodeficiency virus (HIV) infections that have spread to the CNS. The third route is a consequence of the highly vascular nature of the respiratory and olfactory epithelia.

This pathway provides access of intranasally applied agents to the systemic circulation and thus to the brain. Another consequence of the extensive vascularity of the olfactory epithelium is that some viruses (Charles et al., 1995; Oliver and Fazakerley, 1998) and a variety of organic compounds (Brandt et al., 1990; Brittebo, 1988; Feng et al., 1990) are concentrated in the olfactory epithelium following systemic application. Subsequently, there is either transport to the brain or destruction of the epithelium. The substances that have been reported to be internalized, transported, and accumulated through the olfactory system are quite varied, including lectins, dyes, solvents, metals, viruses, amino

Exogenous Agent Uptake

551

Table 1 Substances Analyzed for Transport Through Olfactory Receptor Cells to the Brain Substance

Species studied

Application method

Metals Aluminum lactate

Rabbit

Intranasal

Transneuronal transport

Aluminum silicate Aluminum acetylacetonate Cadmium Cadmium chloride

Rabbit Rat

Bedding Inhalation

Indirect pathological evidence No Yes

Pike Rat

Intranasal Intranasal

No No

Cadmium oxide

Rat

Aerosol

No

Cobalt Gold

Salmon Squirrel Monkey Rabbit Rat

Intranasal Intranasal

Possible Yes

Mucosal Intranasal

No Yes

Pike Rat

Intranasal Intranasal

No No

Rat

Intranasal

Yes

Mouse Mouse Rat

Intranasal Genetic Intranasal

Yes Yes No

Rat

Intranasal

Yes

Borna disease Equine Herpes Herpes simplex

Rat Pig Rat

Intranasal Intranasal Intranasal

Yes Yes Yes

Hepatitis

Mouse Mouse Mouse

Corneal Facial skin Intranasal

Yes Limited Yes

Rabies St. Louis encephalitis Sendai Semliki forest Venezuelan equine

Mouse Hamster Mouse Mouse Mouse

Intranasal Intraperitoneal Intranasal Intranasal Subcutaneous

Yes Yes Limited Age-dependent Yes

Catfish Eel

Intranasal Intranasal

No No

Manganese

Mercury Mercury Lectins WGA-HRP

Barley Concanavilin A Viruses Adeno (recombinant)

Dyes Procion yellow

Ref. Perl and Good, 1987 Hayek and Waite, 1991 Zatta et al., 1993 Gottofrey and Tjälve, 1991 Tjälve and Henriksson, 1999; Tjälve et al., 1996 Hastings and Evans, 1988; Hastings, 1990 Bazer et al., 1987 DeLorenzo, 1970 Czerniawska, 1970 Gianutsos et al., 1997; Tjälve and Henriksson, 1999; Tjälve et al., 1996; Borg-Neczak and Tjälve, 1996. Henriksson and Tjälve, 1998 Baker and Spencer, 1986; Broadwell and Balin, 1985; Balin et al., 1986; Itaya, 1987; Shipley, 1985; Stewart, 1985; Thorne, 1995 Baker, 1995 Horowitz et al., 1999 Wiley et al., 1984 Draghia et al., 1995; Zhao et al., 1996 Morales et al., 1988 Narita et al., 2001 Esiri and Tomlinson, 1984; McLean et al., 1989 Stroop et al., 1984 Stroop et al., 1984 Barnett and Perlman, 1993; Lavi et al., 1988; Perlman et al., 1990 Astic, 1993; Lafay et al., 1991 Monath et al., 1983 Mori et al., 1995 Oliver and Fazakerley, 1998 Charles et al., 1995; Ryshikov et al., 1995 Holl, 1980 Holl, 1981 (continued)

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Table 1 (continued) Substance Lucifer yellow Evans blue Prussian blue Trypan blue Amino acids Leucine

Alanine Taurine Drugs Cocaine Valproic acid Miscellaneous Aflatoxin B1 Ameba Horseradish peroxidase

Immunoglobulins Polychlorinated biphenyls PCB Solvents

Species studied

Application method

Transneuronal transport

Lamprey Frog Mouse Mouse Mouse Bullhead

Intranasal Intranasal Intranasal Intranasal Intranasal Intranasal

No No No No No No

Suzuki, 1984 Suzuki, 1984 Kristensson and Olsson, 1971 Rake, 1937. Seki, 1941 Holl, 1965

Toad Garfish Rabbit Pike Mouse Hamster Mouse

Intranasal Intranasal Intranasal Intranasal Intranasal Intranasal Intranasal

No No No No No No No

Weiss and Holland, 1967 Gross and Beidler, 1973 Land and Shepherd, 1974 Weiss and Buchner, 1988 Margolis and Grillo, 1977 Burd et al., 1982 Brittebo and Ericksson, 1995; Lindquist et al., 1983

Mouse Rat

Intravenous Intravenous

No No

Brittebo, 1988 Hoeppner, 1990

Rat Mouse Rat

Intranasal Intranasal Intranasal

Limited Yes No

Mouse

Intranasal

No

Mouse Ferret

Intranasal Ambient air

No ND

Larsson and Tjälve, 2000 Jarolim et al., 2000 Balin et al., 1986; Kristensson and Olsson, 1971 Meredith and O’Connell, 1988; Stewart, 1985 Baker and Maruniak, 1990 Apfelbach et al., 1998

Mouse

Inhalation

No

Ghantous et al., 1990

Ref.

ND, not determined.

Table 2 Systemically Administered Agents Associated with Olfactory Mucosal Damage in Rodents Compound

Route

Endpoint

Acetaminophen

i.p., oral

OMD

Alachlor

oral

Tumors

Bromobenzene Caffeine-derived N-nitroso compounds Carbimazole Coumarin p-Cresidine 2,6-Dichlorobenzamide 2,6-Dichlorobenzonitrile

i.v., i.p. oral

OMD Tumors

i.p. i.p., oral oral i.p. i.p., i.v., dermal

OMD OMD Tumors OMD OMD

2,6-Dichlorothiobenzamide Dihydropyridines Diethyldithiocarbamate Disulfiram 1,4-Dithiane

i.p. i.p. i.p. i.p. gavage

OMD Protoporphyria OMD OMD OMD

Ref. Jeffrey et al., 1988; Genter, 1998 U.S. Environmental Protection Agency, 1986; Genter et al., 2000 Brittebo et al., 1990 Ivankovic et al., 1998 Genter, 1998 Gu et al., 1997 National Cancer Institute, 1979 Brittebo et al., 1991 Brandt et al., 1990; Deamer et al., 1994 Brittebo et al., 1991 Reed et al., 1989 Deamer and Genter, 1995 Deamer and Genter, 1995 Schiferstein et al., 1988

Exogenous Agent Uptake

553

Table 2 (continued) Compound

Route

Endpoint

Ref.

N-bis(2-Hydroxypropyl)nitrosamine N-Hydroxy-IDPN IDPN Methimazole 3-Methylindole Methylsulfonyl-2,6-dichlorobenzene N-Nitrosodiethylamine NNK 2-Pentenenitrile

s.c. i.p. i.p. i.p., oral i.p. i.p. i.p. s.c. i.p.

Tumors OMD OMD OMD OMD OMD, metaplasia OMD OMD, tumors OMD

Phenacetin

oral

OMD, tumors

Koujitani et al., 1999 Crofton et al., 1996 Genter et al., 1992 Genter et al., 1995 Turk et al., 1986 Bahrami et al., 2000 Jensen and Sleight, 1987 Belinsky et al., 1987 Genter and Crofton, 2000 Bogdanffy et al., 1989 Isaka et al., 1979

Routes of administration: i.p.
intraperitoneal injection; i.v.
intravenous injection; s.c.
subcutaneous. OMD
olfactory mucosal degeneration; IDPN
3,3ⴕ iminodipropionitrile; NNK
4-(N-methyl-N-nitrosamino)-1-(3-pyridyl)-1-butanone.

Table 3 Drugs Administered Via the Nasal Cavity for CNS and Systemic Effects Purpose Drugs administered via the nasal cavity for CNS effects Relief of migraine headaches

Drug

Model

Ref.

Sumatriptan (Imitrex®) BMS-181885 Ergotamine derivatives

Dog, human Monkey Human Rabbit

Butorphanol Lidocaine

Human Human

Sedation

Capsaicin Midazolam

Human Human

Seizure control

Diazepam Midazolam

Rabbit Human

Oxycodone Oxymorphone Acetylsalicylic acid 5-Fluorouracil Lipophilic analog of vasoactive intestinal peptide ([St-Nle17]VIP) Dextromethorphan Neostigmine Nerve growth factor Synthetic acetylcholine receptor epitopes Zidovudine (AZT) D4T Cephalexin Arginine-vasopressin

Human Dog Rat Rat Rat

Barrow, 1997; Dahlof et al., 1998 Srinivas et al., 1998 Gallagher, 1996; Lipton, 1997; Ziegler et al., 1994 Marttin et al., 1997 Melanson et al., 1997 Lane, 1996; Mills and Scoggin, 1997; Sachs, 1996 Levy, 1995 Björkman et al., 1997; Louon and Reddy, 1994 Bechgaard et al., 1997 Jeannet et al., 1999; Wallace, 1997 Takala et al., 1997 Hussain and Aungst, 1997 Hussain et al., 1992 Sakane et al., 1999 Gozes et al., 1996, 1997

Rat Human Rat Mouse

Char et al., 1992 DiCostanzo et al., 1993 Frey et al., 1997 Karachunski et al., 1998

Rat Rat Rat Human

Seki et al., 1994 Yajima et al., 1998 Sakane et al., 1991 Perras et al., 1997; Pietrowsky et al., 1996

Analgesia

Cancer chemotherapy Treatment/prevention of neurodegenerative diseases

Treatment/prevention of CNS infections Increased brain activity/ memory improvement

(continued)

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Table 3 (continued) Purpose Appetite control

Drug

Model

Ref.

Cholecystokinin (CCK) agonists

Dog

Pierson et al., 1997

Insulin

Human, rabbit

Smoking cessation

Nicotine nasal sprays

Human

Vaccines

Cold-adapted influenza viruses. Outer membrane vesicles (N. meningitides). Diptheria-tetanus boosters Hydroxycobalamine

Human

Fernandez-Urrusuno et al., 1999; Hilsted et al., 1995; Valensi et al., 1996 Gourlay and Benowitz, 1997; Jones et al., 1998; Schneider et al., 1995 Liu, 1998; Maassab and DeBorde, 1985 Haneberg et al., 1998

Human Human

Aggerbeck et al., 1997 Slot et al., 1997; Swain, 1995

Scopolamine Promethazine Desmopressin

Human Dog Human

Gonadotrophin-releasing hormone agonist Nafarelin, buserelin

Human

Putcha et al., 1996 Ramanathan et al., 1998 Butler et al., 1998; Kallas et al., 1999 Bergquist et al., 1979

Buserelin

Human

Calcitonin Oxytocin Erythropoietin

Human Human Rat

Drugs administered via the nasal cavity for systemic effects Diabetes

Normalization of plasma vitamin B12 Motion sickness Nocturia Contraception Endometriosis, controlled ovarian hyperstimulation Prostate cancer, oligoasthenozoospermia Prevention of bone resorption Enhanced lactation Anemia

acids, and drugs. Table 1, a compilation of the range of substances transported, also indicates those for which the evidence suggests transneuronal transport. The consequences of such transport have only begun to be assessed (see below). One hypothesis suggests that the olfactory system may be a route by which some, as yet undefined, pathogen or toxin reaches the brain to produce degenerative disorders such as Alzheimer’s disease (Baker and Margolis, 1986; Crapper McLachlan, 1986) and Parkinson’s disease (Tjälve et al., 1996), as well as learning and behavioral abnormalities (Becker, 1995) (see Chapter 24). This internalization may have effects reaching through the food chain as some fish apparently can accumulate materials such as metals found in water by this route (Bazer et al., 1987). The significance of accumulation in the food chain is apparent in Table 2, a list of the compounds that when administered systemically either destroy or produce tumors in the olfactory epithelium. There are, however, benefits to the nasal route of application since many drugs have been admin-

Human

Dantas et al., 1994; Jacobson et al., 1994; Lemay et al., 1984 Matsumiya et al., 1998; Tolis et al., 1983 Silverman, 1997 Ruis et al., 1981 Shimoda et al., 1995

istered by this route (see Table 3) for either direct delivery to the CNS or systemic access. First to be discussed will be the specific substances and pathogens transported through olfactory receptor cells. Second, the proposed mechanisms of internalization and transport will be reviewed with special reference as to why transneuronal transport occurs only in limited instances. Intranasal application of drugs will be discussed as a means of administering a number of agents for direct entry to the brain. Finally, the consequences of xenobiotic access to the brain will be assessed from the disease perspective. II.

NATURE OF SUBSTANCES TRANSPORTED

A.

Metals

Aluminum, cadmium, cobalt, gold, and manganese are among the metals internalized in the olfactory receptor epithelium and transported either directly to the brain via olfactory axons or perhaps, as reviewed by Jackson et al.

Exogenous Agent Uptake

(1979), through either the subarachnoid space or perineurally. The evidence, both direct and circumstantial, for their transneuronal transport to brain has supported the controversial hypothesis that some of these metals, for example, aluminum, may be etiologically significant in pathological syndromes such as Alzheimer’s and Parkinson’s disease (Crapper McLachlan, 1986; Roberts, 1986; Tjälve et al., 1996). Described below is the method of application and evidence for uptake and transport of each of the metals. (Table 1)

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et al., 1996). In rats, exposure to both cadmium oxide and chloride, in aerosol form, resulted in accumulation of cadmium in the olfactory bulb (Evans and Hastings, 1992; Hastings, 1990; Hastings and Evans, 1988). Cadmium oxide did not produce anosmia (Hastings, 1990; Hastings and Evans, 1988). Hastings (1990) suggested that the lack of a functional deficit in rodents following cadmium exposure, in view of the purported effects in humans, might reflect a requirement for protracted exposure to produce anosmia. 3.

1.

Aluminum

Several studies have shown that aluminum can reach the central nervous system after intranasal application (Hayek and Waite, 1991; Perl and Good, 1987; Zatta et al., 1993). Aluminum silicate, presented as a powder in the bedding, was enriched specifically in the rabbit olfactory epithelium and olfactory bulb, but not in cerebellum (Hayek and Waite, 1991). Perl and Good (1987) demonstrated that, following intranasal application of aluminum lactate, granulomas were observed in the olfactory bulb and cerebral cortex. The pathological changes observed in brain suggested that aluminum reaches these brain regions by transport through olfactory neurons. Inhalation of aluminum acetylacetonate resulted in widespread dissemination of the metal complex in diverse brain regions (Zatta et al., 1993). Aluminum was also observed in the neurofibrillary tangles associated with Alzheimer’s disease (for review see Crapper McLachlan, 1986; Good et al., 1992), and on intraventricular administration was shown to produce neurofibrillary degeneration in rabbit and rat brain (Kowall et al., 1989; Shigematsu and McGeer, 1992). Additionally, the demonstration that brain regions having direct connections to the olfactory system are primarily affected in Alzheimer’s disease has suggested the provocative hypothesis that transport of aluminum via the olfactory system may play a role in this degenerative syndrome (Roberts, 1986). 2.

Cadmium

Cadmium transport from the olfactory epithelium to the olfactory bulb has been demonstrated in both fishes and rodents. A number of reports has suggested an association of cadmium exposure in humans with anosmia, and by inference, accumulation and transport of the metal (Hastings, 1990) (see Chapter 27). In the pike (Esox lucius), cadmium appeared to move at a velocity consistent with processing by the fast axonal transport system (Gottofrey and Tjälve, 1991). Cadmium accumulated in anterior regions of the olfactory bulb, but did not appear to move transsynaptically to mitral cell dendrites (Tjälve and Henriksson, 1999; Tjälve

Cobalt

Intranasal irrigation with cobalt lysine in king salmon fry (Oncorhynchus tshawytscha, Walbaum) resulted in transport of the conjugate to the olfactory bulb as well as to both ventral and lateral regions of the ventral telencephalon (Bazer et al., 1987). The latter projections may represent transneuronal transport of the metal conjugate (Bazer et al., 1987), as no direct epithelial connections to this brain region have previously been reported. There is evidence that cobalt is able to cross neuronal membranes (Fredman and Jahan-Pawar, 1980). 4.

Gold

Electron microscopic studies demonstrated that colloidal gold was internalized by olfactory receptor cells within 15 minutes of intranasal application in squirrel monkeys (De Lorenzo, 1970). Gold was thought to be incorporated into receptor cells by the pinocytotic vacuoles normally observed in the olfactory rod region, more commonly referred to as the dendritic knob (De Lorenzo, 1970). Subsequently, gold particles were observed in the cytoplasm of olfactory axons and within 1 hour in the olfactory glomerulus. Evidence was also presented for transneuronal transport of the gold particles from receptor cell axon terminals within the glomeruli to the dendrites of mitral cells (De Lorenzo, 1970). Within the olfactory bulb the particles were preferentially associated with mitrochondria. The rate of appearance in the glomeruli was consistent with the movement of the gold by fast axonal transport (estimated in these experiments at about 2.5 mm/hr). Czerniawska (1970) also demonstrated uptake of gold into the cerebrospinal fluid, especially in the region of the cribriform plate and the olfactory bulbs, following either mucous membrane or intravenous administration of the metal. 5.

Manganese

A recent series of papers documents both possible routes of entry of manganese into the CNS and the neurological consequences of exposure. Definitive evidence now exists in rats for transport of manganese from the

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olfactory epithelium into the brain (Tjälve and Henriksson, 1999; Tjälve et al., 1996). In contrast to other metals applied by intranasal irrigation (e.g., nickel and cadmium), manganese was transported to all regions of the CNS including the spinal cord and could still be detected 12 weeks after initial exposure. Manganese may also reach the CNS when administered by parenteral routes but in much lower concentrations. Manganese has been shown to produce a neurological syndrome with features of Parkinson’s disease (PD). Occupational exposure occurs in the mining industry, in steel manufacturing, and in welding and is primarily through inhalation of metal-containing dusts and fumes (Gorell et al., 1999; Tjälve and Henriksson, 1999). Taken together with the widespread CNS distribution following nasal exposure, the association with PD is consistent with specific toxicity towards dopamine neurons. One proposed mechanism (Tjälve and Henriksson, 1999) suggests that the metal may be involved in the formation of free radicals in the presence of catecholamines. 6.

Other Metals

Several studies investigated the transport of nickel and mercury presented intranasally. Inorganic mercury exhibited limited transneuronal transport restricted to the olfactory bulb following nasal application in rats (Henriksson and Tjälve, 1998). Long-term occupational exposure was not associated with increased risk for Parkinson’s disease (Gorell et al., 1999). Transneuronal transport of nickel was studied in rats and pike. The transport rate for this metal was 20 times slower than for cadmium and manganese. Nickel could be demonstrated in several forebrain regions, suggesting that the metal is transported slowly transsynaptically (Tallkvist et al., 1998; Tjälve and Henriksson, 1999). Nickel exposure has been associated with cell loss in the olfactory epithelium but did not alter olfactory function as indicated by measurement of either odor threshold or discrimination (Evans et al., 1995). B.

Lectins

Although native and derivatized lectins have been utilized by a number of investigators to delineate neuronal connectivity, especially in the visual system (Itaya and Van Hausen, 1982; Spencer et al., 1982), transport studies of these oligosaccharide-specific proteins in the olfactory system have been limited primarily to wheatgerm agglutinin (WGA). A brief report showed that concanavalin A was also transported in the olfactory system, but suggested that transneuronal transport did not occur (Wiley et al.,

1984). A number of investigators (Baker and Spencer, 1986; Balin et al., 1986; Broadwell and Balin, 1985; Itaya, 1987; Shipley, 1985; Stewart, 1985; Thorne et al., 1995) have shown that WGA conjugated to horseradish peroxidase (WGA-HRP) is internalized by olfactory receptor cells following binding to surface receptors and transported to the olfactory bulb. Uptake and transport occurred primarily ipsilaterally following intranasal application (Baker and Spencer, 1986; Shipley, 1985). Both anterograde and retrograde transneuronal transport have been observed from the olfactory bulb. Labeled primary olfactory axons containing WGA-HRP reaction product were observed in the nerve and glomerular layers of the main olfactory bulb within 6 hours of intranasal irrigation (Broadwell and Balin, 1985; Itaya, 1987; Shipley, 1985). At longer survival times (4–7 days) labeled neurons and terminals have been reported in a number of brain regions known to either receive input from or send axons to the olfactory bulb. Anterograde and retrograde label were observed in the anterior olfactory nucleus (Itaya, 1987; Shipley, 1985). The piriform and entorhinal cortices, as well as the olfactory tubercle, also contained significant label (Baker and Spencer, 1986; Itaya, 1987; Shipley, 1985). Brain regions that send centrifugal afferent innervation primarily to the glomerular region of the olfactory bulb also contained retrogradely labeled cells. These regions included the anterior olfactory nucleus, the horizontal limb of the diagonal band, the raphe nucleus, and, in some studies, the locus ceruleus (Baker and Spencer, 1986; Shipley, 1985). These data indicate the extent of transport that occurs from the olfactory epithelium to those brain regions with connections to the olfactory bulb. However, the significance, generality, and specificity of lectin transport remain to be delineated. C.

Viruses

From a historical perspective, a neural route of viral invasion of the CNS was postulated as a mechanism for the development of rabies as early as the eighteenth century and demonstrated in the late nineteenth century (see Stroop, 1995, for review and references). The nasal route as a site of viral entry to the CNS was established during the 1920s and 1930s (Clark, 1929; Hurst, 1936). Poliomyelitis virus was subsequently shown to reach the olfactory bulbs through the olfactory neurons following intranasal application in primates (Bodian and Howe, 1941a,b). Reports that monkeys could be protected against intranasal inoculation with poliomyelitis virus by nasal lavage with solutions of alum, picric acid, or zinc sulfate (Armstrong and Harrison, 1935; Schultz and Gebhardt, 1936) led to prophylactic treatment of children with nasal

Exogenous Agent Uptake

zinc sulfate during poliomyelitis epidemics (Peet et al., 1937; Schultz and Gebhardt, 1937; Tisdall et al., 1937). An undesirable consequence of these treatments was longlasting anosmia in many individuals (Tisdall et al., 1938), presumably produced by irreversible destruction of the olfactory sensory epithelium. Subsequently, transport to the central nervous system has been reported for several types of viruses following intranasal inoculation, including herpes simplex (Esiri and Tomlinson, 1984; McLean et al., 1989; Stroop et al., 1984), murine coronavirus (Barnett et al., 1993; Perlman et al., 1988, 1989), Borna disease virus (Morales et al., 1988), Nile virus (Nir et al., 1965), Venezuelan equine encephalitis virus (Charles et al., 1995), vesicular stomatitis virus (Huneycutt et al., 1994), Semliki virus (Oliver and Fazakerley, 1998), Sendai virus (Mori et al., 1995), and rabies virus (Astic et al., 1993; Lafay et al., 1991). Olfactory bulb ablation prevented the spread of the neurotropic coronavirus mouse hepatitis virus into the brain (Perlman et al., 1990). Two neurotropic viruses, herpes simplex virus (HSV) type 1 and mouse hepatitis virus, spread along different neural pathways following intranasal inoculation, suggesting that uptake in specific neurotransmitter systems influences viral distribution in the CNS (Barnett et al., 1993). Selective lesions of neural pathways also contributed to spatial learning deficits (McLean et al., 1993). Learning and behavioral deficits have recently been associated with HSV-1 brain infection, presumably by the olfactory nerve route (Becker, 1995). Olfactory labeling was observed in several instances where the viruses were applied at nonolfactory sites, including intraperitoneal (Monath et al., 1983) and subcutaneous routes (Charles et al., 1995). Virus titers were observed first in the olfactory epithelium (4 days) and then in the olfactory bulb (5 days) followed by the rest of the brain. Olfactory labeling was observed following corneal inoculation and intradermal application in the mouse (Stroop et al., 1984). In the latter instances the virus may have reached the olfactory epithelium through tears entering the naris or nasopharynx and then entering the nasal receptor epithelium. Applying sensitive immunohistochemical techniques, herpes simplex virus produced Golgi-like, transneuronal labeling in the CNS (Blessing et al., 1994; McLean et al., 1989), and the authors suggested that virus injection may be useful for tracing neuronal pathways. Many recent reports demonstrate that pseudorabies virus also produced significant transneuronal transport and can be used to label CNS pathways (Graf et al., 2002; Sams et al., 1995; Strack and Loewy, 1990; Ugolini, 1995). These and many other studies demonstrate that the olfactory system may serve as an efficient route of entry of viruses into the brain.

557

D.

Dyes

1.

Procion and Lucifer Dyes

Several dyes, including procion, lucifer, and Evans blue, were internalized into olfactory epithelial cells. In those species tested, procion and lucifer dyes specifically labeled olfactory receptor neurons. Two procion dyes, brilliant yellow M4RAN and yellow M4R, labeled the receptor neurons of the catfish, Ictalurus nebulosus (Holl, 1980). Similar intravital staining was observed in olfactory receptor neurons of the eel, Anguilla anguilla (Holl, 1981). In both the lamprey, Entosphenus, and the grass frog, Rana, procion yellow MX4R and lucifer yellow VS exhibited internalization specifically by olfactory receptor neurons in contrast to either supporting cells or the glandular components of the epithelium (Suzuki, 1984). Since the stained receptor neurons retained their dye through dissociation procedures, Suzuki (1984) suggested that these fluorescent dyes may be useful cell markers for identifying viable receptor cells in dissociated epithelial preparations. Interestingly, not all dyes showed specific uptake. Lucifer yellow CH stained the receptor epithelium nonspecifically, and procion red H3B at high concentrations resulted in dye aggregations on the surface of the olfactory epithelium. The basis for the difference in labeling between the dyes was thought to reflect binding to specific membranous molecules. 2.

Evans Blue

Inhalation of a complex of Evans blue dye with albumin resulted in strong red fluorescence within numerous cells of the olfactory epithelium of mice (Kristensson and Olsson, 1971). After 24 hours, the filia olfactoria of the mice exhibited fluorescent label. Although labeling was found in the olfactory bulbs, especially in young mice, there was no evidence of transneuronal transport to secondary neurons such as mitral cells. Evidence for label within the submucosal area and leptomeninges surrounding the olfactory bulb suggested that some of the dye could reach the CNS by other routes in addition to transport through the olfactory nerves (Kristensson and Olsson, 1971). 3.

Trypan Blue

Trypan blue dye was studied in both the brown bullhead, Ictalurus natalis (Holl, 1965), and the mouse (Seki, 1941). The investigations were undertaken to demonstrate the time course and conditions for specific uptake of the dye into olfactory receptor cells and not into other cell types in the olfactory epithelium. Transneuronal transport was not investigated.

558

E.

Baker and Genter

Amino Acids

1. Leucine The transport of leucine in the olfactory system has been studied by a number of investigators (for review, see Weiss and Buchner, 1988). For the most part, uptake, metabolism, and transport of the amino acid were used to characterize the kinetics of protein movement in normal and regenerating nerve fibers. The olfactory system was an ideal model since in many fish species the nerve is long and regenerates readily. One of the pioneering studies used histochemical techniques to demonstrate axoplasmic transport of labeled proteins following intranasal application of tritiated leucine in the toad, Bufo americanus (Weiss and Holland, 1967). Gross and Beidler (1973) studied fast axonal transport in C-fibers of the garfish, Lepisosteus osseus. Experiments performed in the pike, Esox lucius, demonstrated similar dynamics of fast axonal transport (Gross and Kreutzberg, 1978; Weiss et al., 1978). The long olfactory nerve of this species was used to study temperature dependence of axonal transport, especially slow axonal transport in normal and regenerating olfactory nerve (Cancalon, 1979a, b, 1988; Cancalon et al., 1988). The relative uniformity of transport in both the antero- and retrograde direction was shown in the pike olfactory nerve (Weiss and Buchner, 1988). Transport of tritiated leucine was also utilized to analyze the projections of olfactory receptor neurons in the adult rabbit (Land and Shepherd, 1974). In all studies the majority of the leucine was incorporated into amino acids following a short delay. Transport was monitored either autoradiographically or biochemically within dissected nerve segments. Although both slow (Cancalon, 1979 a,b) and fast axonal transport (Gross and Beidler, 1973, 1975) were demonstrable using these techniques, transneuronal transport was not observed under the experimental conditions utilized (Land and Shepherd, 1974; Weiss and Holland, 1967). 2. Alanine In the olfactory system the dipeptide carnosine (alanyl-L-histidine) may be either a neurotransmitter or a neuromodulator of the primary olfactory neurons. Therefore, -alanine presented at the mouse external naris was incorporated specifically into carnosine (Margolis and Grillo, 1977). Subsequent experiments in hamsters demonstrated that the radioactivity associated with the -alanine was converted to carnosine ( 82% of the radioactivity was in the carnosine fraction at 24 hr) and transported to the olfactory bulb (Burd et al., 1982). The rate of appearance in the olfactory bulb was consistent with movement of the -alanine by the fast axonal transport system.

Tritiated -alanine exhibited a pattern of transport consistent with a less specific internalization in the olfactory epithelium and incorporation into a number of proteins after intranasal administration. Both sensory and nonsensory cells and the lamina propria were labeled. Peak labeling in the olfactory bulb was less intense and delayed as compared to -alanine, suggesting that the protein was carried by slow axonal transport. Trans-neuronal transport was not observed for either - or -alanine (Burd et al., 1982). 3.

Taurine

Taurine accumulated specifically in the olfactory bulb after intravenous injection (Lindquist et al., 1983). The ability of the olfactory system to concentrate this amino acid was even more pronounced following intranasal irrigation. An intra-axonal transport mechanism, as opposed to a perineural one, was suggested by the restricted accumulation in the bulb ipsilateral to the application. F.

Drugs

Valproic acid (Hoeppner, 1990), cocaine (Brittebo, 1988), and a number of other drugs (Dahl and Hadley, 1991) were concentrated in the olfactory epithelium following intravenous administration. The relative enrichment of cocaine in the epithelium was maintained for at least 24 hours. The mechanism for the concentration of these drugs in the epithelium is unknown. The presence of D2 dopamine receptors in the receptor epithelium (Guthrie et al., 1991; Shipley et al., 1991) suggests that cocaine, a dopamine uptake blocker, binds to a specific receptor site in the epithelium. The presence of numerous drug-metabolizing enzymes (Dahl and Hadley, 1991) (see also Chapter 3) also may contribute to the binding and metabolism of drugs in the olfactory epithelium. Transport has not been studied following intranasal administration of the drugs. G.

Miscellaneous Substances

1.

Horseradish Peroxidase

The transport of HRP differed from that observed for the conjugate, WGA-HRP. HRP, like WGA-HRP, was internalized by receptor cells and labeled the olfactory glomeruli (Balin et al., 1986; Kristensson and Olsson, 1971; Meredith and O’Connell, 1988; Stewart, 1985). However, transneuronal transport was not observed. Movement also occurred through the intercellular junctions to label the leptomeninges (Balin et al., 1986). HRP

Exogenous Agent Uptake

uptake was not observed in the accessory olfactory system unless a large dose of epinephrine was administered to activate the vomeronasal organ pumping mechanism (Meredith and O’Connell, 1988). Mechanistic considerations discussed below may underlie the differences between WGA-HRP and HRP. 2. Immunoglobulins Only one report exists documenting the uptake of immunoglobulins (Baker and Maruniak, 1990). The study demonstrated that mouse olfactory receptor neurons internalized IgG molecules from a number of species. Twentyfour hours after intranasal application, receptor neurons, microvillar cells, and nerve bundles in the olfactory mucosa contained IgG, but transport was not observed into the olfactory bulbs.

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there is partial to nearly full recovery of olfactory mucosa that has undergone chemically induced degeneration (see chemicals labeled “OMD,” referring to those causing olfactory mucosal degeneration in Table 2). However, a toxicant that causes persistent metaplasia of the olfactory mucosa has recently been described (Bahrami et al., 2000). A number of drugs (e.g., phenacetin), industrial and agricultural chemicals (e.g., p-cresidine, alachlor), and environmental pollutants (e.g., NNK) are associated with the development of nasal cancer in rodents (Table 2). It is interesting to note that humans are exposed to low levels of many of these, or related, agents every day, but none has been linked definitively to human nasal cancer; the most widely recognized risk factors for human nasal cancer are exposure to wood dust or nickel, both via the inhalation route (Barceloux, 1999; Hayes et al., 1986).

3. Solvents

III.

Uptake and metabolism were investigated for several solvents, including benzene, toluene, xylene, and styrene (Ghantous et al., 1990). All the aromatic hydrocarbons were found in the olfactory epithelium, but only toluene, xylene, and styrene were found in the olfactory bulb. Transport of the three solvents in olfactory receptor cells was thought to require conversion to either their aromatic acids or their conjugates. In support of this hypothesis, benzene does not exhibit biotransformation to aromatic acids and was not transported (Ghantous et al., 1990). The cytochrome P-450 enzymes necessary for these biotransformations are known to be present in the olfactory epithelium (Dahl, 1988; Dahl and Hadley, 1991).

A.

H. Olfactory Toxicants Delivered via the Bloodstream Multiple xenobiotics that are systemically administered (i.e., by noninhalation routes of exposure) have toxic effects on the olfactory mucosa of experimental animals (Table 2). For example, oral administration of the analgesic acetaminophen or the antihyperthyroid drugs methimazole or carbimazole to rodents causes degeneration of the olfactory mucosa (Genter, 1998; Genter et al., 1995, 1998; Jeffery and Haschek, 1988). The antihyperthyroid drugs have been associated with impaired olfaction in humans (Schiffman, 1983). The herbicide dichlobenil (2,6-dichlorobenzonitrile) is particularly interesting from the point of view that intravenous (i.v.), intraperitoneal (i.p.), or dermal exposure results in olfactory mucosal degeneration, with dermal exposure representing an occupationally relevant route of exposure (Bergman et al., 2002; Brandt et al., 1990; Deamer et al., 1994). In general,

MECHANISMS OF TRANSPORT Uptake

The preceding discussion demonstrates that olfactory receptor cells can internalize many types of substances. The issue is how and why these cells should internalize a wide range of xenobiotics. The answer to both these questions may be the same. Since the olfactory mucosa comes in contact with many substances in inspired air, a means of either elimination or detoxification has likely evolved as a protective mechanism. In fact, the olfactory epithelium contains high concentrations of a wide variety of xenobiotic-metabolizing enzymes, even when compared to levels in the liver (Dahl, 1988; Dahl and Hadley, 1991; ThorntonManning and Dahl, 1997). A number of enzymes have been found in the olfactory epithelium, including numerous isozymes of P450 monooxygenases, aldehyde dehydrogenases, alcohol dehydrogenase, carboxyesterases, epoxide hydrolases, UDP-glucuronyl transferase, glutathione S-transferase, and rhodanese (Dahl and Hadley, 1991) (see also Chapter 3). For some of these enzymes, isozymes have been found that are specific to the olfactory mucosa (Dahl, 1989; Jones and Reed, 1989; Lazard et al., 1991; Nef et al., 1989; Zupko et al., 1991). Thus, uptake into receptor, supporting, and glandular cells of xenobiotics, including odorants, may serve a detoxification as well as a clearance function. These enzymes also may play a role in defining the characteristic odor of a substance through the production of active metabolites, thereby creating new odorants (Dahl, 1988; Dahl and Hadley, 1983; Getchell et al., 1984). The specificity of the uptake mechanism has not been established. For WGA-HRP, receptor-mediated processes have been suggested, since the conjugate, which is known

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to bind to surface membrane oligosaccharides, is found associated with vesicles (Baker and Spencer, 1986; Broadwell and Balin, 1985). Unconjugated HRP, on the other hand, is thought to be internalized via bulk endocytosis (Broadwell and Balin, 1985; Kristensson and Olsson, 1971). Surprisingly, one study reported that some preparations of WGA-HRP were not transneuronally transported, suggesting that the method of HRP conjugation may alter the ability of WGA to bind with specific membrane receptors (Russell et al., 1991). The association of gold particles with vesicles suggested that uptake of gold occurred subsequent to the formation of vesicles by pinocytosis (De Lorenzo, 1970). Demonstration of a vesicular association of several intranasally applied tracers indicated that the olfactory epithelium possesses a well-developed system of endocytic vesicles, perhaps as a result of a rapid rate of membrane turnover (Bannister and Dodson, 1992). These authors suggested that materials may be trapped during normal membrane processing. The internalization of materials into the olfactory receptor cells thus may take place by either receptor- or non–receptor-mediated processes. In the former case, internalization may occur at low xenobiotic concentrations, and in the latter, high concentrations may be necessary. Some evidence exists for the presence of specific cell surface receptors in the olfactory system. A superfamily of putative odorant receptors has been cloned (Buck and Axel, 1991). The large number of these receptors indicates the presence of cell type–specific surface molecules that interact with odorants (see Chapter 4). The binding of odorants to the receptor proteins may result in their internalization. Specific transport of odorant receptor mRNA to the olfactory bulb was used to demonstrate that cells expressing a specific odorant receptor project to a single glomerulus (Ressler et al., 1993, 1994; Sullivan et al., 1995; Vassar et al., 1994). Olfactory marker protein mRNA shows similar transport to the glomerular layer but no transneuronal transport (Wensley et al., 1995). Interestingly, transneuronal transport was demonstrated for barley lectin (a close relative of WGA) synthesized in olfactory receptor cells of a transgenic mouse expressing the lectin under control of the olfactory marker protein gene promoter (Horowitz et al., 1999). The function of the intra-axonal mRNA is unknown, but the ability of olfactory receptor neurons to transport numerous other molecules may be a consequence of this endogenous transport capacity. Also, the D2 dopamine receptors reported in olfactory receptor neurons may play a role in the uptake of molecules that bind to this receptor (Guthrie et al., 1991; Mansour et al., 1990; Shipley et al., 1991). Glycoproteins, as indicated by lectin binding, also have been demonstrated on olfactory receptor cells using

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both in vivo and in vitro techniques (Allen and Akeson, 1985; Barber, 1989; Foster et al., 1991; Key and Akeson, 1990; Key and Giorgi, 1986 a,b; Lundh et al., 1989; Mori et al., 1985; Polak et al., 1989; Shirley et al., 1983). Uptake of anti IgG antibodies into receptor cells has been reported (Baker and Maruniak, 1990; Baker and Spencer, 1986). However, immunoreactivity for constituents of the immune system was not found in receptor cells, but primarily in Bowman’s glands, the mucociliary complex, lymphocytes clustered near the basement membrane and scattered in the lamina propria, as well as in mast cells of the human olfactory mucosa (Mellert et al., 1992). A similar distribution of the secretory immune system components was observed in salamanders and rats (Getchell and Getchell, 1991). Monoclonal antibodies also recognize different cell types within the olfactory mucosa (Hempstead and Morgan, 1985), suggesting differences in cell surface constituents that might direct differential binding to olfactory receptor as opposed to either supporting or glandular cells. Finally, odorant-binding proteins synthesized by mucus-secreting glands of the olfactory and respiratory mucosa have been demonstrated (Baldaccini et al., 1986; Dal Monte et al., 1991; Pevsner et al., 1986). The binding proteins may be necessary for either the transport of odorants to olfactory receptors or their clearance after signal transduction. The odorant specificity of these proteins suggests that a similar binding specificity on the receptor cells may underlie ligand (either odorant or other xenobiotic) uptake into receptor, supporting, and glandular cells. Transfer between these compartments also cannot be ruled out, especially since many of the degradative enzymes described above are found in glandular and supporting cells and not receptor cells (Dahl and Hadley, 1991; Thornton-Manning and Dahl, 1997). B.

Transneuronal Transport

Once internalized, transport in the olfactory receptor cells can occur by either the slow or rapid transport systems characteristic of neuronal systems (Weiss and Buchner, 1988). The distinction between molecules transported only to the olfactory bulb and those that are transneuronally transported may lie in the nature of their internalization and thus the organelles with which the molecules are associated. For example, WGA-HRP, which is internalized by receptor-mediated endocytosis, is transported intra-axonally in tubulovesicular profiles (Baker and Spencer, 1986) and transneuronally following processing in the transmost Golgi saccule (Baker and Spencer, 1986; Broadwell and Balin, 1985). The latter compartment processes vesicles destined for axonal terminals (Broadwell and Balin, 1985).

Exogenous Agent Uptake

Gold is vesicle-associated and is transneuronally transported (De Lorenzo, 1970). Viruses are transported in axonal vacuoles within receptor cells and appear to move transsynapically to invade other brain regions synaptically connected to the olfactory bulb (Lavi et al., 1988; Monath et al., 1983). The relative lack of glial labeling for WGAHRP and some viruses (Baker and Spencer, 1986; Lavi et al., 1988) indicates limited release into the extracellular space and accounts for the restricted transfer to olfactoryrelated brain regions. In contrast, HRP, which does not exhibit transneuronal transport, is taken up by bulk endocytosis and is not processed through the Golgi saccule. Leucine, which also does not appear to be transported transneuronally (Land and Shepherd, 1974; Weiss and Holland, 1967), is primarily processed into cellular protein components (Weiss and Buchner, 1988). Taken together these data suggest that transneuronal transport requires receptor-mediated uptake into the olfactory sensory cells, followed by vesicular transport of unmodified materials through the axon, with subsequent release and uptake in association with synaptic specializations.

C.

Specificity of CNS Transport

As mentioned above, transport from the olfactory bulb to other brain regions occurs along specific pathways. The ability of centrifugal afferents to support specific transport is suggested by data demonstrating selectivity of retrograde axonal transport of radioactive transmitters from the olfactory bulb to their nuclei of origin (Araneda et al., 1983; Bonnet-Font and Bobillier, 1990; Watanabe and Kawana, 1984). Intrabulbar application of radioactive serotonin or norepinephrine results in specific labeling, respectively, of the dorsal raphe nucleus and the locus coeruleus (Araneda et al., 1983; Bonnet-Font and Bobillier, 1990). Similarly, the receptor-mediated retrograde transfer of nerve growth factor from the olfactory bulb to forebrain cholinergic nuclei indicates the ability of these neurons to transport exogenously applied substances (Altar and Bakhit, 1991).

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route of administration can be particularly useful for drugs that are readily degraded in the gastrointestinal tract (e.g. small, readily digestible peptides) or drugs that are extensively inactivated by liver metabolism following oral ingestion. This is not to say that the nasal respiratory and olfactory epithelia are metabolically inert; in fact, there is a vast body of literature documenting the presence of multiple Phase I and Phase II metabolic enzymes in rodent, dog, and human nasal mucosa (Dahl and Hadley, 1991; Gervasi et al., 1991; Sarkar, 1992; Thornton-Manning and Dahl, 1997). Of the three major routes of entry of drugs from the nasal cavity and into the brain or systemic circulation, the first, transneuronal transport, has been dealt with to this point in this chapter. Another putative mechanism for transport of agents from the nasal cavity and into the CNS has been termed the olfactory epithelial pathway (Mathison et al., 1998) or paracellular transport (McMartin et al., 1987). Regardless of the designation, the concept is that molecules access the CNS from the nasal cavity via supporting cells, cell-to-cell junctions, and/or spaces between cells. A great deal of research effort has gone into our understanding of the factors that are important in this process. For example, molecular weight, lipophilicity, and ionization state/pH of the agent are all variables that appear to contribute to the ability of agents to access the central nervous system from the nasal cavity (McMartin et al., 1987; Sakane et al., 1991a, 1994; Shimoda et al., 1995). The concentration of sulfonamides in the cerebrospinal fluid (CSF) of the rat resulting from intranasal administration was demonstrated to increase with the lipophilicity of the sulfonamide derivative (Sakane et al., 1991a). In contrast, lipophilicity did not enhance the absorption of a series of peptides from the nasal cavity (Donnelly et al., 1997; Sakane et al., 1991). Using fluorescein isothiocyanate–labeled dextran with molecular weights ranging from 4.4 to 40 kDa, it was determined that drugs with a molecular weight of 20 kDa or less could reasonably be expected to be transported from the nasal cavity to the CSF (Sakane et al., 1995). B.

IV. THE NASAL CAVITY AS A ROUTE OF ADMINISTRATION FOR THERAPEUTIC DRUGS A.

Background

There is currently considerable interest in the concept of administration of therapeutic drugs via the nasal cavity for delivery into the brain or into the systemic circulation. This

Enhancement of Delivery of Intranasally Administered Drugs

Transport of therapeutic drugs across the nasal epithelia and into the blood is also a desired outcome, as is evidenced by the increasing number of drugs being developed for this route of administration. Considerable effort has gone into identifying agents that can enhance the absorption of drugs from the nasal cavity without causing clinically relevant damage to the mucosal surface of the nasal cavity. The putative absorption enhancers, sodium

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tauro-24,25-dihydrofusidate (STDHF) and dimethyl-cyclodextran (DM CD), exhibited some epithelial toxicity, whereas ethyleneamine diamine tetraacetic acid (EDTA) was not associated with enhanced absorption or epithelial toxicity (Donnelly et al., 1997). Methylated cyclodextrins were less toxic to nasal epithelia than sodium glycocholate, STDHF, laureth-9 (a detergent) and L--phosphatidylcholine (Marttin et al., 1998). Free amine chitosans and soluble chitosan salts were evaluated for their efficacy and safety as nasal enhancers of peptide absorption and were found to be comparable in their action, if not more potent, than cyclodextrins (Tengamnuay et al., 2000). Cyclodextrins are believed to enhance absorption of intranasally administered drugs by transiently opening tight junctions between nasal epithelial cells (Marttin et al., 1998). High concentrations of certain cyclodextrins are associated with ciliary damage, an endpoint also associated with benzalkonium chloride, which has been used as a preservative in many nasal formulations (Bernstein, 2000; Uchenna Agu et al., 2000). C.

Examples of Intranasally Administered Therapeutic Drugs

Intranasally administered drugs can be roughly divided into three main categories: (1) drugs administered via the nasal cavity for central nervous system effects; (2) drugs administered via the nasal cavity for systemic effects; and (3) drugs administered via the nasal cavity for nasal and/or respiratory tract effects. Although the development of drugs for treatment of respiratory tract symptoms is undoubtedly the strategy most intuitively associated with intranasal drug delivery, it will not be dealt with in this chapter because of our interest here in transport. The development of drugs administered via the nasal cavity for central nervous system and/or systemic effects has exploded in recent years (Table 3). 1.

Drugs Administered via the Nasal Cavity for Central Nervous System Effects

a. Antimigraine Medicines. Therapeutic drugs formulated for intranasal administration for CNS effects include migraine headache treatments, sedatives/antiepileptics, neuroprotective agents, and drugs to suppress cravings for smoking and eating. While some efforts are still largely in the experimental stages, migraine patients using medications administered via the intranasal route have realized significant relief. Due to the high incidence of nausea, vomiting, and visual disturbances during migraine attacks, intranasal administration of migraine medicines is proving to be superior to pills or self-injections. While ergotamine derivatives have been extensively used in the past

(Gallagher, 1996; Lipton, 1997; Marttin et al., 1997), newer generation drugs which act as 5-HT1D receptor agonists (e.g., sumatriptan or Imitrex®) are also proving to be potent, highly specific antimigraine agents when administered via the intranasal route. The bioavailability of intranasally administered sumatriptan in dogs was equivalent to an oral dose (Barrow et al., 1997). Similarly, BMS-181885, another 5-HT1-like receptor agonist, was demonstrated to be quantitatively absorbed from the nasal cavity of cynomolgus monkeys, with the highest observed plasma concentration attained in 30 minutes (Srinivas et al., 1998). There has also been smaller-scale use of intranasal lidocaine or butorphanol as abortive treatments for migraines, as well as a case study reporting the effectiveness of intranasal capsaicin in migraine relief (Levy, 1995; Melanson et al., 1997; Mills and Scoggin, 1997; Sachs, 1996). b. Sedatives and Analgesics. The intranasal route is also a practical alternative to injections/intravenous administration of sedatives and medicines for chronic pain. Midazolam is administered in several medical situations, including induction of anxiolysis and sedation, particularly for endoscopic and dental procedures. Midazolam absorption from the nasal mucosa was nearly complete in a study involving adult surgical patients who were administered midazolam in small intranasal doses to preclude swallowing. This study was important in that it revised previous observations that the bioavailability following intranasal administration was approximately 55%, thus reducing the risk of accidental overdoses (Björkman et al., 1997). Nasal midazolam also appeared to be safe and effective in treatment of epileptic seizures in children (Jeannet et al., 1999). Intranasally administered oxycodone and oxymorphone are effectively absorbed from the nasal cavity for treatment of chronic pain (Hussain and Aungst, 1997; Takala et al., 1997). Experiments to investigate the feasibility of administering acetyl salicylic acid by the nasal route have also been conducted in rat with promising results (Hussain et al., 1992). c. Antivirals and Antibiotics. Drugs with activity toward the HIV, namely zidovudine (AZT; 3ⴕ-azido-2ⴕ, 3ⴕ-dideoxythymidine) and D4T (2ⴕ,3ⴕ-didehydro3ⴕ-deoxythymidine), are absorbed from the rat nasal epithelia and transported into the CSF (Seki et al., 1994; Yajima et al., 1998). Under normal administration regimens, these drugs cannot cross the blood-brain barrier and therefore are ineffective against acquired immunodeficiency syndrome (AIDS) dementia when administered orally or by injection. The antibiotic cephalexin appeared rapidly in the CSF following intranasal administration (Sakane et al., 1991b).

Exogenous Agent Uptake

d. Treatment/Prevention of Neurodegenerative Diseases. Vasoactive intestinal peptide (VIP) is widely distributed in regions of the brain associated with learning and memory. VIP antagonists are associated with learning impairments in experimental systems, and in vitro studies have demonstrated that VIP exerts neuroprotective effects, including protection of cortical neurons from the cytotoxic effects of -amyloid peptide. Intranasal administration of a lipophilic analog of VIP ([St-Nle17]VIP) to rats has been shown to deliver unmetabolized ([St-Nle17]VIP to the brain and to prevent impairments in spatial learning and memory associated with cholinergic blockade in rats (Gozes et al., 1996, 1997). Similarly, dextromethorphan and neostigmine were found to be well absorbed from the rat and human nasal cavities, with the latter more effective in relieving symptoms of myasthenia gravis as a nasal spray than when administered intravenously (Char et al., 1992; Di Costanzo et al., 1993). Administration of vasopressin to humans to increase brain activity and improve memory has resulted in reports of marginal improvement (Perras et al., 1997; Pietrowsky et al., 1996). e. Smoking Cessation and Appetite Suppression. Nicotine nasal sprays have been evaluated as agents to assist individuals in smoking cessation. It is possible that the sprays are efficacious due to the direct delivery of nicotine to the brain as well as maintenance of blood nicotine levels such that the majority of symptoms of withdrawal are avoided. While there have been reports that the irritation associated with nicotine nasal sprays precluded their use to attain therapeutic blood levels (Gourlay and Benowitz, 1997), another study showed that successful “quitters” who most frequently used nicotine nasal sprays as part of a smoking cessation program had higher blood nicotine and cotinine levels than those using the sprays occasionally or not at all (Jones et al., 1998). Experimentally, cholecystokinin (CCK) agonists show promise as appetite suppressants (Pierson et al., 1997) 2. Drugs Administered via the Nasal Cavity for Systemic Effects There is a similarly long and varied list of therapeutic drugs which are designed for intranasal delivery to achieve a desired systemic effect (see recent reviews by Jones et al., 1997; Mathison et al., 1998). a. Peptides. Delivery of peptides such as insulin and calcitonin via the nasal route would in theory improve their bioavailability over oral administration. There have been mixed results with insulin administration via the nasal route (Hilsted et al., 1995), possibly because of peptidases in nasal secretions that have high activity toward insulin peptides. Methods to encapsulate insulin peptides appear

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promising in the effort to improve systemic delivery of insulin following intranasal administration (FernandezUrrusuno et al., 1999). Intranasal calcitonin is a potent agent for reducing bone resorption (Silverman, 1997). b. Hormone Analog. Buserelin and nafarelin are being used as nasal sprays for treatment of endometriosis, prostate cancer, and low sperm count and show favorable results as contraceptives (Bergquist et al., 1979). Oxytocin nasal sprays are associated with high blood levels of the drug and enhanced lactation (Landgraf, 1985; Ruis et al., 1981). c. Vaccines. The intranasal route is also becoming recognized as a route of immunization against agents such as influenza viruses, Neisseria meningitides, diphtheria, and tetanus (Liu, 1998; Maassab and DeBorde, 1985). d. Others. Vitamin B12 derivatives are well absorbed following intranasal administration, as are drugs for treatment of motion sickness and bedwetting (Butler et al., 1998; Kallas et al., 1999; Putcha et al., 1996; Ramanathan et al., 1998; Slot et al., 1997; Swain, 1995).

V.

CONSEQUENCES OF TRANSPORT

One of the early changes associated with several neurodegenerative disorders, including Parkinson’s and Alzheimer’s diseases, is a substantial deficit in olfactory function of a discriminatory nature (Doty et al., 1987, 1988, 1991) (see Chapters 23 and 24). These data, in conjunction with recent findings of pathological changes in the olfactory epithelium (Tabaton et al., 1991; Talamo et al., 1989) and olfactory bulb (Esiri and Wilcock, 1984; Ohm and Braak, 1987), as well as brain areas with close synaptic relationships to the olfactory bulb (FerreyaMoyano and Barragan, 1989; Saper et al., 1987), formed the basis for the hypothesis that the olfactory system may be a route of entry for either a toxin or a virus that is important to the etiology of these diseases. For most substances, the evidence is at best circumstantial. Several reviews have outlined the data, suggesting that environmental aluminum could contribute to the loss of olfactory functioning and the ensuing disease process in Alzheimer’s disease. The findings of high levels of aluminum in plaques and the ability to produce lesions following inhalation of aluminum support this hypothesis. The data could reflect, however, other abnormalities in Alzheimer’s disease, such as changes in the blood-brain barrier that permit accumulation of this metal in neurons (Crapper McLachlan, 1986; Roberts, 1986). Recent studies have

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provided anatomical demonstration of transport to the CNS of intranasally applied manganese (Tjälve and Henriksson, 1999) as well as epidemiological evidence for a Parkinson’slike syndrome following environmental exposure to the metal (Gorell et al., 1999). While there is no definitive evidence that the olfactory system serves as a route of entry for metals and other toxins in humans, there is strong support for an environmental role in sporadic Parkinson’s disease (Betarbet et al., 2001; Engel et al. 2001; Langston, 1998; Tanner and Langston, 1990). Currently, there is no evidence for viral involvement in neurodegenerative disorders such as Alzheimer’s or Parkinson’s diseases. To postulate that the olfactory system serves as a route of entry for a virus in these diseases is only conjectural. The fact that in some degenerative disorders the affected neuronal populations appear to be restricted to interconnected pathways has been clearly established (Saper et al., 1987), and transneuronal transport of viruses does occur along some of these routes especially in the olfactory system. Trophic substances also were shown to be transported along these same pathways. In the cholinergic system, for example, which innervates the olfactory bulb, nerve growth factor is retrogradely transported (Altar and Bakhit, 1991). Thus, an abnormality in transport of a trophic factor, either inborn or toxin (viral?)-induced, could result in neuronal degeneration. Alternations in peripheral afferent innervation of the olfactory bulb, both denervation and odorant deprivation, produce profound anatomical and biochemical consequences. Both neonates and adults show reductions in bulb size (Maruniak et al., 1989 a,b; Meisami, 1976), granule cell spine densities (Benson et al., 1984), number of granule cells (Frazier and Brunjes, 1988), and neurotransmitter expression, including substance P and dopamine, in juxtaglomerular cells (Baker et al., 1983; Kream et al., 1984). The latter changes occur without apparent cell death, indicating the presence of phenotypic plasticity (Baker et al., 1984, 1993; Cho et al., 1996; Stone et al., 1990, 1991) (See also Chapter 29). These data indicate the importance of afferent innervation as a trophic regulator of bulb function and suggest that either exogenous agents or viral internalization may alter this trophic balance and result in the loss of olfactory function with eventual consequences in synaptically interconnected brain regions.

VI.

CONCLUSIONS

There is no question that olfactory receptor cells can internalize and transport a variety of materials, both inorganic and organic, including viruses. However, a number of issues remain to be clarified. First, how general is the phe-

nomenon? What are the characteristics of cell surface receptors that lead to binding and transport of specific xenobiotics and viruses? Is internalization concentrationdependent, and are the concentrations utilized in experimental application overwhelming mechanisms that normally remove odorants and other substances? Is there age-dependence? Recent data showed that viral spread following inhalation exposure was influenced by neuronal maturity (Oliver and Fazakerley, 1998) and that in the olfactory mucosal damage induced by 3,3ⴕ-iminodipropionitrile is age-dependent (Genter and Ali, 1998). What happens to the receptor cells following uptake? There is experimental evidence to suggest that excessive uptake would lead to neuronal degeneration and replacement since exposure to normal laboratory environments produced greater turnover of receptor cells than exposure of animals to purified air such as in a laminar flow hood (Hinds et al., 1984). In contrast, intranasal WGA-HRP resulted in a reversible thinning of the olfactory epithelium produced primarily as a result of apoptotic receptor cell loss (Moon and Baker, 1998, 2002) (see also Chapter 5). What is the role of the immune system in protecting the epithelium and brain from viruses and exogenous agents? Are the drugmetabolizing enzymes present in sufficient concentrations to protect the epithelium against toxin exposure, and, in fact, is this the role of these enzymes? We appear to survive exposure to many substances without disease. However, the human olfactory epithelium appears to degenerate with age, so the question remains as to how effective the epithelium is at protecting itself and the brain from whatever enters the nares. From a positive perspective, recent experimental and clinical data show that nasal application may provide an ideal route for delivery of drugs to the CNS. ACKNOWLEDGMENTS Supported by in part grant number ES08799 from NIEHS to MBG and grant number AG09686 from NIA to HB. REFERENCES Aggerbeck, H., Gizurarson, S., Wantzin, J., Heron, I. Intranasal booster vaccination against diphtheria and tetanus in man. Vaccine 1997; 15:307–316. Allen, W. K., Akeson, R. Identification of a cell surface glycoprotein family of olfactory receptor neurons with a monoclonal antibody. J. Neurosci 1985; 5:284–296. Altar, C. A., Bakhit, C. Receptor-mediated transport of human recombinant nerve growth factor from olfactory bulb to forebrain cholinergic nuclei. Brain Res. 1991; 541:82–88. Apfelbach, R., Engelhart, A., Behnisch, P., Hagenmaier, H. The olfactory system as a portal of entry for airborne

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27 Influence of Environmental Toxicants on Olfactory Function Lloyd Hastings University of Pennsylvania, Philadelphia, Pennsylvania, U.S.A.

Marian L. Miller University of Cincinnati College of Medicine, Cincinnati, Ohio, U.S.A.

I.

INTRODUCTION

In this chapter, we review the literature noting diminution or loss of the sense of smell from occupational exposure to airborne chemicals. Additionally, we provide an overview of the mucosal pathophysiology that occurs after exposure to a number of toxic compounds.

The sense of smell, besides being essential for the enjoyment of food and contributing to various aspects of quality of life, serves the sentinel function of warning the organism of dangerous elements in the air. Hence, damage to this system is of considerable consequence. Among the causes of such damage is exposure to volatile toxic chemicals, most commonly in industrial environments. Unfortunately, it is often difficult to establish a causal relationship between such exposure and dysfunction. This is due, in part, to the paucity of sound research concerning the effects of specific chemical agents on the ability to smell. Exposure to toxic volatiles at levels sufficient to rapidly impair olfactory function usually occurs by accident. In most such cases, the acute exposure is at a sufficiently high concentration that physical damage to the olfactory epithelium and related tissues rapidly ensues. More insidious are situations where subtle cumulative damage follows chronic exposure to relatively low levels of compounds that produce no noticeable irritation or untoward sensations. In such cases, the pathological changes may be so gradual that inferences of causality are difficult to make. Moreover, the chemical exposure may exacerbate damage to the olfactory system from other causes (e.g., viral infections) and vice versa, obscuring the specific influences of the exposure.

II.

HISTORICAL BACKGROUND

Ramazzini (1713), in one of the first books written on occupationally related diseases, noted only 1 of over 50 professions as being associated with “loss of smell” —that of painters. Exposure to extremely purulent odors was an integral part of many of the occupations examined, as was exposure to excessive concentrations of heavy metals, dusts, and toxic chemicals. The often morbid or lethal effects resulting from exposure to these toxic compounds, such as emphysema after exposure to mine dusts, perhaps overshadowed any concern about the viability of the sense of smell. In the centuries since Ramazzini’s early work, more than 100 anecdotal reports and/or case histories of olfactory loss resulting from exposure to toxic compounds have been published. However, fewer than two dozen studies have even attempted to address this question quantitatively, and these studies only appeared within the last 25 years. This dearth of knowledge, and perhaps interest, is reflected in a recent review of the cognitive, psychomotor, and sensory 575

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effects resulting from exposure to solvents, in which the sense of smell was not assessed in any of the 15 studies cited (Gamble, 2000). This is in spite of the fact that the nasal passages are an obvious target site for airborne solvents and that sensory measurement is usually less ambiguous in terms of outcome than cognitive measurement. Two relatively recent reviews have listed numerous volatile chemicals or manufacturing processes that reportedly adversely influence olfaction (Amoore, 1986; Naus, 1975). Those listed in Amoore’s review, which subsumes most of those reviewed by Naus, are presented in Table 1. Of the 75 or more reports noted by Amoore of relationships between exposure to toxic compounds and loss of olfactory function, most were descriptive accounts occurring before 1970. Rarely were industrial hygiene data pertinent to airborne exposures available, and even when available, they were, at best, crude estimates. Furthermore, the indications of smell dysfunction were largely subjective, as accurate olfactory measurement was generally lacking. In many of these studies, workers were clearly exposed to exceedingly high concentrations of toxicants in the workplace. Such nonhygienic factory conditions are much less likely to occur today in developed industrial societies because of improved worker education and increased governmental regulations. Unfortunately, this is not the case in many developing countries. Three additional reviews have appeared in the decade after the account of Amoore that have focused on more contemporary studies (Doty and Hastings, 2001; Schwartz, 1991; Shusterman and Sheedy, 1992). Shusterman and Sheedy reviewed the effects of toxic compounds on the special senses, including olfaction, while Schwartz focused on the use of epidemiology to discern the relationship between workplace exposure to toxic agents and olfactory sensory dysfunction. Doty and Hastings focused on nasal anatomy, methods of assessment, and more contemporary empirical studies. In the early 1990s, the first of a series of studies investigating the effects of exposure to airborne toxicants on upper airway function appeared (Cometto-Muniz and Cain, 1991), examining in detail the effects of airborne contaminants on trigeminal (CN V) free nerve endings within the nose and, in some cases, the eye. Exposure to irritants often causes a reflexive depression in respiratory rate, and this reflexive response has frequently been used as a guide in establishing permissible exposure limits (PELs) and threshold limit values (TLVs). The TLV, much like the PEL, reflects limits to airborne concentrations of substances “under which it is believed that nearly all workers may be repeatedly exposed day after day without adverse effect” (American Conference of Governmental Industrial Hygienists, 1994). It has not been adequately determined, however, whether protracted exposure under the TLV standards produces olfactory deficits.

Hastings and Miller Table 1 Substances Affecting Olfaction for Which There Are Some Supportive Scientific Data Metallic compounds cadmium compounds cadmium oxide dichromates nickel hydroxide zinc chromate alum arsenic compounds chromic acid silver nitrate copper arsenite Metallurgical processes chromium plating lead smelting magnet production mercury (chronic intoxication) nickel plating nickel refining (electrolytic) silver plating steel production zinc production aluminum fumes copper fumes manganese fumes tin fumes arsenic Nonmetallic inorganic compounds ammonia carbon disulfide carbon monoxide chlorine hydrazine nitrogen dioxide sulfur dioxide fluorides sulfuric acid bromine flue gas hydrogen chloride hydrogen fluoride nitric acid phosgene selenium dioxide sewer gas Organic compounds acetaldehyde Source: Adapted from Amoore, 1986.

acetophenone benzene benzine butyl acetate chloromethanes ethyl acetate menthol pentachlorophenol trichloroethylene acetic acid acetonitrile benzoic acid benzaldehyde chloroform dimethyl sulfate furfural formaldehyde trichloroethane m-xylene Dusts cement chemicals hardwoods lime printing cotton cyanides flax flour potash silicon dioxide Manufacturing processes acids asphalt cutting oils fragrances lead paints paprika coal tar fumes rubber vulcanization perfumes (concentrated) spices tobacco varnishes wastewater tanning

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The problem of acquiring reliable data to be used to discern cause and effect, as well as to set standards, is onerous. Severe limitations are present in human research and epidemiological studies, and animal research, which can be more tightly controlled, fails to provide an easy extrapolation to humans, because of differences in the structure and function of the nasal passages and olfactory epithelium. Nevertheless, there is increasing awareness that olfaction should be examined whenever there is airborne exposure to toxic compounds (Suruda, 2000).

larger airborne particles, volatile toxic vapors can still reach this vulnerable area. Even when active sniffing does not occur, e.g., during conditions of normal resting inspiration (0.15–0.25 L/sec), 10–15% of the airflow is shunted towards the olfactory cleft (Swift and Procter, 1977). The remainder flows over the lower turbinates, through the nasopharynx, and into the trachea. This flow results in not only warming, cleansing, and humidifying the major portion of the airstream, but also exposes the turbinates and the olfactory mucosa to xenobiotics contained in the air.

III. A.

OLFACTION AND TOXIC INSULT The Olfactory System

In recent years, much progress has been made in understanding the basic mechanisms of olfaction, especially in the area of molecular biology (see Chapters 3–9). However, the effects of toxic compounds on this system are still poorly understood. Even less well known are the specific compounds that actually pose a risk to humans, as well as the actual prevalence of incidents of toxicantinduced dysfunction occurring in the workplace. Toxic compounds can affect olfactory function either indirectly, e.g., by causing irritation in the nasal passage, blocking the flow of air to the olfactory epithelium, or directly, e.g., by altering the viability of the sensory epithelium. Either way, airflow patterns within the nasal cavity are important factors. In the first case, the air carrying the odorant stimuli is prevented, at least to some degree, from reaching the sensory receptor cells; in the second, the distribution of the toxic agents within the inspired air onto the neuroepithelium is dependent on the local airflow patterns. In the human, the olfactory neuroepithelium lines the cribriform plate and sectors of the superior turbinate, superior septum, and middle turbinate.* The total area of the neuroepithelium has been estimated to be approximately 2 cm2 (for both sides) and is positioned well away from the main airstream. However, the total surface area of the receptive elements, the sensory cilia, is, perhaps, 10 times as great (Doty, 1998). Even though the epithelium is situated well away from the major flow of air, affording it some degree of protection from exposure to

*The olfactory system (CN I) is not the only sensory system present in the nasal cavity that responds to chemical stimuli. Besides the fine nerve endings of CN V, which respond to chemical irritants, there is also the vomeronasal organ (Jacobson’s organ), the septal organ, and the nervus terminalis (CN 0). Other than the CN V, the exact functions of the remaining systems are ill defined, as are their contributions to olfactory function.

B.

Mechanisms of Toxic Insult

As pointed out previously, the process of nasal inspiration ensures that the olfactory receptor neurons in the nasal mucosa will come into contact with deleterious agents in the inspired air. To counter this vulnerability, the system employs a number of defense mechanisms. As a first line of defense, free nerve endings of the trigeminal nerve detect and respond to airborne sensory irritants. Usually a flight response is initiated by the organism upon detection of an irritant. If escape is not possible, the breathing rate and/or pattern are altered in an attempt to minimize the entry of the irritant into the airways. As a second line of defense, the nasal passages actively participate in the metabolism of these xenobiotics through the presence of phase I and phase II enzymes—cytochrome P-450s, glutathione, and related enzymes—which are found there in concentrations that can be greater, on a per gram basis, than in the liver or lungs (Reed, 1993). Since the nasal passages are the first site of internal exposure, these tissues are understandably involved in the detoxification process. However, it should be pointed out that, in some cases, the resultant metabolites can be toxic themselves, especially to the olfactory receptor neurons (Brandt et al., 1990) The nasal mucosa is also capable of secreting antibodies, as well as antimicrobial proteins such as lactoferrin and lysozyme, to deal with inhaled pathogens (Mellert et al., 1992) (see also Chapter 3). Unfortunately, these protective mechanisms in the olfactory epithelium may fail or be circumvented, as evidenced by the occurrence of olfactory deficits after some toxic exposures. It should also be acknowledged that while the peripheral olfactory mucosa may be the site of insult for many airborne toxins, the possibility exists that, in some cases, the site of damage may be more central. A variety of compounds, including metals and solvents, can pass from the nasal lumen via the olfactory receptor neurons to the olfactory bulb and even, in some cases, to higher brain regions. The effects of entry of toxic compounds into the brain via

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this route on olfactory function and higher brain functions are largely unknown (see Chapter 26). Lesions in the olfactory epithelium induced by toxins have distribution patterns that reflect the patterns of chemical uptake, local clearance processes, and cell types present within, around, and beneath the epithelium, as well as a host of other factors. Some of these conditions are physical, such as air-phase delivery of chemicals and airflow patterns, which can exhibit considerable across-and within-species variation. Some are related to the interactions between the olfactory tissue and the chemical agent, and others are related to the metabolic fate of the chemicals, indirectly affecting the surrounding tissue (Brenneman et al., 2000). To assess potential effects of nasal airflow on lesion location and severity, Kimbell and coworkers (1993, 1997) used computational fluid dynamics to map the regional dosimetry of inhaled chemicals in the upper respiratory tract. By using two physiochemical principles, water solubility and reactivity, in conjunction with simulated regional flow velocities, they developed models that predict if and where lesions will develop in the nasal cavity after exposure to a specific agent. The development of such models holds great promise, as it allows rapid testing of many compounds without the expense associated with conducting animal studies. To determine the effects of toxic compounds on the peripheral olfactory system, the olfactory epithelium should be examined, ideally, after exposure to the toxicant. In the case of humans, two square centimeters of tissue would appear, on the surface, to provide sufficient olfactory epithelium to permit biopsy and examination. However, because of its relative inaccessibility, its irregular and often spotty distribution, and inherent surgical risks (penetrating the cribriform plate), human olfactory epithelial biopsy has rarely been done to address histopathological issues. To worsen matters, histological examination of biopsied material can be hampered by the limited amount of material sampled and the loss of associated structures and orientation. Similarly, physiological measures in the nasal cavity, such as mucociliary flow, are influenced by all elements of the nasal epithelium, of which the olfactory epithelium contributes only a small portion, and therefore they do not provide specific data on the structural and functional status of the olfactory epithelium. For these reasons, experimental animals have been used to determine mechanisms of action of toxic compounds in the nasal cavity. Many specific “olfactotoxins” have been identified using animals. Some act immediately and directly (e.g., methyl bromide), causing the sloughing of the olfactory epithelium down to the basement membrane within 24 hours of exposure (Hastings et al., 1991). Others (e.g., 3-methylfuran and dimethylamine) act indirectly only after

Hastings and Miller

being metabolized into reactive intermediates (Reed, 1993). Genetic variations in the ability to metabolize such compounds, and the state of induction of constituent enzymes in the olfactory epithelium, play major roles in their ultimate toxicity. The competency of the host also modulates the severity of the response to toxins through genes regulating such systems as immune responses, chemokine production, vascular integrity, age, gender, and growth factors, etc., which govern regeneration and wound healing. A typical toxic insult to the rodent epithelium damages the cells that lie above the basement membrane (see Fig. 1). The degree and depth of this injury is specific for each compound and dose. Recovery of the epithelium is in part related to the degree of damage and in part to the cell types that have been damaged. For example, olfactory epithelium can be denuded down to the basement membrane, while adjacent respiratory epithelium remains intact. In cases of complete damage to the olfactory epithelium, save the basal cell layer, regeneration may begin within 24 hours, with the covering of the naked basement membrane with squamous cells from surrounding areas. By 4 days this epithelium becomes more cuboidal, and by 30 days it increases in differentiation and thickness to become a more or less mature olfactory epithelium (Hastings et al., 1991). The rapidity with which the rodent olfactory epithelium regains its architectural integrity and functionality depends to some degree upon how heavily the tissues beneath the basement membrane, i.e., in the lamina propria, are damaged. In this respect, when there is a loss of Bowman’s glands beneath the olfactory epithelium, the regeneration is considerably slower. The participation of Bowman’s glands and ducts (which surface above the basement membrane of the olfactory epithelium) appears to be critical in the efficient and complete regeneration of the epithelium in animals exposed to olfactotoxins (Uraih et al., 1987). A factor that may influence the outcome from exposure of the olfactory epithelium to toxins is the health status of the host. For example, an upper respiratory infection that, in and of itself, may not inflict severe damage to the olfactory epithelium may do so when present at the same time as a toxic exposure. In other words, synergism can produce greater damage than that which would result from either insult alone. Conversely, just as exposure to a toxic compound may work in a synergistic fashion with weakened defense mechanisms to cause greater than normal damage, induction of some enzymes by exogenous agents (e.g., as a result of cigarette smoking) may protect against olfactory insult resulting from exposure to some chemicals. Clearly, the specific factors that increase the susceptibility of the olfactory

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few patients of a larger number who were treated with vitamin A seemed to regain function. The latter observation failed to take into account the fact that spontaneous recovery occurs in a number of patients without treatment. The development of an effective treatment strategy for olfactory dysfunction is one of the most pressing needs that faces clinicians working in the area of smell and taste disorders. D.

Figure 1 (Left) Olfactory mucosa from a control rat shows a normal olfactory epithelium (E) and lamina propria. Bowman’s glands (G) and nerve bundles (N) are located below the basement membrane (dotted line). (Middle) After methyl bromide exposure, the epithelium (E) is almost completely denuded, but even at the point of most severe damage, the lamina propria remains essentially intact with glands (G) and nerve bundles (N) being present. (Right) After 3-methyl indole however, the endothelium (E) is reduced in height, but the lamina propria remains severly atrophic, with a loss of glands and neural elements, and recovery is protracted; (V) blood vessel. (250 .)

epithelium to permanent damage from exposure to toxic compounds require further investigation. C.

Therapeutic Approaches

Therapeutic approaches to ameliorate olfactory dysfunction resulting from upper respiratory infections, head trauma, etc., are very limited. The same is true when dysfunction results from exposure to toxic agents. Once the olfactory mucosa has been severely traumatized, neither oral nor topical steroid therapy has proven to be particularly beneficial. While zinc supplementation has been suggested as a therapeutic remedy, no positive value has been substantiated in double-blinded, placebo-controlled studies of patients suffering from olfactory dysfunction (Henkin et al., 1976). Another suggested, but unproven, therapeutic strategy is intramuscular injections of betacarotene (vitamin A) (Duncan and Briggs, 1962) or oral retinoid therapy (Roydhouse, 1988). This therapy arose, however, from two questionable sources: the mistaken notion that pigmentation plays a role in olfactory transduction in humans and a physician’s observation that a

Nomenclature of Olfactory Dysfunction

The degree of severity of olfactory dysfunction varies along a continuum. Based upon quantitative testing (see Chapter 10), olfactory loss can range from mild hyposmia (decreased sensitivity) to total anosmia (complete loss) (see Chapter 22). Although anosmia can occur as a result of exposure to toxic compounds, hyposmia is the more frequent outcome. A toxicant can also produce altered or distorted perception of odors (dysosmia) that occurs either in response to a stimulus (parosmia) or independently (phantosmia). Among the particularly annoying dysosmias are those described as being like the odor of feces or rot (cacosmia), medicine, or burnt toast. Although rare, there are anecdotal reports of some dysosmias resolving by removing a specific chemical from the patient’s environment. For example, Emmet (1976) described the case of a pipefitter exposed chronically to tetrahydrofuran-containing pipe cement who complained of a constant unpleasant smell distinct from pipe cement (parosmia); recovery occurred after removal from occupational exposure to the cement. Shusterman and Sheedy (1992) reported that a woman exposed to chloramine gas complained of a “stinging” in the nasopharynx in response to normally unoffensive household odors; recovery occurred without therapy. IV.

TOXIC METALS

A.

Cadmium

Cadmium is often cited as the “textbook” example of a toxic compound to which exposure results in anosmia (World Health Organization, 1986). In one of the first cadmium/olfactory studies, Friberg (1950) reported that 37% of workers in an alkaline battery factory showed impaired olfaction after an exposure history of 20 years or more. Thus, cadmium-exposed workers scored significantly poorer on tests of olfactory function than age-matched, nonexposed controls; 27% of these workers required 200 times the stimulus concentration required by normals to detect the odor of phenol. Interestingly, about half of these workers were unaware of their dysfunction, conceivably reflecting its slow development. Physical examination of

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the nasal respiratory mucosa failed to yield any relationship between degree of irritation and the olfactory dysfunction. Potts (1965) found olfactory deficits in approximately 60% of workers exposed to cadmium for 10–29 years and deficits in over 90% of those exposed for more than 30 years. These workers were exposed to extremely high concentrations of cadmium in a setting of poor industrial hygiene, where measurements of airborne cadmium ranged from 0.600 to 236.0 mg/m3. A contemporaneous study of cadmium smelter workers reported no change in olfactory function, although exposure was sufficient to result in proteinuria (Tsuchiya, 1967). Both the duration (less than 12 years) and the exposure concentration (0.133 mg/m3), however, were significantly less than in previous studies. In a more recent study of a cohort of individuals who had worked for 5 years or longer in a cadmium-refining plant, 28% reported being anosmic (Yin-Zeng et al., 1985). The classification of anosmia, however, was limited to self-report, as no qualitative measures of olfactory function were obtained. At the time of this study, the average concentration of airborne cadmium was between 0.004 and 0.187 mg/m3; earlier concentrations of cadmium ranged between 0.21 and 0.95 mg/m3. This work suggests the possibility that even moderate exposure to cadmium adversely affects olfactory function. In a relatively extensive quantitative study, 55 workers exposed to cadmium fumes (from brazing refrigerator coils) for a duration of approximately 12 years were evaluated using both a butanol odor–detection threshold and odor discrimination tests (Rose et al., 1992). Thirteen years after production began, the first measurements of airborne cadmium in the workplace were determined to be 0.300 mg/m3. Those workers with the highest body burden of cadmium, determined by the measurement of 2-microglobulin in their urine, showed moderate to severe hyposmia, but not anosmia. No deficits in odor discrimination, however, were observed in these workers. Since the cadmium-related hyposmia was specific to detection threshold, and not to discrimination behavior, it was concluded that the peripheral olfactory receptor neurons were damaged, but the more central components of the system were not. While this interpretation is often evoked when deficits are found in threshold, but not identification, measures, the rationale for doing so is questionable (see Chapters 10 and 21). The olfactory function of 73 workers at a plant in Poland involved in the production of nickel-cadmium batteries was recently compared with that of 43 nonexposed, age- and smoking-matched controls (Sulkowski et al., 2000). While the exposure levels exceeded the maximum allowable concentration (Polish MAC
20 g/m3), the

Hastings and Miller

actual exposure levels were still relatively low. Olfactory function was assessed using the controversial blast injection procedure (Elsberg and Levy, 1935). Anosmia or hyposmia was found in 45.2% of the exposed group but in only 4.6% of the controls. The authors presented some evidence that cigarette smoking, which is a considerable source of cadmium in itself, may intensify the dysfunction associated with occupational cadmium exposure. This is contrary to what is seen with exposure to solvents and irritant gases, where smoking appears to elicit some protective mechanism (Schwartz et al., 1989). The results of the last three studies, despite involving exposure concentrations that are much lower than those in studies that took place in the 1950s and 1960s, are in accord with the results of earlier ones and further imply that, even at low concentrations, cadmium exposure damages olfactory function. This conclusion is further supported by a recent case report of anosmia in two patients after cadmium exposure that emphasized the need for qualitative olfactory testing after exposure to toxic compounds (Suruda, 2000). B.

Chromium

Chromium, like cadmium, is often employed with nickel and other metals in industrial situations, most notably in the manufacture of high-quality steel alloys. Although chromium has long been suspected as having an adverse influence on olfaction, there has been relatively little research on the effects of chromium on the ability to smell. Using the T & T olfactometer (Oka, 1981) (see Chapter 10), Watanabe and Fukuchi (2000) examined odor detection and recognition thresholds of 26 male and 7 female employees of a chromate-producing factory who had worked there for at least 7 years. Over half of the subjects (51.4%) were noted as having perforated nasal septa; 54.5% exhibited elevated smell thresholds to the five odorants evaluated (phenyl ethanol, cyclopentenolone, isovaleric acid, r-undecalactone, and skatole), with two reportedly being anosmic. While the degree of olfactory function was not related to the presence of the septal perforations, a common symptom associated with chromium exposure, it was related to the duration of employment in the factory. C.

Mercury

Metals other than cadmium and chromium can produce olfactory deficits. Exposure to high levels of organic mercury in utero, as in Minamata disease, both raised detection thresholds for serial dilutions of phenyl ethyl alcohol and decreased olfactory identification ability, as measured by the University of Pennsylvania Smell Identification Test

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(UPSIT) (Furuta et al., 1994). The olfactory epithelium and olfactory bulb were reported as being grossly normal at autopsy in these patients, although some gliosis and neuronal degeneration were evident in the bulb. Since most bulb alterations are secondary to epithelial damage, quantitative assessment of the epithelium of such patients is sorely needed.

not considered by the authors as meaningful, since they concluded that olfactory function was unaffected by lead exposure, (Bolla et al., 1995).

D.

Lead

Exposure to inorganic or organic lead can perturb the central nervous system in many ways, from subtle changes in cognitive function to frank encephalopathy. While the effects of lead exposure on various sensory systems, including the visual, auditory, and somatosensory, are well documented (Chuang et al., 2000; He et al., 2000; Wu et al., 2000), little attention has been paid to whether such exposure effects olfactory function. Even more surprising is the fact that no studies have been undertaken to assess the effects of pediatric lead exposure on olfaction, since children are much more sensitive than adults to the neurotoxic effects of low level lead exposure. Schwartz et al. (1993) tested 222 employees involved in the manufacture of tetraethyl lead on a neurobehavioral test battery and on their ability to identify odors employing the UPSIT. The testing was performed on site and before a work shift to minimize any acute effects that the lead might exert. Comprehensive personal industrial hygiene data were available, allowing accurate exposure histories to be reconstructed. Mean differences in the test scores were estimated by comparing the average scores of moderate, high, and highest exposure groups to those of the low exposure (reference) group, and adjusting for the confounding influences of age, intellectual ability, race, and alcohol consumption. No influence of lead on UPSIT scores was apparent, even though lead exposure was associated with decrements in a number of the neuropsychological measures. In a companion study, Bolla et al. (1995) compared a subset of the tetraethyl lead–exposed workers discussed above to workers exposed to solvents as well as to workers exposed to neither compound. The same olfactory test and neurobehavioral test battery were administered to all participants. As before, the overall results showed that, after appropriate adjustments for confounding variables, the UPSIT scores of lead-exposed workers did not differ significantly from those of the reference group (respective mean scores
36.4 and 36.9). However, when duration of exposure was included in the analysis, the UPSIT scores of the middle exposure group (11–17 years) were significantly lower than the UPSIT scores of the reference group. This was not the case, however, for the other two groups (11 and 17 years). This nonlinear effect was apparently

E.

Other Metals

Over a dozen metals or metallurgical processes are considered as adversely influencing olfaction (Tables 1, 2), although, as noted above, only cadmium has received extensive investigation. There is circumstantial evidence that several other metals, such as aluminum and manganese, need to be investigated in detail. Thus, workers involved in aluminum reclamation were found to selfreport a reduction in the sense of smell twice as often as controls (Kilburn, 1998). Odor-detection thresholds for n-butanol were paradoxically reported in one study to be lower (i.e., higher sensitivity) in a group of 115 manganese-exposed iron alloy workers than in controls (Mergler et al., 1994). The overall airborne manganese concentration in total dust ranged from 0.014 to 11.48 mg/m3 (mean
0.225 mg/m3; TLV
1 mg/m3). The authors suggested that this enhanced olfactory acuity was temporary and may reflect an excitatory phase of early manganese intoxication, which might be followed by a subsequent impairment of olfactory perception. In contrast, a second study examining n-butanol detection thresholds of 35 manganese workers exposed to roughly comparable levels and durations of manganese (0.026–0.750 mg/m3, mean
0.193 mg/m3) as the previous one found no effects of the metal on smell function (Lucchini et al., 1997). Nevertheless, a significant negative correlation was found between manganese levels in the urine and olfactory perception threshold.

F.

Utility of Animal Studies

Animal studies, necessitated by the ethical issues surrounding human exposure studies, allow for the assessment of histopathological changes in the olfactory epithelium after known exposures to specific compounds. In rats exposed to 0.500 mg/m3 cadmium for 20 weeks, cardiac and pulmonary toxicity was evident, but the exposure regimen was insufficient to produce an effect on respiratory or olfactory tissues (Sun et al., 1996). Nor was there any evidence of olfactory dysfunction when assessed using an operant detection threshold measure. Similar studies have been conducted using nickel and chromium. Although exposure to nickel sulfate at 6 times the TLV produced damage in the rodent olfactory epithelium, olfactory function was not affected (Evans et al., 1995). In a similar paradigm, no apparent consequences of exposure

Table

2 Effects of Toxic Agents on Olfactory Mucosa of Common Laboratory Rodents (Mice, Rats, Hamsters)

Compound, concentration

Duration

Effects on histology and function

Acetaldehyde, 400–5000 ppm

6 hr/d 5 d/wk 1–28 mo

Degeneration, metaplasia, loss of Bowman’s glands and nerve bundles, adenomas, squamous cell carcinomas

Acrolein, 1.7 ppm Acrylic acid, 5–75 ppm

6 hr/d 5d

Benomyl, 50–200 mg/m3 Bromobenzene, 25 mol/kg ip Cadmium, 250–500 g/m3 Chlorine gas, 0.4–11 ppm

6 hr/d 6 d/wk 13 wk [5 min–3 d]

Hypertrophy, hyperplasia, erosion, ulceration, necrosis inflammation Degeneration, replacement with respiratory epithelium, inflammation, hyperplasia of Bowman’s glands Degeneration

Chloroform, 300 ppm Chloropicrin, 8 ppm Coumarin, 50 mg/kg ip Chlorthiamid, 6–50 mg/kg ip

6 hr/d 7 d

6 hr/d 5 d/wk 13 wk

5 hr/d 5 d/wk 20 wk 6 hr/d 5 d/wk 16 wk

6 hr/d 5 d [48 hr] [8 hr-7 d]

Degeneration of olfactory epithelium and Bowman’s glands Little change Degeneration, septal perforations, intracellular deposits of eosinophilic material, mucus cell hypertrophy Degeneration of Bowman’s glands, cell proliferation in periosteum and bone Hypertrophy, hyperplasia, ulceration, necrosis, inflammation Necrosis, cell loss, and basal cell metaplasia in the olfactory mucosa Degeneration of olfactory epithelium and Bowman’s glands, replacement with respiratory epithelium fibrosis in lamina propria Degeneration, sustentacular cells injured initially, cell proliferation

Ref. Appelman et al., 1982; Woutersen et al., 1984 Woutersen et al., 1986 Buckley et al., 1984 Miller et al., 1981

Warheit et al., 1989 Brittebo et al., 1990 Sun et al., 1996 Wolf et al., 1995

Mery et al., 1994 Buckley et al., 1984 Gu et al., 1997 Brittebo et al., 1991

Dibasic esters, 20–900 mg/m3

4 hr/d 7–13 wk

1,2-Dibromo-3chloropropane, 5–60 ppm 1,2-Dibromoethane, 3–75 ppm Dichlobenil 12–50 mg/kg ip 1,3-Dichloropropene, 30–150 ppm Dimethylamine 10–511 ppm 1,4-Dithiane, 105–420 mg/kg ig Epichlorohydrin, 687 ppm Ferrocene, 3–30 mg/m3 Formaldehyde, 0.25–15 ppm

6 hr/d 5 d/wk 13 wk

Degeneration, metaplasia, hyperplasia

Keenan et al., 1990; Bogdanffy and Frame, 1994 Reznik et al.,1980

6 hr/d 5 d/wk 13 wk

Degeneration, metaplasia, hyperplasia

Reznik et al.,1980

[8 hr-7d]

Degeneration of olfactory epithelium and Bowman’s glands Degeneration and /or metaplasia

Brandt et al., 1990

Furfural, 250–400 ppm

7 hr/d 5 d/wk 52 wk

6 hr/d 5 d/wk 6–24 mo 6 hr/d 5 d/wk 6–12 mo [90 d]

Degeneration, loss of nerve bundles, hypertrophy of Bowman’s glands Anisotrophic crystals in giant cells (undetermined chemical composition)

Scott et al. 1988, Lomax et al., 1989 Buckley et al., 1984, 1985 Schieferstein et al., 1988

6 hr/d 5 d

Ulceration, necrosis

Buckley et al., 1984

6 hr/d 5 d/wk 13 wk 6 hr/d 5 d 4 mo

Iron accumulation, necrotizing inflammation, metaplasia Decrease number of bipolar cells, increase number of basal cells, degeneration of nerve bundles, reduced odor discrimination) Disorientation of sensory cells, degeneration of Bowman’s glands, cyct-like structures in lamina propria

Nikula et al., 1993 Apfelbach et al., 1991, 1992 Feron and Kruysse, 1978

Table 2 (continued) Compound, concentration

Durationa

Furfural alcohol, 2–250 ppm

13 wks

Hexamethylene diisocyanate, 0.005–.175 ppm ,ⴕ-Iminodipropionitrile, 200–400 mg/kg ip Methyl bromide, 200 ppm 3-Methylfuran, 148–322 mol/l 3-Methylindole, 100–400 mg/kg ip

6 hr/d 5 d/wk 12 mo

Methyl isocyanate, 10, 30 ppm Napthalene, 400–1600 mg/kg ip Nickel subsulfide, 0.11–1.8 mg/m3 Nickel sulfate, 3.5–635 mg/m3 N-Nitrosodimethylamine, 20–80 mg/kg ip N-Nitrosopyrrolidine, 30–100 mg/kg ip Propylene glycol monomethyl ether acetate, 3000 ppm Propylene oxide, 0–525 ppm Pyridine, 5–444 ppm RP 73401, 1 mg/kg/day Sulfur dioxide, 10–117 ppm Sulfuryl fluoride, 0–600 ppm Tetramethoxysilane 1–45 ppm 2,4-Toluene diisocyanate, 0.4 ppm 3-Trifluoromethyl pyridine, 0.1–329 ppm aDuration

Effects on histology and function

Ref.

Squamous and respiratory metaplasia of olfactory epithelium, inflammation, hyaline droplets, squamous metaplasia of ducts Degeneration, mucus hyperplasia

Miller et al., 1991

[6 hr-56 d]

Degeneration of axon bundles, increase of glial fibrillary acidic protein

Genter et al., 1992

4 hr/d 4 d/wk

Degeneration, decreased carnosine, behavioral deficits Degeneration, more severe in rats than hamsters Degeneration, fibrous adhesions, osseous remodeling, Bowman’s gland hypertrophy, behavioral deficits Degeneration of the respiratory and olfactory epithelium

Hastings et al., 1991

1 hr [7–90d]

2 hr

[24 hr]

Foureman et al.,1994

Morse et al., 1984 Turk et al., 1987; Peele et al.,1991 Uraih et al., 1987

Cytotoxicity (mice and hamsters), necrosis (rats) Atrophy

Plopper et al., 1992

Atrophy, degeneration, decrease in carnosine consecutive Degeneration of olfactory epithelium and Bowman’s glands

Evans et al., 1995 Rangga-Tabbu and Sleight, 1992

[6 hr-30 d]

Degeneration of olfactory epithelium and Bowman’s glands

Rangga-Tabbu and Sleight, 1992

2 weeks

Slight to moderate degeneration of olfactory epithelium

Miller et al., 1984

4 weeks

Degeneration of the olfactory epithelium

Eldridge et al. 1995

6 hr- 4 d

Degeneration of olfactory epithelium

1 hr-5 d

Degeneration of olfactory epithelium and Bowman’s glands Necrosis, edema, destruction, hyperplasia, hypertrophy

Nikula and Lewis, 1994 Pino et al., 1999

6 hr/d 5 d/wk 13 wk 6 hr/d 12–16 [6 hr-30 d]

72 hr, or 6 hr/d 5d 6 hr/d 5d 2 wk 6 hr/d 5 d 28 days 6 hr/d 5d

6 hr/d 10–90 d

in brackets are times postexposure until killed.

Inflammation Ulceration, inflammation, and necrosis of epithelium Ulceration, necrosis, inflammation, degeneration Degeneration, reduced Bowman’s activity

Dunnick et al., 1989

Giddens and Fairchild,1972; Buckley et al., 1984 Eisenbrandt and Nitschke, 1989 Kolesar et al., 1989 Buckley et al., 1984

Gaskell et al., 1988

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to chromium (sodium dichromate) at six times the TLV were observed (Hastings et al., 1994). Other rodent studies have found numerous industrial compounds that damage the olfactory mucosa (Table 2). In general, the rodent olfactory system is both robust and resilient; while exposure to many toxic compounds can nearly obliterate the olfactory mucosa, it almost always reconstitutes itself, at least to some degree. In the majority of the studies conducted, however, the emphasis usually has been on structural or biochemical/molecular changes; only rarely has a focus been on behavioral indices of olfactory function (Barrow, 1986; Miller, 1995) (Table 2). More studies, in addition to the ones cited above, are sorely needed that assess in greater detail the relationship between structural damage and functional effects. While the animal data are important in determining which toxins have the potential for causing damage to the olfactory system in humans, the resilience of the rodent system leads to valid skepticism as to whether the human olfactory epithelium reacts similarly.

V.

IRRITANT GASES

There is little doubt that accidental exposure to high concentrations of irritant gases can have an adverse effect on human olfactory function. However, the toll taken on the olfactory system by chronic exposure to low levels of airborne contaminants that are sufficient to at least mildly activate CN V afferents is not known. Such exposure routinely occurs in some workplaces and, in the case of such agents as ozone and formaldehyde, are common components of both indoor and outdoor air pollution. Such exposure greatly expands the potential population that may be exposed to such compounds, including those that may be more sensitive to adverse effects, i.e., the very young, the sick, the elderly, and those with genetic predispositions. Animal studies, primarily rodent ones, support the idea that, in fact, chronic exposure to a number of irritants can seriously damage the olfactory epithelium if present at high enough concentration (see Table 2). In a study investigating the effects of exposure to SO2 and/or ammonia (concentrations unknown) on olfactory function, 25% of the workers surveyed reported an increase in subjective complaints involving olfactory disorders (Harada et al., 1983). When tested empirically using a T & T olfactometer (Oka, 1981), odor-detection thresholds for five different odors were elevated, but only for the group exposed to SO2. As with cadmium (Rose et al., 1992), the deficit was assumed to be peripheral in nature, i.e., related to injury of the nasal mucous membrane. Although workplace exposure data were lacking, this

assumption seem reasonable, since exposure of mice to SO2 has been shown to cause extensive damage in the olfactory epithelium (Giddens and Fairchild, 1972). The authors also pointed out that deficits were found in workers who had not been exposed in recent years, suggesting that the damage was more or less permanent in nature. In one of the few human laboratory studies on this topic, volunteers were exposed to ozone (0.400 ppb, 4 hr/day for 4 days), a component of both indoor and outdoor pollution. This resulted in an initial increase in the olfactory threshold to the one compound they examined, butyl alcohol (Prah and Benignus, 1979). However, by the end of the 4 days of exposure, thresholds had returned to normal, suggesting that the system may have compensated for the initial insult. This situation is quite different from most occupational chronic exposures, where the exposure may span years. In the latter situation, there may be an early adaptive phase, which is followed, after longer continued exposure, by loss of the ability of the system to compensate, perhaps as the result of cumulative damage to the epithelium. Thus, both short-and long-term exposure data from various concentrations of airborne chemicals are needed to accurately establish their toxicity. Probably the most comprehensive investigation addressing the effects of exposure to toxic compounds on olfaction was a study by Schwartz et al. (1989). Olfactory function of 731 chemical workers exposed to acrylic acid and a variety of acrylates and methacrylates, at levels below the TLVs, was assessed using the UPSIT. While analysis of the cross-sectional data revealed no relationship between chemical exposure and olfactory deficits, a nested case-control study designed to examine cumulative effects of exposure uncovered several associations: first, olfactory dysfunction increased with cumulative exposure; second, the effects appeared to be reversible; and third, the highest relative risk of olfactory dysfunction occurred in the group of workers who had never smoked. The more pronounced effects found in the never-smoking group may be due to the fact that smoking induces metabolic enzymes in the nasal mucosa that may provide protection against the toxic effects of exposure to other chemicals (Reed, 1993). Recall that, in contrast, smoking in conjunction with cadmium exposure appeared to exacerbate the olfactory deficit (Sulkowski et al., 2000). One final point stressed by Schwartz et al. (1989) was that although the study suggested that exposure to acrylates and methacrylates was associated with a significant decrease in olfaction, the impairment may not always be of sufficient magnitude to be clinically important. Another well-designed study investigated the effects of styrene exposure on olfactory function (Dalton et al., 2000).

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Styrene exposure in animals has been shown to cause extensive damage to the olfactory epithelium (Ohashi et al., 1986), but whether this was also true in humans was not known. Dalton et al. examined olfactory function in a group of workers with a minimum of 4 years of exposure to styrene and a historic mean dose of 154.1 ppm-years (range: 13.8–328 ppm-years) with that of a group of age- and gender-matched unexposed controls. Olfactory function was assessed using a detection threshold tests for phenylethyl alcohol and styrene, as well as identification tests employing ortho- and retronasally presented odorants. Individual exposure histories were reconstructed, and olfactory function was examined with regards to the individual exposure profiles. Contrary to the findings from the animal literature in which comparable exposure concentrations had been used, there was no evidence of any long-term alteration in olfactory function due to styrene exposure. This discrepancy in findings could be due to a number of factors. For example, rats and mice are obligate nose breathers, and thus the olfactory epithelium may experience a much higher exposure than that which occurs in humans. Furthermore, the ability to metabolize styrene in olfactory tissue may differ between the two species. Since odor-detection thresholds for styrene, but not for phenyl ethyl alcohol, were significantly but reversibly elevated among the exposed workers, exposure-induced adaptation may have been present. Another toxic compound for which there exists both animal and human data is methyl bromide. Methyl bromide has been widely used as a fumigant and is one of the most potent olfactotoxins identified in the animal literature. The olfactory function of 123 structural fumigation workers exposed to either methyl bromide or sulfuryl fluoride (another fumigant) was tested by Calvert et al. (1998) using the UPSIT. While methyl bromide appeared to have no effect on olfactory function, high sulfuryl fluoride exposure caused a significant decrease in the UPSIT score—33.1 compared to 34.4 for the controls—representing a drop from normal (34 and above) to mild hyposmia (33 and lower) (Doty, 1995). The lack of effect seen with the methyl bromide may reflect enhancement of the metabolic activity of the phase 1 enzymes from low exposure, thereby preventing damage from occurring when higher, more toxic concentrations were encountered. Thus, only if the initial exposure was high will damage likely result. Formaldehyde, a ubiquitous and highly reactive gas, is employed widely in a variety of manufacturing processes and is also frequently encountered in biology classes, histology departments, and mortuaries. It is absorbed mainly in the nose, particularly in the anterior portion. While exposure to formaldehyde is often alleged to decrease olfactory acuity (Spearman, 1954), the supportive data are very sparse. In one study, workers exposed to 0.075–0.750

ppm formaldehyde (alone or in combination with wood dust) showed a slight but significant elevation in detection thresholds (serial dilutions of pyridine) when compared to controls (14.2 vs. 15.6) (Holstrom and Wilhelmsson, 1988). In a questionnaire study, 68% of histology technicians exposed to 0.200–1.900 ppm formaldehyde reported decreased odor perception; only 9% of the control group reported the same (Kilburn et al., 1985).

VI.

SOLVENTS

Many solvents have been found to be neurotoxic, producing effects on cognitive function as well as on such sensory systems as vision and audition (Dick, 1995). Due to their lipophilic nature, most solvents readily penetrate the underlying cellular membranes after traversing the mucus. Workers exposed to petroleum products (fuel oil vapors) have been found to be less able to detect decreasing concentrations of n-butanol and fuel oil than controls, but were no different than controls in detecting thresholds for pyridine and dimethyl disulfide (Ahlstrom et al., 1986). The elevated thresholds, however, were still within the “normal” range. These workers also displayed “odor intensity recruitment,” i.e., normal perception of strong stimuli but impaired perception of weak stimuli. Not surprisingly, the largest decrement was found for detection of fuel oil vapor, which supports the concept of “industrial anosmia”—the phenomenon whereby exposure to strong odors in the workplace results in a reduction in sensitivity for those specific odors, while sensitivity to other odors remain the same. Unlike the deficits associated with exposure to cadmium, the effects due to solvents were transient and reversible. Schwartz et al. (1990) investigated the effects of solvent exposure on olfactory function in 187 workers at a paint formulation plant. Historical industrial hygiene sampling data were available, which provided accurate exposure histories. Olfactory function was measured using the UPSIT, and the data revealed a subtle, dose-related effect of solvent exposure on olfactory function, but only in neversmokers. The similarity of these results to those found for the chemical workers (Schwartz et al., 1989), but not for the cadmium workers (Sulkowski et al., 2000), illustrates how ancillary factors can influence the toxicity of compounds on olfactory function. Once again, although the results were statistically significant, the deficits were, overall, very subtle in nature. In spite of the many limitations inherent in field studies, these results also suggest that chronic low-level exposure to solvents adversely affects olfactory function. In some cases, painters may be exposed to higher concentrations of solvents than chemical plant workers.

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This is due to the fact that airborne contaminants are usually closely monitored in commercial plants, as required by government regulations. On the other hand, painters work in confined spaces and without monitoring equipment. Using the UPSIT and a threshold test based on serial dilutions of pyridine, Sandmark et al. (1989) compared the olfactory function of a group of 54 painters exposed to organic solvents with that of 42 unexposed controls. No differences in olfactory function were found between the two groups. The authors attributed the lack of effect to a lowto-moderate degree of exposure to solvents. In contrast, floor-layer workers, who are exposed to high levels of toluene, reported significantly more smell disturbances than controls when responding by questionnaire (Hotz et al., 1992). Airborne contaminants found in the workplace almost invariably consist of a mixture of compounds, making it difficult to link specific compounds with identified effects. Testing volunteers in laboratory studies under tightly controlled conditions circumvents many of these limitations, but such studies, in turn, are compromised by restrictions on duration and concentration of exposure, as well as safety and ethical constraints (many compounds are suspected carcinogens). Mergler and Beauvais (1992) exposed volunteers for 7-hour periods to either toluene, xylene, or a mixture according to a Latin square design, and evaluated their olfactory function. Detection thresholds for toluene or phenylethyl methylethyl carbinol were measured. A sixfold shift in the detection threshold for toluene was found immediately following exposure to toluene, xylene, or a mixture of the two. No differences were found in phenylethyl methylethyl carbinol thresholds after exposure to any of the compounds. As previously noted, exposure to a compound may affect detection of the same compound but have no effect on detection of other odorants. This would imply that thresholds for several different compounds should be obtained when testing for olfactory deficits or, at the very least, a test such as the UPSIT, which incorporates a large number of different odorants, should be employed. The fact that the alterations in olfactory function seen in most of these studies were reversible suggests the possibility that the shift in detection threshold was due to short-term effects such as olfactory fatigue or adaptation, rather than to toxic insult to the olfactory receptor neurons.

VII. ODORS, ANNOYANCES, AND MULTIPLE CHEMICAL SENSITIVITY In the absence of exposure levels that produce irritation or other traditional toxicological endpoints, acute odor-

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related symptoms can nonetheless occur. Probably the most widespread problem related to odors, in general, is annoyance. Annoyance can range from the discomfort experienced by an individual upon being exposed to a strong perfume or malodor to the distress experienced by a whole community located downwind from a chemical plant, paper mill, waste treatment plant, or similar facility. Such exposures often involve some form of sulfur-containing compound, e.g., hydrogen sulfide, mercaptans, thiopenes, etc. (Shusterman, 1992). These compounds often produce such effects at concentrations greater than olfactory detection threshold, which may or may not exceed the TLV. However, for some individuals, the response is much greater than mere annoyance and can become highly aversive and debilitating. The olfactory system may play a role in mediating or triggering a constellation of symptoms collectively known as idiopathic environmental intolerances (Staudenmayer, 1999). These symptoms include autonomic arousal (lightheadedness, nausea, anxiety, tachycardia), upper and lower respiratory tract irritation, neurological symptoms (shortterm memory loss), and others. A more limited version of this phenomenon with a well-defined set of criteria is known generally as multiple chemical sensitivity (MCS) (see Chapter 25). MCS is characterized by an aberrant response elicited primarily by an odorant stimulus, in contrast to the situation where chemical exposure causes abnormal perception of odor stimuli. Exposure to a solvent or pesticide is usually the precipitating event (a “sensitizing” experience) that leads to MCS. In general, in individuals experiencing MCS, exposure to many airborne contaminants including solvents, perfumes, cleaning agents, gasoline, pesticides, and paint, called “triggers”, elicits the multimodal symptoms. Numerous complications exist in understanding the etiology of MCS, if indeed it is a homogeneous disease or disorder. One is that within an individual, a similar set of symptoms is caused by a diverse range of chemically and structurally dissimilar synthetic compounds. Conversely, exposure to the same chemical can elicit different symptoms in different MCS patients. Most problematic is the fact that the exposure levels of the chemicals that “trigger” the responses are usually far below the TLVs for the compounds and have no effect on the vast majority of people. In most circumstances, the level of exposure sufficient to induce the symptoms needs to be only at the threshold of odor perception. Since the etiology of these symptoms does not appear to based on traditional toxocological mechanisms, a number of nontoxicological odor-related mechanisms have been proposed to explain the elicitation of such acute, odor-triggered symptoms (Shusterman, 1992; Siegel and Kreutzer,

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1997). These include odor-related aversive conditioning, and odor-related, stress-induced illness; both rely heavily on learned associations mediated by classical conditioning processes. Usually odor-related aversive conditioning involves a traumatic exposure episode, while the odorrelated, stress-induced illness paradigm represents the conditioning of an odor cue with autonomic arousal symptoms resulting from stress. The stress is usually chronic in nature and may result from the workplace environment (job pressures, employment insecurity, etc.) or from perceived dangers, such as working with “unknown chemicals” or living near a toxic waste site. Boxer (1990) stated, “It has been observed clinically that psychologic reactions to exposure to neurotoxins can be more serious than the direct neurotoxic effects.” In any event, odors are very salient sensory cues, and it is postulated in such thinking that they quickly become associated with the subjective symptoms. After conditioning, the odor cue alone becomes sufficient to elicit the symptoms. Other proposed mechanisms include innate odor aversion and odor-related exacerbation of underlying conditions. The latter is suggested by the finding that MCS patients display an increased prevalence of depression and psychosomatic disorders compared to persons from the normal population (Stewart and Ruskin, 1985). Experimental data exist that suggest, contrary to patient complaints, that odor sensitivity is not altered in most MCS patients. In an odor-detection threshold experiment, Doty et al. (1988) found that odor thresholds were no lower among MCS symptomatics than among control subjects. In a similar study, Hummel et al. (1996) found no differences in odor-detection thresholds between MCS symptomatics and normal controls. Paradoxically, they found that the MCS patients were impaired, relative to controls, in identifying and discriminating among suprathreshold odors, yet exhibited smaller odor-induced event-related potentials. If these counterintuitive findings are valid (no control group was tested in the event-related potential phase of the study), the suprathreshold olfactory function of MCS patients may, in fact, be decreased, not increased. In contrast to the notion that MCS results from a primary emotional (psychogenic) response to perceived chemical exposure is the view that the symptoms result from pathological interactions between chemicals and the biological system. Ziem and McTamney (1997) have suggested that toxic porphyria may be the basis for MCS. Meggs (1993; see also Bascom et al, 1997) has advanced the concept that neurogenic inflammation arising from stimulation of chemical irritant receptors may explain the occurrence of chemical sensitivities. Bell et al. (1992) have proposed that increased sensitivity to chemical exposure seen in MCS patients results from increased limbic

system reactivity resulting from time-dependent sensitization. A recent review of these conflicting views (psychogenic vs. biological) concluded that sufficient information is not currently available to say unequivocally whether either is correct and that neither should be dismissed in determining both etiology and treatment strategies (Gots, 1996). The role played by perceived olfactory cues and the olfactory system in the etiology of MCS is still largely unknown. VIII.

CONCLUSIONS

Although many reports in the literature suggest that exposure to a wide variety of toxic compounds can have a deleterious effect upon olfactory function, there is little specific information relating to dose effect, duration, mechanisms of action, etc., for these compounds. Only in recent years have there been reliable industrial hygiene data of value for linking exposure to specific compounds with olfactory dysfunction. Similarly, practical tests for assessing olfactory function in the workplace have only recently been developed. Consequently, very few studies have demonstrated, with any certainty, altered olfactory function as a result of toxic exposure. Furthermore, most of these show that under normal circumstances exposure to high levels of a toxic compound is required to produce observable deficits. However, the number of compounds actually tested for potential olfactotoxicity is minuscule compared to the large number of chemicals used in the workplace. Many more epidemiological studies are needed which examine the relationship between workplace exposure to specific toxic agents and olfactory toxicity. Animal studies, while suggestive, cannot be definitive, given the major species differences in the deposition and fate of chemicals that enter the nose. REFERENCES Ahlstrom, R., Berglund, B., Berglund, U., Lindvall, T., and Wennberg, A. (1986). Impaired odor perception in tank cleaners. Scand. J. Work Environ. Health 12:574–581. American Conference of Governmental Industrial Hygienists. (1994). Threshold Limit Values for Chemical Substances and Physical Agents and Biological Exposure Indices. ACGIH, Cincinnati. Amoore, J. E. (1986). Effects of chemical exposure on olfaction in humans. In Toxicology of the Nasal Passages, Barrow, C. S. (Ed.). Hemisphere Publishing, Washington, DC, pp. 155–190. Apfelbach, R. (1991). Sensitivity to odors in Wistar rats is reduced after low-level formaldehyde-gas exposure. Naturwissenschaften 78:221–223.

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590 Miller, F. J. (1995). Nasal Toxicity and Dosimetry of Inhaled Xenobiotics. Taylor & Francis, Washington, DC. Miller, R. R., Ayres, J. A., Jersey, G. C., and McKenna, M. J. (1981). Inhalation toxicity of acrylic acid. Fundam. Appl. Toxicol. 1:271–277. Miller, R. R., Hermann, E. A., Young, J. T., Calhoun, L. L., and Kastl, P.E. (1984). Propylene glycol monomethyl ether acetate (PGMEA) metabolism, disposition, and short-term vapor inhalation toxicity studies. Toxicol. Appl. Pharmacol. 75:521–530. Miller, R. A., Mellick, P. W., Leach, C. L., Chou, B. J., Irwin, R. D., and Roycrott, J. H. (1991). Nasal toxicity in B6C3F1 mice inhaling furfuryl alcohol for 2 or 13 weeks. Toxicologist 11:669A. Morse, C.C., Boyd, M.R., and Witschi, H. (1984). The effect of 3-methylfuran inhalation exposure on the rat nasal cavity. Toxicology 30:195–204. Naus, A. (1975). Olphactoric properties of industrial matters, Charles University, Prague. Nikula, K. J., and Lewis, J. L. (1994). Olfactory mucosal lesions in F344 rats following inhalation exposure to pyridine at threshold limit value concentrations. Fundam. Appl. Toxicol. 23:510–517. Nikula, K. J., Sun, J. D., Barr, E. B., Bechtold, W. E., Haley, P. J., Benson, J. M., Eidson, A. F., Burt, D. G., Dahl, A. R., and Henderson, R. F. (1993). Thirteen-week, repeated inhalation exposure of F344/N rats and B6C3F1 mice to ferrocene. Fundam. Appl. Toxicol. 21:127–139. Ohashi, Y., Nakai, Y., lkeoka, H., Koshimo, H., Nakata, J., Esaki, Y., Horiguchi, S., and Teramoto, K. (1986). Degeneration and regeneration of respiratory mucosa of rats after exposure to styrene. J. Appl. Toxicol. 6:405–412. Oka, T. (1981). [Studies on the olfactory fatigue by T & T olfactometer (author’s transl)]. [Japanese]. Nippon Jibiinkoka Gakkai Kaiho 84:850–857. Peele, D. B., Allison, S. D., Bolon, B., Prah, J. D., Jensen, K. F., and Morgan, K. T. (1991). Functional deficits produced by 3methylindole-induced olfactory mucosal damage revealed by a simple olfactory learning task. Toxicol. Appl. Pharmacol. 107:191–202. Pino, M. V., Valerio, M. G., Miller, G .K., Larson, J. L., Rosolia, D. L., Jayyosi, Z., Crouch, C. N., Trojanowski, J. Q., and Geiger, L.E. (1999). Toxicologic and carcinogenic effects of the type IV phosphodiesterase inhibitor RP 73401 on the nasal olfactory tissue in rats. Toxicol. Pathol. 27:383–394. Plopper, C. G., Suverkropp, C., Morin, D., Nishio, S., and Buckpitt, A. (1992). Relationship of cytochrome P-450 activity to Clara cell cytotoxicity. I. Histopathologic comparison of the respiratory tract of mice, rats and hamsters after parenteral administration of naphthalene. J. Pharmacol. Exp. Ther. 261:353–363. Potts, C. L. (1965). Cadmium proteinuria—the health of battery workers exposed to cadmium oxide dust. Ann. Occup. Hyg. 8:55–61. Prah, J. D., and Benignus, V. A. (1979). Decrements in olfactory sensitivity due to ozone exposure. Percept. Motor Skills 48:317–318. Ramazzini, B. (1713). Diseases of Workers (De Morbis Artificum). Hafner Publishing Company, New York.

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28 Evaluation of Olfactory Deficits by Structural Medical Imaging Cheng Li, Richard L. Doty, and David W. Kennedy University of Pennsylvania, Philadelphia, Pennsylvania, U.S.A.

David M. Yousem The Johns Hopkins University School of Medicine, Baltimore, Maryland, U.S.A.

I.

INTRODUCTION

1996, 1997, 1998, 1999). In this chapter we comprehensively review the pertinent medical literature on this general topic and detail our own experience.

Olfactory dysfunction can generally be classified into (1) conductive disorders caused by interference with the access of odorants to the olfactory receptors, (2) peripheral sensorineural disorders resulting from injury to the olfactory receptors (within the olfactory mucosa), and (3) central neural disorders of the olfactory bulb or tract or related parts of the central nervous system such as the prefrontal lobe, septal nuclei, amygdala, and temporal lobe. For medical imaging and the anatomical approach, we categorize olfactory dysfunction into two major groups: peripheral causes—sinonasal tract disorders—and central causes— intracranial disorders. It is important to relate olfactory deficits to the appropriate anatomical and pathological changes. Unfortunately, clinical olfactory testing, whether psychophysical or electrophysiological, is rarely capable of localizing the source (aside from determining whether it is on the right or left) or identifying the specific cause of decreased smell function. Modern medical imaging techniques offer a valuable means for assessing the basis of some disorders of olfaction. Although revolutionary changes in medical imaging techniques have occurred in the last few decades, only a few articles have dealt with imaging studies related to chemosensory disorders (Doty et al., 1999; Goodspeed et al., 1987; Kimmelman, 1991; Klingmuller et al., 1987; Li et al., 1994; Schellinger et al., 1983; Yousem et al.,

II.

IMAGING MODALITIES AND TECHNIQUES

Major advances in pinpointing the anatomical and pathological changes of many disorders of the sinonasal cavity and brain have become possible as a result of the development and refinement of imaging techniques (Carter and Runge, 1988; Healy, 1992; Jagust and Eberling, 1991; Jolles et al., 1989; Reiman and Mintun, 1990; Shapiro and Som, 1989; Vogl, 1990; Yousem et al. 1996a, 1997b, 1998). Even though the imaging evaluation is not the diagnostic equivalent to histological study, anatomical imaging, such as highresolution computed tomography (CT) and magnetic resonance imaging (MRI), can not only map regional lesions, but may also suggest a differential diagnosis (Carter and Runge, 1988; Shapiro and Son, 1989; Som and Shapiro, 1988). On the other hand, functional imaging (PET, SPECT, fMRI), which is reviewed in Chapter 12, affords one the potential to explore regional pathophysiological changes in the living brain (Healy, 1992; Jagust and Eberling, 1991; Jolles et al., 1989; Reiman and Mintun, 1990; Yousem et al., 1997b, 1999b, c). The relevant imaging modalities which may be helpful in the evaluation of common causes of olfactory deficits are reviewed in this section. 593

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A.

Li et al.

Plain Radiographs

Plain film radiography, i.e., the “sinus series,” including the Caldwell view, the Waters view, the lateral view, and the base view, has long been a standard method of diagnosing nasal and paranasal sinus inflammatory disease. Problems of overlap and nonspecific findings are impossible to avoid with plain films, and thus the study has been largely replaced by CT. The most important deficit of the plain film is its inability to provide the road map of the ostiomeatal complex, which may guide endoscopic surgical intervention (Zinreich et al., 1987). Plain radiographs and conventional plain film tomography have virtually no role in the imaging evaluation of olfactory dysfunction. B.

Computed Tomography

CT is well suited to the investigation of the sinonasal cavities. Because CT scanning is as sensitive to soft tissue disease as to bony changes, each scan can be photographed at an appropriate window width and level to optimally see insidious soft tissue differences in attenuation and fine bony detail. To study soft tissue, the window widths range from 150 to 400 Hounsfield units. Conversely, the bony detail is best observed at wide window settings—from 2000 to 4000 Hounsfield units. The basic CT scanning protocol should include all of the nasal cavity, paranasal sinuses, hard plate, anterior skull base, orbits, and nasopharynx. The brain should be included if central causes of olfactory dysfunction are suspected. The scans are commonly performed in both the axial and coronal planes for optimal assessment of the complex paranasal anatomy, but coronal scans are the most valuable for the anterior naso-ethmoid (ostiomeatal) region. Alternatively, thin sections in one plane with multiplanar reconstructions may be adequate. For practical purposes, slice thicknesses of 3–5 mm are often employed. For the evaluation of the ostiomeatal complex (the maxillary sinus ostium, infundibulum, uncinate process, and middle meatus), 3-mm-thick coronal sections are fairly standard unless three-dimensional (3D) reconstructions are requested. The quality of the 3D images is improved by utilizing 1-mmthick sectioning, which is rapidly performed with the new spiral scanners (minutes) and multidetector scanners (seconds). Intravenous contrast enhancement is usually reserved for the identification of vascular lesions, tumors, meningeal or parameningeal processes, and abscess cavities (Carter and Runge, 1988). Intrathecal contrast may be employed when cerebrospinal fluid leaks accompany the olfactory deficits. High-resolution CT is the most useful and cost-effective screening tool for the evaluation of sinonasal tract inflammatory disorders.

C.

Magnetic Resonance Imaging

MRI’s multiplanar capability is especially advantageous in the evaluation of sinonasal tract neoplasms and brain disorders. MRI, however, is less sensitive for the detection of bony cortical abnormalities and landmarks. Soft tissue discrimination, on the other hand, is more clearly illustrated by MRI than by CT. Most soft tissue disease processes can be accurately localized with a minor degree of tissue differentiation, i.e., infection vs. tumor vs. hemorrhage. The anatomical discrimination of the brain is much better using MRI than CT. One can use thin sections, large matrices, and smaller fields of view to improve resolution, yet maintain, high contrast to noise using T1-weighted scans (T1W) or fast spin echo T2-weighted (T2W) images. T2-weighted scans can better delineate the contrast between normal and inflammatory or neoplastic tissue (Shapiro and Som, 1989). New phase sensitive inversion recovery pulse sequences or standard spoiled gradient echo sequences can highlight the gray-white matter differentiation and allow better assessment of the hippocampus, parahippocampus, gyrus rectus, and entorhinal cortex regions. Segmentation of images to separate cortical volume from whole brain volume is customary for volumetric studies nowadays. For the evaluation of skull base invasion by sinonasal tumors, MRI is superior to CT (Paling et al., 1987). Gadolinium enhanced scans are particularly useful at the skull base to detect dural or leptomeningeal involvement. Gadolinium-DTPA, a paramagnetic contrast agent, has been widely utilized for distinguishing solidly enhancing tumor from rim-enhancing inflammatory processes (Brasch, 1992; Vogl et al., 1990). With regard to the olfactory system, CT and MRI play complementary roles in evaluating sinonasal tract neoplasms (Shapiro and Som, 1989; Som et al., 1990). However, MRI is the study of choice to directly visualize the olfactory bulbs, olfactory tracts, and intracranial causes of olfactory dysfunction (Klingmuller et al., 1987; Suzuki et al., 1989; Yousem et al., 1993, 1998, 1999a). D.

Nuclear Medicine

In general, conventional radionuclide imaging plays no significant role in the diagnostic work-up of patients with suspected sinonasal tract disease (peripheral causes of olfactory deficits), except in the case of cerebrospinal fluid (CSF) leaks. Functional imaging studies, such as positron emission tomography (PET) and single photon emission computed tomography (SPECT), are valuable in detecting alterations of regional brain function and biochemistry in vivo (Alavi & Hirsch, 1991; Fowler et al., 1988; Jagust and Eberling, 1991; Jolles et al., 1989; Reman and Mintun,

Medical Imaging of Olfactory Deficits

1990). Recent studies have suggested that functional imaging is more sensitive than anatomical imaging in detecting abnormalities of the brain related to disorders such as Alzheimer’s disease and Parkinson’s disease—conditions associated with loss of olfactory function (Jagust and Eberling, 1991; Jolles et al., 1989). III. BASIC ANATOMY AND PHYSIOLOGY OF THE OLFACTORY SYSTEM Since the anatomy and physiology of the olfactory system is discussed elsewhere in this volume (see Chapters 1–9), we only briefly mention this topic here. The sensation of smell is induced by the stimulation of olfactory receptor cells by volatile chemicals. The olfactory receptor cells, i.e., the primary olfactory neurons, are encompassed in the neuroepithelium, which is located at the top of the nasal vault, the upper portion of the nasal septum, the superior surface of the superior nasal turbinate, sectors of the middle turbinate, and the region of the cribriform plate. Afferent information from the receptors is transmitted by the olfactory nerves, which course through the cribriform plate of the ethmoid bone to terminate in the glomeruli of the olfactory bulb. In the olfactory bulb, the olfactory nerves make synaptic contact with dendrites of mitral and tufted cells. From there, the efferent neurons of the olfactory bulb give rise to fibers forming the olfactory tracts, which lie just under the gyrus rectus region in the olfactory sulcus of the frontal lobes. Axons from mitral and tufted cells project to central brain limbic system components including the pyriform cortex and adjacent corticomedial amygdala (which together form the uncus), the ventral striatum, the parahippocampus area, and the anterior olfactory nuclei. From these areas there are widespread interconnections with many parts of the brain, including the mediodorsal thalamus, hypothalamus, orbitofrontal and dorsolateral frontal cortex, temporal cortex, and other areas of the limbic system (see Chapters 8 and 9). IV. PERIPHERAL CAUSES OF OLFACTORY DISTURBANCE Sinonasal tract disease is one of the common causes of olfactory disturbance (Deems et al., 1991; Doty and Mishra, 2001). The etiology of the olfactory deficits among patients with nasal and paranasal sinus disease is due, in many cases, to nasal airway obstruction. The influence of nasal obstruction on olfaction has been comprehensively reviewed (e.g., Doty and Frye, 1989; Doty and Mirshra, 2001) (see also Chapter 21). Any cause of bilateral obstruction can decrease smell sensations by limiting

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airflow to the olfactory receptors. Besides the obstructive effect, lesions located in the upper nasal vault and/or cribriform plate region may also directly damage the olfactory epithelium and olfactory neurons (Kern, 2000). The common peripheral sinonasal tract causes of olfactory deficits include infections, tumors, allergic rhinosinusitis, congenital or developmental abnormalities, etc. A.

Sinonasal Infectious Disease

Paranasal sinusitis is a relatively common disorder affecting approximately 30% of the population at some time in their lives (Allphin et al., 1991). One of the common symptoms of acute and chronic paranasal sinusitis is decreased smell sensation, which is generally reversible. The prompt diagnosis and treatment of sinusitis are important for restoring olfactory function. Though the exact cause of chemosensory dysfunction secondary to sinusitis is elusive, alterations in nasal air flow and mucociliary clearance or obstruction from secretory products, polyps, or retention cysts may contribute to olfactory dysfunction (Loury and Kennedy, 1991). In the diagnosis and evaluation of paranasal sinusitis, medical imaging plays an important role. At present, highresolution CT is the preferred imaging technique, preceded by nasal endoscopic examination. Radiographic manifestations of sinusitis have been well documented. In general, air-fluid levels are usually indicative of acute sinusitis, whereas mucoperiosteal thickening or sinus opacification can be seen in acute and chronic disease. Polyps, mucous retention cysts, sinus expansion, and bony thickening of the walls of the sinus might also indicate chronicity of disease. CT is an excellent modality for the evaluation of bony abnormalities, such as osteitis or remodeling, seen in some inflammatory lesions. CT also will identify the infundibulum, the maxillary sinus ostium, the middle meatus, the uncinate process, and the individual ethmoid air cells that make up the ostiomeatal complex. This will help the functional endoscopic sinus surgeon in his ability to plan effective surgery to restore normal mucociliary clearance. On the other hand, MRI is also highly sensitive for detecting mucosal thickening and other soft tissue abnormalities (Shapiro and Som, 1989). By and large, inflamed mucosa is usually high in signal intensity on T2-weighted MR images and low in intensity on T1-weighted scans. The signal intensity of the sinus secretions will vary with the concentration of protein within the sinus (Barat, 1990; Drutman et al. 1991; Shapiro and Som, 1989). B.

Tumors of the Nasal Cavity and Paranasal Sinuses

Neoplasms of the sinonasal tract are uncommon. Malignant tumors of the nasal cavity and paranasal sinuses account for

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only 0.2–0.8% of all human malignancies (Som, 1991). Early symptoms of sinonasal tract tumors, such as nasal discharge, unilateral nasal obstruction, and minor intermittent epistaxis, may simulate low-grade chronic infection. Subsequent symptoms depend on the tumor’s location and pattern of growth. Neoplasms arising in the upper nasal cavity and extending through the cribriform plate or into the ethmoid sinuses are often accompanied by frontal headache, visual disturbances, and decreased smell sensation. Almost all sinonasal tract tumors and tumor-like conditions that grow to a large size may cause a decline in olfactory acuity by interfering with patency of the nasal airway or directly destroying the olfactory receptors. The most common malignancies of the sinonasal system are squamous cell carcinoma and adenocarcinoma, but lymphoma, melanoma, adenoid cystic carcinoma, and chondrosarcomas also populate the nasal cavity. Two examples of intrinsic sinonasal tract tumors relatively unique to the sinuses (the olfactory neuroblastoma and the inverted papilloma, both of which often cause hyposmia or anosmia) may serve as prototypes for masses in this region. 1. Olfactory Neuroblastoma Olfactory neuroblastoma, or esthesioneuroblastoma, is a rare nasal tumor originating from the olfactory neuroepithelium lining the roof of the nasal vault and in close proximity to the cribriform plate. There have been less than 300 reported cases in the world literature. Olfactory neuroblastomas occur in all age groups with a peak incidence in the 11–20 and 51–60 year groups. There is a slight preponderance of the tumor in women. The incidence of olfactory neuroblastoma has been estimated to range from 2 to 3% of all malignant intranasal neoplasms. The most common symptoms are unilateral nasal obstruction and recurrent epistaxis. Hyposmia or rhinorrhea is not unusual. Extension into the orbit, paranasal sinuses, or anterior cranial fossa may cause vision disturbances and headache (Elkon et al., 1979; Li et al., 1993; Newhill et al., 1985). In the detection and staging of olfactory neuroblastoma, CT and/or MRI play an important role. Generally speaking, MRI is more accurate than CT in showing the tumor’s intracranial extent. MRI is also exquisitely useful for differentiating neoplasm from postobstructed secretions because of the difference in the signal intensity (secretions are bright on T2, tumor intermediate). Unfortunately, signal intensity characteristics of various sinonasal tract tumors overlap each other, so MRI cannot usually predict specific tumor histology. However juvenile angiofibroma can usually be distinguished from other tumors on the basis of its high vascularity and marked enhancement.

Li et al.

A recently described imaging finding characteristic of olfactory neuroblastomas is the presence of peripheral peritumoral cysts along the intracranial portion of the tumor. If stippled calcifications are also seen on CT, the diagnosis is assured. 2.

Inverted Papillomas and Other Sinonasal Tumors

The inverted papilloma is a relatively rare and locally aggressive sinonasal tumor. It constitutes 0.5–4% of primary nasal tumors and occurs predominantly in males in the fifth and sixth decades of life (Phillips et al., 1990). The most common presenting symptoms are nasal obstruction, epistaxis, and hyposmia. Subsequent sinusitis and tumor extension into the sinuses and orbits can cause purulent nasal discharge, pain, and diplopia (Som, 1991). Radiographic findings of inverted papilloma can vary from a small nasal polypoid nodule to an expansile large mass, which may remodel the nasal vault and extend into the sinuses, orbits, or even the anterior skull base. CT and MRI are very useful in defining the location and extension of the tumor (Buchwald et al., 1990; Yousem et al., 1992) (Fig. 1.). Calcification is not uncommon in this tumor. Other sinonasal tract tumors, such as squamous cell carcinoma, adenocarcinoma, melanoma, etc., can also cause hyposmia or anosmia during their late stage. Squamous cell carcinoma accounts for 80% of paranasal sinus malignances, is most commonly seen in the maxillary sinus, and usually demonstrates bone destruction at the time of presentation. Adenocarcinomas occur most frequently in the ethmoid sinus while melanoma is usually seen intranasally. Additional benign neoplasms known to affect the sinonasal cavity include osteomas, enchondromas, schwannomas, and juvenile angiofibromas. Osteomas are usually identified in the frontal sinus and may be a source for recurrent headache and /or recurrent sinusitis. The classic story of a frontal sinus osteoma narrowing the sinus opening is a patient who has severe sinus pain associated with takeoffs from airplane flights. This is a benign mass, which is often completely invisible on MRI due to the presence of dense compact bone making up the mass. On the other hand, it is easily identified on CT as a markedly hyperdense bony mass protruding in the sinus. Occasionally, the osteoma will result in mucocele formation and/or pneumocephalus as the posterior wall of the frontal sinus is breached. Enchondromas are less common neoplasms of the sinonasal cavity which, on CT, often have a popcorn calcification appearance different from the stippled calcification of inverted papillomas. This lesion, because of its characteristic calcification, is best evaluated with CT. Schwannomas of the fifth cranial nerve are the most common to affect the sinonasal cavity. They will typically

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Figure 1 A 40-year-old woman with 3-month history of decreasing smell sensation and left nasal obstruction. (A) Bone-targeted coronal CT shows an expanded opacified left nasal cavity with bowing of the lateral nasal wall (arrows) and opacification of the left maxillary and both sphenoid sinuses. (B) Axial contrast-enhanced CT scan shows erosion through the left lamina papyracea (arrow) with displacement of the medial rectus and globe laterally. The differentation between tumor and obstructed secretions is not readily apparent with CT. Histological diagnosis: nasal cavity carcinoma arising within a dysplastic inverted papilloma.

follow the course of the nerve and can expand skull base foramina through which they travel. The signal intensity of schwannomas varies according to the content of the dense Antoni A tissue or loose Antoni B tissue, the latter being brighter on T2W scans. Schwannomas enhance avidly, although they may have inhomogeneity to the enhancement. Finally, one has the juvenile angiofibroma, a fascinating benign neoplasm, which appears to arise in the region of the sphenopalatine foramen and/or the pterygopalatine fossa The lesion accounts for 0.5% of head and neck masses and is typically seen in adolescent males who present with epistaxis and/or a nasal mass (Mehra, 1989). The lesion is highly vascular as exemplified on MRI by the signal flow voids within the lesion and its marked contrast enhancement. Because of its propensity for spreading via the canals and foramina at the skull base, MRI is probably the study of choice for the evaluation of this neoplasm. Embolization of these lesions will assist the surgeon in limiting blood loss if resection is considered. 3. Malignant Neoplasms CT and MRI probably play complementary roles in the evaluation of sinonasal malignancies because of CT’s superiority in defining bony margins and MRI’s superior soft tissue resolution and ability to define intracranial or

intraorbital spread. One of the advantages of MRI is the ability to distinguish sinus neoplasm from postobstructive secretions. This may be difficult by CT if the secretions are isodense to the mass and if the malignancy does not enhance dramatically. If one was forced to study the patient with a single modality, the literature supports MRI as the best study for the staging of sinonasal malignancies (Hunink et al., 1990; Kraus et al., 1992; Paling et al., 1987; Sisson et al., 1989). Som et al. (1991) noted that squamous cell carcinoma (low in T2 intensity) could be distinguished from inflammation (high in T2 intensity). They compared CT to MRI for mapping sinonasal tumors. They found that MRI and CT were equivalent in 23 of 53 patients in defining tumor extent and that MRI was superior to CT in 26 patients. Of the 4 cases in which CT was superior, subtle bony erosion (2) and osteo(1)-cartilaginous(1) lesions accounted for the “misses” on MRI. Of 60 inflammatory lesions, MRI was superior (Bonte et al., 1993) or equivalent (Everall et al., 1991) to CT in all cases. Inflammation (bright) and neoplasm (intermediate) could be distinguished in 95% of cases based on T2W signal intensity. Even when the sinus secretions become increasingly inspissated and the signal intensity on T2W scans decreases, the neoplasm can be distinguished from the obstructed secretions by its typical heterogeneity as opposed to the smooth homogenous

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appearance of sinus secretions. This is also true in the cases of mucoceles, which may occur after or in association with sinus neoplasms. Additionally, MRI has shown that most squamous cell carcinomas of the sinonasal cavity enhance with gadolinium in a solid fashion as opposed to a peripheral rim of enhancement in sinus secretions and/or mucoceles. Unfortunately, lymphomas, undifferentiated carcinomas, inverted papillomas, and some sarcomas may have identical signal intensity and enhancement characteristics as squamous cell carcinoma. Gadolinium is particularly useful for demonstrating epidural or meningeal invasion of neoplasms. Often, postcontrast scans must be combined with fat suppression techniques in order to identify enhancement amidst the abundant skull base fat. In one series, 75% of patients with intracranial extension of sinonasal malignancies had additional information about tumor extent demonstrated with postcontrast MRI studies (van Tassel et al., 1991). Subtraction MRI of pregadolinium scans from postgadolinium scans may improve visibility of such subtle enhancement (Lloyd and Barker, 1991). It should be noted that meningeal enhancement need not necessarily imply neoplastic invasion; just as in cases of meningioma, the dura may enhance because of reactive fibrovascular changes alone. When one encounters a sinonasal mass that is eroding intracranially, one must consider carcinoma, olfactory neuroblastoma, sarcomas, lymphomas, sinonasal polyposis, and inverted papillomas. Twelve percent of patients with polyposis and mucoceles eventually erode the skull base (Som et al., 1991). The pattern of bone destruction may be similar between malignant and benign lesions at the non–sinus bearing skull base. Bone remodeling in this location is a rarity; a permeative pattern is the norm for all lesions. Som et al. (1988) have suggested that a lesion with homogeneous signal intensity invading intracranially is more likely to be a malignancy, whereas heterogeneity suggests an inflammatory cause. Unfortunately, necrosis, hemorrhage, or calcification in carcinomas, olfactory neuroblastomas, or sarcomas may cause signal heterogeneity. Polyps generally enhance in a peripheral pattern; true neoplasms enhance solidly. Malignancies have a broad flat base of skull erosion; benign conditions have a rounded polypoid intracranial excrescence. Squamous cell carcinomas account for 80% of the malignancies to affect the paranasal sinuses and 80% in the maxillary sinus. The hallmark of malignancies of the sinonasal cavity is bony destruction, seen in approximately 80% of CT scans of sinonasal squamous cells carcinoma at initial presentation. The lesion is confined to the maxillary antrum in only 25% of cases at presentation (Lyons and Donald, 1991). In most series documenting sinonasal

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squamous cell carcinoma signal intensity characteristics on MRI, the lesion is characterized by a low signal intensity on T2W scans. This is why differentiation with obstructed secretions which are typically bright in signal intensity on T2W scans is so easy on MRI. Because of Som et al.’s early work depicting sinonasal malignancies as hypointense on T2W scans, people have come to rely on this pulse sequence for mapping cancers (Som et al., 1990). Unfortunately, low intensity on T2W scans is an inconstant finding in sinonasal malignancies in general. Hunick et al. found that over 50% of head and neck malignancies had signal intensity on T2W scans that was brighter than muscle and isointense to brain (Hunink et al., 1990). Approximately 25% of benign tumors had the same intensity pattern. Lanzieri et al. (1991) also reported that the signal intensities of tumors, mucoceles, schwannomas, and obstructed secretions may show some overlap. Som et al. (1991) have found that minor salivary gland masses and schwannomas may have T2W signal intensity similar to that of inflammatory lesions. Minor salivary gland tumors and melanoma are the next most common malignancies to affect the sinonasal cavity after squamous cell carcinoma (van Tassel et al., 1991). The minor salivary gland tumors represent a wide variety of histological types including adenoid cystic carcinoma, mucoepidermoid carcinoma, adenocarcinoma, and undifferentiated carcinoma. Of these tumors, adenoid cystic carcinoma is the most common variety. Its signal intensity may be high or low on T2W scans, possibly related to the degree of tubular or cribriform histological pattern as well as cystic spaces, necrosis, and tumor cell density. Tissue specificity is not readily achievable with MRI or CT. Gadolinium is of particular use with adenoid cystic carcinomas, which have a propensity for perineural spread (Graamans and Slootweg, 1989). With sinonasal cavity malignancies one should always attempt to trace back the branches of the fifth cranial nerve via the pterygopalatine fossa, foramen rotundum, foramen ovale, and orbital fissures in order to identify perineural neoplastic spread. Adenocarcinomas of the paranasal sinuses have a predilection for the ethmoid sinuses and appear more commonly in woodworkers. This tumor also tends to have low signal intensity on T2W MRI images but may have high signal intensity in a small percentage of cases. Sarcomas of the sinonasal cavities are very rare, with chondrosarcoma being the most common. Again, the histological diagnosis is probably better suggested by CT based on the characteristic whorls of calcification. However, for staging, MRI is competitive with CT, and, particularly if repeat examinations are going to be required, follow-up with MRI to avoid the radiation exposure of CT is recommended.

Medical Imaging of Olfactory Deficits

Melanoma is a tumor that is usually identified in the nasal cavity as opposed to the paranasal sinuses. It has been associated with melanosis in which there is field deposition of melanin along the mucosal surface of the sinonasal cavity. Therefore, multiplicity of lesions becomes a problem when dealing with melanomas. Neither CT nor MRI is particularly helpful in identifying the field “cancerization” of melanoma. When melanoma contains melanin there is paramagnetism which causes T1 and T2 shortening accounting for high signal intensity on T1W scans and low signal intensity on T2W scans (Atlas et al., 1990). However, an amelanotic melanoma may have bright signal intensity on T2W scans. The presence of hemorrhage associated with the melanoma, a common occurrence because of the coincidence of epistaxis, may further obfuscate the signal intensity pattern (Yousem et al., 1996c). Lymphoma does occur in the paranasal sinuses and may have variable signal intensity as well. It is characterized by homogeneous signal intensity without necrosis and the association with cervical lymphadenopathy. Metastatic disease to the paranasal sinuses is extremely rare. Of the primary causes of metastases to the sinuses, renal cell carcinoma is probably the most common. This tumor also has a propensity for hemorrhage and may also have a variable signal intensity depending upon the stage of hemorrhage. C.

Allergic Reactions

Allergic rhinitis is a common upper airway condition affecting about 30 million Americans with peak prevalence in the age group from 35–54 years (Baroody and Naclerio, 1991). Hyposmia or anosmia is common with allergic rhinitis, mainly caused by nasal obstruction by polyps or inflamed mucosa, which limit access of inspired air to the roof of the nasal vault (Cowart et al., 1993). The diagnostic work-up begins with a careful history, which attempts to identify offending allergens. Skin testing of specific antigens is often used to confirm the diagnosis. Medical imaging studies play a supplementary role in the evaluation of sinonasal airway status and differential diagnosis. CT and MRI are also important for detecting any complications such as sinusitis, mucoceles, and aggressive polyps in patients with allergic rhinitis. Rounded excrescences and enlargement of ostia are seen in the airway of patients with polyposis. D.

Congenital or Developmental Abnormalities

It is generally accepted that normal variations in the nasal anatomy may play a role in preventing the access of an

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odorant to the olfactory receptor area. The sense of smell is probably less than normal in many patients with craniofacial anomalies (Crysdale, 1981). Congenital developmental abnormalities include choanal atresia, hereditary nasal septal deviation, facial hypoplasia, cleft palate, nasal dermoids and epidermoids, cephaloceles, and gliomas, etc. Medical imaging techniques, especially high-resolution CT, play a key role to detect and evaluate the facial and bony changes (Barkovich et al., 1991; Klein et al., 1987). CT is most useful because surgical correction requires identification of and closure of the osseous abnormalities. MRI is most effective in defining soft tissue masses such as cephaloceles and nasal gliomas. Congenital anosmia can be associated with a number of developmental and inflammatory conditions. Kallmann’s syndrome, also known as hypogonadotrophic hypogonadism with anosmia, is a congenital X-linked disorder in which the olfactory bulbs and tracts are not formed. This is not associated with holoprosencephaly, and the usual deficits are related to hormonal abnormalities in the pituitary gland with the loss of sense of smell. Infertility often coexists. In 1993, an MR study of the olfactory system in Kallmann’s disease showed absence of the olfactory bulbs and tracts in 17 of 18 patients while confirming the presence of the olfactory bulbs and tracts in all 10 studied patients with idiopathic hypogonadotropic hypogonadism (Yousem et al., 1993, 1996a). Some patients have absence of the olfactory bulbs and tracts without Kallmann’s syndrome. It is unclear whether this represents congenital absence or whether an inflammatory condition early in infancy destroys the olfactory bulbs and tracts. Certain viruses have a propensity for injuring the olfactory system. A recent study has noted the incomplete formation of olfactory sulci in patients with congenital anosmia as well as a variable percentage of aplastic olfactory bulbs, tracts, and tubercles (Di Rienzo et al., 2002). Still others may have congenital absence of sense of smell on the basis of early head trauma where the ciliary nerves as they crossed the cribiform plate may be sheared and the olfactory system is affected. Infectious causes may also affect the sense of smell in early childhood, usually secondarily to viruses. In these cases one sees the olfactory bulbs and tracts; but they are not functional. Holoprosencephaly is a congenital, multiple midline malformation disorder that has a known association with sensory deficits of vision and olfaction. Although variable amounts of aplasia and hypoplasia of the olfactory apparatus may be identified, the most common MR finding is complete absence of the olfactory bulbs, occurring in 92% of patients. A high association with absence of the olfactory nerves and tubercles is also seen. There does appear to be some, albeit poor, differentiation of the olfactory sulci and gyri recti, which were absent only in a little over half of the subjects (Barkovich and Quint, 1993).

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Other Peripheral Causes

It is estimated that 30 million Americans have used cocaine and 5 million use it regularly (Gregler and Mark, 1986). Intranasal use of cocaine and heroin has reached epidemic proportions in the United States. Although hyposmia or anosmia has been suggested to occur often in cocaine abusers, few studies using quantitative measures of olfactory function have confirmed such reports. A sole study on this topic reported that of 11 cocaine abusers who underwent detailed olfactory testing, only one was found to be anosmic and another had mild olfactory discrimination dysfunction (Gordon et al., 1990). These authors note that most cocaine abusers do not develop permanent olfactory dysfunction. If, in fact, olfactory disturbance occurs as a result of heavy cocaine use, it could be due to associated conductive disorders, nasal airway obstruction, alteration in sinonasal aerodynamics, damage to the olfactory epithelium, damage to the central olfactory system, or osteolysis of the cribriform plate (Kuriloff, 1989). Concerning the conductive disorders, several reports of osteolytic sinusitis and extensive osteocartilaginous necrosis of the nasal septum in cocaine abusers have been described (Newman, 1988; Schweitzer, 1986). Erosion of nasal septal cartilage is a known complication of cocaine abuse. Within the differential diagnosis for cartilaginous destruction, one should include Wegener’s granulomatosis, syphilis, leprosy, lymphoma, rhinoscleroma (a klebsiella infection), and fungal invasion. CT, preferably in the coronal plane, can provide excellent views of septal perforation, osteolysis, and sinusitis. To evaluate intracranial disorders associated with cocaine, MRI is the study of choice. Vasculitic infarcts, hypertensive hemorrhages, and white matter ischemic foci may be seen with MRI. Recently Tumeth and colleagues demonstrated multifocal cortical deficits with a special predilection for the frontal and temporal lobes on SPECT perfusion brain scans in chronic cocaine abusers (Tumeth et al., 1990). Similar findings have been reported by others (Holman et al., 1991; Kolow et al., 1988). These findings may suggest a central basis for some cases of cocainerelated decreased olfaction. Some studies also have revealed that cerebral atrophy develops in chronic cocaine abusers and that the severity correlates with the duration of abuse (Pascual-Leone et al., 1991). Anosmia or hyposmia is a frequent sequela of highlevel midface fractures in which the olfactory nerves may be severed at the level of the cribriform plate (Kassel, 1988; Mathog, 1992). Because ethmoid complex and cribriform plate fractures are difficult to detect on plain radiographs, thin-section coronal CT is the best

measure to assess naso-ethmoid trauma (Daly et al., 1990; Kassel, 1988). V. CENTRAL CAUSES OF OLFACTORY DISEASES There are numerous CNS disorders that are associated with olfactory dysfunction. The most common types fall in the categories of degenerative neuropsychiatric disorders, hereditary conditions, trauma, and central neoplasms. Of course, in some disorders the involvement of both peripheral and central neural processes may occur. A.

Alzheimer’s Disease

It has been well documented that olfaction is significantly altered in Alzheimer’s (AD). Nearly all studies of olfactory function in patients with AD have reported decreased smell relative to age-matched controls (see Chapter 23). These studies demonstrate marked impairment of smell function in early AD, whether measured by identification, discrimination, or threshold sensitivity (Doty, 1991; Doty et al., 1987; Serby et al., 1991). Recent neuropathological studies have correlated well with these clinical findings. The anterior olfactory nuclei in AD patients contain senile plaques, neurofibrillary tangles, granulovascular degeneration, and cell loss (Averback, 1983; Esiri and Wilcock, 1984). The olfactory bulbs also show involvement (Esiri and Wilcock, 1984; Ohm and Braak, 1987), as does nasal sensory epithelium (Jafek et al., 1992). In addition, central olfactory structures, especially the amygdala and the entorhinal, pyriform, and temporal cortices, are frequently affected by Alzheimer’s disease (Harrison, 1986; Pearson and Powell, 1989). Besides the above findings, devastating nerve cell loss and gliosis in the region of the hippocampal formation have been observed at autopsy in AD patients (Ball et al., 1985; Hyman et al., 1984). Neuroimaging has played an important role in detecting some of the pathological changes of AD patients in vivo, and its uses are growing, both for clinical evaluation and as a research tool. Early CT studies in AD patients demonstrated generalized enlargement of the ventricular system and sulci (George et al, 1981; Naser et al., 1980). Several reports have noted that ventricular and sulcal enlargement correlate with the severity of AD (Albert et al., 1984; George et al., 1983). However, these findings are not specific and have relatively weak correlations. de Leon and colleagues (1989) have emphasized the rate of change in ventricular size with repeated CT scans as an important index in the diagnosis of AD. Recently, several investigators have recog-

Medical Imaging of Olfactory Deficits

nized that CT and/or MRI delineation of atrophic changes in the temporal lobe and the hippocampus with enlargement of hippocampal-choroidal fissures strongly support the diagnosis of AD (de Leon et al., 1988; George et al., 1990; Kesslak et al., 1991; Kido et al., 1989). McDonald and colleagues (1991) reviewed MRI scans in 22 patients with early-onset AD. The results showed that patients with AD were significantly more likely than agematched controls to have MR evidence of periventricular hyperintensities on T2W scans. This study suggested that the increased frequency of periventricular hyperintensities may have a relationship to the disease process. Our own experience with MRI studies of AD patients is that most of the cases with AD have, in addition to ventriculomegaly and sulcal widening, significantly reduced volume of the temporal lobe and slight atrophy of olfactory bulbs. (Fig. 2). Besides CT and MRI, SPECT and PET techniques are also useful for evaluating regional cerebral blood flow,

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regional oxygen, and glucose metabolism, which may provide evidence supportive of the diagnosis of AD (Jagust and Eberling, 1991). The above-mentioned structural atrophic changes by CT and MRI are also supported by functional imaging studies (McDonald et al., 1991; Ohnishi et al., 1991). The major findings of functioning imaging studies in patients with AD are abnormal regional cerebral blood flow pattern and flow reduction. The common sites of blood flow reduction are in the temporoparietal region and the frontal areas. In one report (Bonte et al, 1993), seven patients with possible diagnosis of AD studied by SPECT showed only frontal flow abnormalities. Is this an early imaging finding which may suggest a pathophysiologic basis to explain the decreasing smell sensation in AD? Of course, more studies are needed for further discovering the nature of AD. We believe that early and correct diagnosis of AD in vivo by neuroimaging techniques will be possible in the near future. There is a dose-related association between apolipoprotein E-4 (APOE-4) allelic frequency and the development of AD (APOE-2 may confer protection). Recent studies have shown a decline in resting parietal, temporal, and prefrontal PET glucose metabolism in cognitively intact patients with APOE-4. It remains to be seen whether this, and/or an analogous fMRI study, may serve to be a predictor of development of AD. Recently some investigators have used dynamic contrast susceptibility contrast imaging MR to try to duplicate the nuclear medicine flow studies. Indeed they have found that relative values of temporoparietal regional cerebral blood volume (as a percentage of cerebellar rCBV) were reduced by a factor of 20% bilaterally in the patients with Alzheimer disease compared to normals. Using left and right temporoparietal rCBV as index measures, specificity was 96% and sensitivity was 95% in moderately AD and 88% in mild AD (Harris, 1998).

B.

Figure 2 A 60-year-old woman with Alzheimer’s disease. UPSIT scores revealed severe bilateral anosmia. (A) Normal olfactory bulbs are seen (arrows) on coronal MR. Dilation of the olfactory sulci (arrowheads) reflects generalized atrophy. (B) Coronal MR scan through the temporal lobes shows temporal horn enlargement and atrophic changes, slightly worse on the right side.

Parkinson’s Disease

Odor detection and identification are significantly impaired in Parkinson’s disease (PD) patients (Doty et al., 1988, 1995; Montgomery et al., 2000) (see Chapter 23). It is unclear whether the olfactory deficits in PD and AD share the same cause. Not surprisingly, PD research into the cause of smell dysfunction has focused on dopaminergic changes. Brooks and colleagues (1991) have demonstrated by using PET that patients with PD show significantly reduced mean uptake of 18F-dopa in the caudate and putamen, especially in the posterior part of the putamen. Previous functional imaging studies have also indicated a reduction of striatal dopamine storage in PD.

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PET technique with 18F-dopa in PD patients has also demonstrated reduced basal ganglia activity (Alavi and Hirsch, 1991). However, the olfactory deficit is unrelated to severity of motor or cognitive symptoms and is not improved by L-dopa therapy (Doty et al., 1992), so the underlying causes of olfactory dysfunction in PD still requires more study. CT scanning has little role in establishing the diagnosis of PD other than to exclude mass lesions in the brain. In general, CT shows no specific striatal abnormalities and occasionally only mild, nonspecific ventricular and sulcal enlargement. The major feature of PD on MRI appears to be a trend towards a decreased width of the pars compacta of the substantia nigra (Braffman et al, 1989). There is a lateral to medial gradient of loss of the normal signal of the pars compacta as well as volume loss. Moderate or marked cortical atrophy tends to occur more frequently in PD patients than in controls. MRI may occasionally show abnormal decreased T2W intensity in the putamen and to a lesser degree in the caudate nuclei and substantia nigra, suggestive of iron deposition (Drayer et al, 1986). C.

Huntington’s Disease

Patients with Huntington’s disease (HD) evidence olfactory dysfunction (Doty, 1991; Moberg et al., 1987). Neuropathological studies in HD have demonstrated premature neuronal cell death and reactive gliosis occurring most markedly in the head of the caudate nuclei bilaterally (Myers et al., 1991; Vonsattel et al., 1985). A loss of 70–80% of the striatal neurons may occur before functional impairment is obvious. Similar but less extensive changes also affect the putamen. Later, atrophy of the cerebral cortex occurs as well. All of these progressive atrophic changes can be identified on CT and MRI scans, especially in the caudate nuclei, where the volume of the caudate head decreases and the intercaudate distance increases (Simmons et al., 1986; Starkstein et al., 1989). Increased signal intensity in the putamen and globus pallidus has been described in the juvenile form of Huntington’s disease, and frontal atrophy is usually present. PET studies of patients with HD have consistently demonstrated hypometabolism in the caudate nuclei, often before the development of atrophy on CT (Hayden et al., 1986). SPECT studies involving HD patients have also revealed decreased uptake in the caudate nuclei, including the caudates of one patient with early disease and no evidence of atrophy on MRI (Nagel et al., 1988; Reid et al., 1988). Thus, functional imaging with PET or SPECT may contribute to the early diagnosis of HD. Theoretically, the input of caudate/putaminal fibers to the limbic system and striatum may be altered, leading to

olfactory dysfunction, but the exact mechanism for hyposmia in HD patients remains to be worked out. D.

Korsakoff’s Psychosis

Patients with Korsakoff’s psychosis (KP) exhibit impaired odor detection, identification, and intensity estimation (Jones et al., 1975; Mair et al., 1986). An animal model study has shown that the behavior of rats recovering from pyrithiamine-induced thiamine deficiency share several important features with KP patients, including the impairments observed for smell, hearing, learning, and memory (Mair et al., 1991). The mechanism of hyposia and/or dysosmia in patients with KP is unclear and still under investigation. Olfactory perception may be selectively impaired in KP by the diencephalon lesions that are characteristic of this disease. Degeneration in the mediodorsal thalamic nucleus (the common neuropathological lesion in KP) and atrophy in the prefrontal areas may also cause the olfactory dysfunction (Mair et al., 1986). A quantitative neuropathological study of the human cerebral cortex has shown that the number of cortical neurons in the superior frontal lobe in chronic alcoholic patients is significantly reduced (Happer et al., 1987). Chronic alcoholism is also associated with smaller volumes of cortical white and gray matter relative to controls (Pfefferbaum et al., 1995). Traditional neuropsychological tests and functional imaging studies have also demonstrated disturbances of frontal-lobe function and metabolic deficits in patients with KP (Joyce and Robbins, 1991; Kopelman, 1991; Metter et al., 1989). Brain CT scans have demonstrated that KP patients show more pronounced third and lateral ventricular dilatation and wider interhemispheric fissures than matched groups of normal controls and non-Korsakoff alcoholics (Jacobson and Lishman, 1990; Ron, 1983; Ron et al., 1982). Shrinkage in the frontal lobes and cerebellum appears to be especially pronounced (Jacobson and Lishman, 1990). A MRI study (Jernigan et al., 1991) has revealed that patients with KP show widespread reductions in gray matter volumes in addition to CSF increases, with the greatest reductions observed in diencephalic structures. The volume losses that best differentiate the KP patients from the alcoholic controls included losses in anterior portions of the diencephalon, mesial temporal lobe structures, and orbitofrontal cortices (areas involved in olfaction perception). Several other studies (Donnal et al., 1990; Gallucci et al., 1990; Squire et al., 1990; Victor, 1990) have also demonstrated that MRI is highly sensitive in detecting reversible diencephalon (medial thalamic) and mesencephalic (periaqueductal) lesions. In addition to generalized cortical and cerebellar vermian atrophy seen on CT

Medical Imaging of Olfactory Deficits

and MR, recent reports have noted the presence of high signal intensity areas in the periaqueductal gray matter of the midbrain (40%), the paraventricular thalamic regions (46%), the mamillothalamic tract, and in tissue surrounding the third ventricle on T2W MR scans (T2WI). Reversible thalamic lesions in the dorsal medial nuclei have also been reported. These areas may or may not enhance (in some cases the enhancement may be dramatic, almost sarcoid-like) and may be associated with mamillary body atrophy. Mamillary body enhancement may be the sole manifestation of Wernicke’s encephalopathy. Myelin degeneration, mamillary body volume loss, intracellular edema, and microglial proliferation are seen pathologically (but may be present in alcoholics without Wernicke’s). MRI findings in patients with KP may enable early diagnosis of the disease, which may have a positive effect on both treatment and prognosis (Gallucci et al., 1990). E.

Schizophrenia

Impaired olfactory function has been reported in schizophrenics, especially males (see Chapters 23 and 24). These olfactory deficits, which are not of the same magnitude as those seen in AD and PD, are perhaps not unexpected given the occurrence of olfactory hallucinations as symptoms in a number of patients with schizophrenia and the evidence linking both to temporal lobe dysfunction (Rausch et al., 1977; Roberts, 1988). Neuropathological studies in schizophrenic patients have reported neuronal loss in the entorhinal region and prefrontal cortex, gliosis in the basal limbic structures of the forebrain, and atrophy in temporolimbic structures (Benes et al., 1986; Falkai et al., 1988). Neurophysiological function studies (including regional cerebral blood flow, brain electrical activity mapping, and regional metabolic activity in the brain) in patients with schizophrenia have demonstrated prefrontal cortex and temporal lobe dysfunction (Mesulam, 1990). Functional imaging, such as PET or SPECT, in the study of schizophrenia is limited and inconclusive. However, functional imaging has provided some evidence that certain schizophrenic patients have decreased blood flow and metabolism in the frontal lobes (hypofrontality) (Alavi and Hirsch, 1991). Anatomical imaging findings have basically paralleled the neuropathological changes in the brains of patients with schizophrenia. The most consistent finding on both CT and MRI is an increase in the size of the cerebral ventricular system, especially in the frontal and temporal horns, and corresponding decreases in cerebral tissue, especially in the prefrontal cortex and in medial temporolimbic structures (Mesulam, 1990; Suddath et al., 1989; Young et al., 1991). Suddath and colleagues (1989)

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evaluated the volume of the temporal lobes in schizophrenias by a quantitative MRI study. The results showed that the volume of temporal lobe gray matter was 20% smaller in the patients than in the control subjects, and lateral ventricular volume was 67% larger in the schizophrenia group than in the control group. Schizophrenic patients tend to have smaller hippocampi that matched controls. schizophrenias are also reported to have cavum septum pellucidum more frequently than controls. In a recent study, Turetsky et al. (2000) reported that patients with schizophrenia exhibited 23% smaller olfactory bulb volume bilaterally than comparison subjects by a quantitative MRI study. F.

Congenital Anosmia

Congenital anosmia, which traditionally has been defined as anosmia present from a patient’s earliest recollection, has been recognized for centuries. The most common form of congenital anosmia is Kallmann’s syndrome or olfactory dysplasia, which is characterized by hypogonadotropic hypogonadism and anosmia (Kallmann et al., 1944; Lieblich et al., 1982). The incidence of Kallmann’s syndrome is about 1:100,000 in men and 1:50,000 in women. There has been increasing interest in the pathology, pathophysiology, and genetics of this disorder. Pathological and surgical studies of patients with Kallmann’s syndrome have shown agenesis of the olfactory bulbs (DeMorsier and Gauthier, 1963; Males et al., 1973). Laboratory findings include decreased serum follicle-stimulating hormone and luteinizing hormone as well as decreased urinary gonadotropins (Lieblich et al., 1982). In medical imaging studies, CT is a limited tool for the demonstration of sinonasal and intracranial abnormalities in patients with congenital anosmia (Klein et al., 1987; Moorman et al., 1984). Surface coil MRI is the optimal modality to reveal the intricate details of the olfactory bulbs, tracts, and rhinencephalon in vivo. Klingmuller and colleagues (1987) have clearly demonstrated the olfactory sulci in a normal control group by MRI, but not in the patients with olfactory dysplasia. More recently, the authors have studied two cases with Kallmann’s syndrome by MRI. Both showed no olfactory bulb at all and flattening of the gyrus recti (Yousem et al, 1993, 1996a); frontal and temporal lobe volumes were normal (Fig. 3). In a mixed population of patents with congenital anosmia, we found olfactory bulb and tract absence (68–84%) and hypoplasia (16–32%) in all 25 cases studied. Eight individuals had Kallmann’s syndrome (hypogonadotropic hypogonadism with anosmia). Temporal and/or frontal lobe volume loss were noted in 5 individuals, mild in all but one individual. We concluded that congenital anosmia

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G.

Figure 3 (A) Coronal 500/20 scan from normal volunteer (64-year-old woman with normal smell function) demonstrates normal olfactory bulbs (arrows). (B) Coronal 500/17 scan of 27-year-old woman with congenital anosmia without Kallmann’s syndrome shows extremely atrophic olfactory bulbs (arrows). (C) Coronal 500/14 scan of 29-year-old male patient with Kallmann’s syndrome evidences absence of olfactory bulbs and tracts with flattened gyrus rectus (arrow) on the right side, but with normal-appearing gyrus rectus on the left side.

or hyposmia appears to be an olfactory bulb tract phenomenon rather than a central process (Yousem et al., 1996a).

Head Trauma

Craniofacial trauma can alter olfactory ability through one of several mechanisms: (1) damage to the nose, sinuses, or both with resultant mechanical obstruction to odorants, (2) shearing of olfactory filaments as they course through the cribriform plate, (3) contusion to the olfactory bulb, and (4) contusion or shearing injury of the cerebral cortex, particularly the frontal and temporal lobes (see Chapter 30). The incidence of anosmia or hyposmia after head trauma has been reported quite variably from 2 to 38%, (Deems et al., 1991; Doty et al., 1997; Hagan, 1967; Leigh, 1943; Levin et al., 1985; Schechter and Henkin, 1974; Summer, 1964; Zusho, 1982) and increases with the severity of injury (Levin et al., 1985; Summer, 1964). However, even a minor injury can sometimes result in anosmia or hyposmia (Schechter and Henkin, 1974; Summer 1964). Recent evidence has shown that the location of the hematoma or contusion of the brain after head trauma is one of the most important factors leading to olfactory dysfunction (Costanzo and Zasler, 1991; Doty et al., 1997; Levin et al., 1985; Yousem et al., 1996b). Specifically, diminished olfactory discrimination has been confirmed in patients with prefrontal lesions (Potter and Butters, 1980). Animal studies have shown that the prefrontal olfactory area plays a prominent role in the fine and specific discrimination of odors (Tanabe et al., 1975). Besides prefrontal lesions, temporal lobe structures are also involved in the odor processing of odor perception (Rausch and Serafetinides, 1975; Rausch et al., 1977). Indeed frontal or temporal lobe hematomas or contusions are now believed to be one of the most common causes of olfactory dysfunction after head injury (Costanzo and Zaster, 1991; Doty et al., 1997; Levin et al., 1985; Schellinger et al., 1993; Yousem et al., 1996b) (Fig. 4). It has been established that plain skull radiography plays only a small role in the evaluation of head trauma (Masters et al., 1987). CT currently is the study of choice when diagnostic imaging is necessary after acute head trauma (Cohen, 1990; Kelly et al., 1988). CT can detect subarachnoid hemorrhage, fractures, and intraventricular blood, lesions for which MRI is less sensitive acutely. CT can be performed with close patient monitoring in a rapid fashion. However, MRI is superior to CT in the detection and characterization of subacute injuries, hemorrhage outside the subarachnoid space as in subdural hematomas, cortical contusion, and shearing injuries. MRI is exquisitely sensitive to diffuse axonal injuries leading to demyelination. MRI is also useful in the follow-up of brain contusion and/or hemorrhage, thereby eliminating the radiation exposure associated with CT (Cohen, 1990; Zimmerman et al., 1986).

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tory test scores (Doty et al., 1997b; Yousem et al., 1996b). Abnormalities on MR in patients with posttraumatic olfactory dysfunction occur at a very high rate (88%), predominantly in the olfactory bulbs, tracts, and inferior frontal lobes. Qualitative and quantitative assessments of damage show little correlation with olfactory tests probably due to multifocal injury, ciliary nerve damage, and the constraints of small sample size. H.

Brain Tumors

The incidence of chemosensory changes caused by intracranial tumors has rarely been investigated. In a study of 750 consecutive patients presenting with chemosensory disorders to the University of Pennsylvania Smell and Taste Center, only two cases (0.3%) were induced by brain tumors (Deems et al., 1991). In one study anosmia was reportedly present in 19 of the 26 cases of FosterKennedy syndrome (retrobulbar optic neuritis, central scotoma, optic atrophy on the side of the lesion and contralateral papilledema usually occurring in tumors of the frontal lobe of the brain which press downward) (Jarus and Feldon, 1982). Bakay (1984) emphasized that loss of smell perception is one of the first signs of olfactory meningiomas. In general, tumors or other destructive lesions involving the olfactory bulb, tract, or prefrontal lobe may cause olfactory deficits. Temporal lobe tumors usually cause olfactory hallucinations. It is estimated that approximately 20% of the tumors of the temporal lobe produce some form of olfactory disturbance (Furstenberg et al., 1943). The presence of olfactory deficits correlates more with the location of tumors than the histology (Fig. 5). I. Figure 4 A 20-year-old woman with posttraumatic anosmia. (A) A small olfactory tract is seen on the right side (arrow), but none is seen on the left. Severe inferior frontal lobe encephalomalacia is soon on this coronal T1W MR scan. (B) Encephalomalacia is well seen on the T2W MR scan where hyperintense signal (S) has replaced the inferior frontal lobes (where smell processing occurs).

At at our institution, 25 patients with posttraumatic smell dysfunction were evaluated by olfactory testing and MR. Quantitative and qualitative gradings for olfactory bulb, tract, subfrontal region, hippocampus, and temporal lobe damage correlated with olfactory test results. Twelve patients were anosmic, 8 had severe impairment, and 5 were mildly or impaired. Olfactory bulb and tract (88% of patients), subfrontal (60%), and temporal lobe (32%) injuries were found but did not correlate well with olfac-

Acquired Immunodeficiency Syndrome

Olfactory deficits of patients with human immunodeficiency virus (HIV) infection have been reported (Brody et al., 1991; Heald et al., 1998). These authors suggest that impaired olfaction might serve as a marker of early central nervous system HIV involvement. The principal histopathological abnormalities in the brain of acquired immunodeficiency syndrome (AIDS) patients are in the subcortical structures, predominantly in the central white matter, deep gray structures including the basal ganglia, the thalamus, and the brain stem (Petito et al., 1986; Price et al., 1988). Everall et al., (1991) have found that the neuronal numerical density in the frontal cortex is significantly lower in HIV patients than in controls—a loss of about 38% of neurons in the superior frontal gyrus in AIDS patients. This may account for the olfactory deficits in these patients.

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Therefore, the possibility of peripheral cause of olfactory deficits in AIDS patients also has to be taken into account in certain cases. J.

Multiple Sclerosis

Multiple sclerosis (MS), a markedly debilitating neurological disease, affects millions of Americans in the prime of their lives. Though the influence of MS on the sense of smell has long been controversial, recent MRI studies (Doty et al., 1997, 1999) have demonstrated that the olfactory function in patients with MS is closely correlated with the number of demyelinating plaques within central olfactory processing areas of the brain, as determined by MRI (Fig. 6, 7). A strong negative relationship (Spearman r
0.94) was found between scores on the University of Pennsylvania Smell Identification Test (UPSIT) and the number of plaques within the inferior frontal and temporal lobe regions (Doty et al., 1997). A close association was present, longitudinally, between the remission and exacerbation of plaque numbers and UPSIT scores, with lower UPSIT scores occurring during periods of exacerbation (Doty et al., 1999). K.

Other Central Causes

There are also reports of olfactory dysfunction in hypochondriasis, amyotrophic lateral sclerosis, epilepsy, and migraine (Doty et al., 1991b; Mott and Leopold, 1991). Although the pathogenesis of olfactory dysfunction in these disorders is still unclear, it appears that a central mechanism is involved, rather than a peripheral one. VI. Figure 5 Temporal lobe mass in a 62-year-old woman with olfactory hallucinations. (A) T2W MR scan reveals a relatively well-defined right temporal lobe mass with mild mass effect. (B) Contrast-enhanced T1W MR image shows peripheral enhancement of the tumor with a satellite nodule laterally. Sulci are effaced and the temporal horn is obliterated.

Neuroradiological study has found that patients with HIV infection show widened cortical sulci, enlarged ventricles, cerebral atrophy, and brain stem atrophy when compared with controls (Brun et al., 1986; Elovaara et al., 1990; Post et al., 1988). Opportunistic infections and CNS lymphoma may be superimposal on these changes. The pathogenesis of the olfactory deficits of AIDS patients needs further investigating but most likely will relate to disease in the prefrontal lobe. In addition to CNS changes, sinusitis in HIV-infected patients is common and severe.

OVERVIEW AND DISCUSSION

It is apparent from the studies reviewed in this chapter and the information presented elsewhere in this volume that olfactory dysfunction can be due to numerous causes. Once an olfactory disorder has been recognized, the most important step in the diagnostic process is to determine the site of the lesion, i.e., anatomical localization. Unfortunately, current clinical olfactory testing is unable to localize the site of morphological changes (Doty et al., 1984). Modern medical imaging techniques can be of great value in the anatomical classification and localization of the common causes of olfactory dysfunction (Li et al., 1994). The most common source of olfactory dysfunction is the peripheral pathway (Goodspeed et al., 1987; Mott and Leopold. 1991). In the evaluation of peripheral causes, the “sinus series” radiographs offer limited information. At present, high-resolution CT, especially coronal scans, is

Medical Imaging of Olfactory Deficits

Figure 6 A 55-year-old MS patient with no significant olfactory dysfunction, as measured by the UPSIT. Axial T2W MRI scan shows no obvious plaques in the inferior frontal and temporal lobe regions (A). Numerous plaques were identified in supraperiventricular regions (B).

the study of choice to look at the bony sinonasal structures and the ostiomeatal complex. CT can also provide important information as a road map, which may be needed for surgical treatment. MRI possesses special ability in soft tissue discrimination and offers multiplanar capabilities. In the evaluation of the central causes of olfactory disturbances, MRI has a paramount role. Neuroimaging studies of patients with olfactory deficits related to neuropsychiatric problems have revealed interesting findings and possibly clues for understanding some of the links between olfactory deficits and pathophysiological changes of the brain. The neuroimaging findings of patients with AD, KP, or schizophrenia share some similarities. Thus, almost all of the

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Figure 7 Axial T2W MRI scans from a severely micrsomic (UPSIT
20) 50-year-old man with an 8-year history of MS. The place of section in (A) is 6 mm below that of (B). Note the prominent plaques (10  5 mm each) within the posterior part of the white matter of the gyrus rectus of the L and R subfrontal lobe regions (arrows 1 and 2, respectively), and the relatively high signal intensity plaques in the subtemporal lobe regions (arrows 3 and 4).

abnormalities of the brain parenchyma revealed by neuroimaging studies in patients with AD, KP, or schizophrenia involve central brain areas that contain netrons of olfactory projections including areas of the prefrontal lobe, temporal lobe, hippocampus, and thalamus. Recent studies have provided a clear physiological explanation for decreased olfactory function in patients with MS (Doty et al., 1997, 1998, 1999). Current studies from our laboratory suggest that MS, with its relatively discrete focal regions of demyelination lesion, may be of value in studying brain regions involved in sensory perception in addition to olfaction. It is much more difficult to explain the olfactory dysfunction in PD patients, and presently imaging studies have been of little use in clarifying this matter. Loss of olfaction in these patients may be related to factors with dopamine

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and dopamine receptors, although, as noted earlier, no return of function accompanies L-dopa treatment. In addition, pathological changes in the areas of putamen and caudate nuclei, which have fibers connected with limbic system and striatum, may contribute to the loss of the sense of smell. In this hypothesis, the olfactory dysfunction in PD patients might share a similar etiology to patients with HD. In congenital disorders, such as Kallmann’s syndrome, the cause of anosmia can be seen on MRI studies as the absence of olfactory bulbs (Yousem et al., 1993, 1996a). Other congenital abnormalities, such as choanal atresia and meningoencephaloceles, also can be detected by imaging studies (Klein et al., 1987; Moorman et al., 1984). In the categories of head trauma and brain tumors, imaging studies have shown strong links between olfactory dysfunction and the location of the damaged brain. The histology of the tumor or traumatic injury is less critical than its location (Costanzo and Zasler, 1991; Jarrus and Feldon, 1982; Schellinger et al., 1983; Yousem et al., 1996b). Hyposmia or anosmia induced by occupational or accidental exposure to toxins, as well as that induced by intranasal use of drugs such as cocaine, has been traditionally thought to be due to damage to the peripheral pathways. However, one study has suggested that olfactory deficits caused by occupational exposure to toxins may have both peripheral toxic and CNS effects (Schwartz et al., 1989). Imaging studies have shown CNS complications in cocaine abusers (Holman et al., 1991; Kalkow et al., 1988; Pascual-Leone et al., 1991; Tumeth et al., 1990), and one report of anosmia as a sequela of hydrogen sulfide (H2S) inhalation suggested the loss to be due to central brain damage (Tvedt et al., 1991). VII.

SUMMARY

Medial imaging is an essential part of the evaluation of patients with olfactory disorders. In the assessment of the peripheral causes of olfactory deficits, medical imaging studies, especially CT and/or MRI, can reveal anatomical information and structural changes, suggest differential diagnosis, and provide the road map that may be needed for surgical intervention. On the other hand, in the evaluation of the central causes, MRI, fMRI, PET, or SPECT can provide information elucidating the links between olfactory dysfunction and the structural or functional changes in the living brain. ACKNOWLEDGMENTS Supported, in part, by Grants RO1 DC04278, RO1 AG17496, RO1 DC 02974, and PO1 DC00161 (R. L. Doty, Principal Investigator).

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29 Plasticity Within the Olfactory Pathways: Influences of Trauma, Deprivation, Stem Cells, and Other Factors Joel Maruniak University of Missouri, Columbia, Missouri, U.S.A.

I.

INTRODUCTION

within 90 days. Schultz (1941, 1942) discovered the regenerative properties of the olfactory epithelium while conducting experiments aimed at finding a way to protect humans from polio virus. In those studies he found that destruction of the monkey olfactory epithelium by zinc sulfate conferred protection against intranasal exposure to polio virus. However, a few months later he determined that susceptibility of the monkeys to intranasal virus delivery had returned. He concluded that destruction of the olfactory epithelium prevented infection by removing the pathway into the central nervous system (CNS) but that it must regenerate within a few months. In 1960 he published the first definitive paper documenting the regeneration of the olfactory epithelium after experimental destruction (Schultz, 1960). In the ensuing decade other researchers confirmed Schultz’s findings (Andres, 1965; Takagi, 1969). In the 1970s it was established that the plasticity of the adult olfactory epithelium arose from its property of normal turnover of receptor neurons (Moulton, 1974). For the next 15 years the olfactory epithelium was thought to be unique in being the only part of the mammalian nervous system where neurons were routinely replaced during adulthood (see Chapter 5). The idea that the olfactory bulbs might also possess unusual plasticity arose from Meisami’s early studies of the effects of unilateral naris closure on their development (Meisami, 1976). At the time, the first reports of the

The olfactory pathways of terrestrial mammals are exceptional for their extraordinary plasticity. For most components of the adult nervous system, plasticity is limited to the standard capabilities of individual neurons such as extending and retracting axons and dendrites, changing synaptic strength, and regenerating severed processes. While this ordinary type of plasticity is important and interesting, it pales in comparison to the more global capabilities of the olfactory pathways. For example, the olfactory system of adults shows a unique ability to respond to and recover from trauma and sensory deprivation. This striking plasticity is largely attributable to the presence of stem cells, which normally supply replacements for certain neurons in the olfactory epithelia and bulbs. In response to trauma or deprivation, the production and survival of these stem cells can be increased or decreased. In many ways these properties of the adult olfactory system are reminiscent of those of the other sensory systems during development. The unusual regenerative properties of the olfactory epithelia were known for decades before the plasticity of the bulbs was established. The first indication that the olfactory epithelium might possess remarkable plasticity arose from studies in the early 1940s. Nagahara (1940) cut the olfactory nerves in mice and reported that most cells in the epithelium had degenerated by 3 days and regenerated 615

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negative effects of early visual deprivation had just been published (Barlow, 1975). In order to test the effect of odor deprivation on postnatal development of the olfactory system, Meisami pioneered a technique for closing a naris in newborne rats (Meisami, 1976). He found that after a month of closure, the deprived-side bulbs were 28% smaller than the nondeprived bulbs. Many subsequent studies have confirmed this striking effect of neonatal naris closure on bulb size (see Brunjes and Frazier, 1986). Because of the normal turnover of receptor neurons, researchers in the 1980s began touting the olfactory epithelium as a part of the adult nervous system in which development could be studied. This concept led my lab to wonder if adult olfactory bulbs might similarly retain the kind of plasticity they had been shown to possess during early postnatal development. In the late 1980s we performed unilateral naris closures on adult mice and found that their bulbs responded almost identically to the bulbs of neonatally closed mice (Maruniak et al., 1989). That study indicated that the olfactory bulbs were unlike any other sensory component of the CNS in retaining inordinate sensitivity to sensory deprivation throughout life. In subsequent studies we showed that the unique plasticity of the adult olfactory bulbs had its origin in the normal turnover of its granule cells (Corotto, et al., 1993). We reported that neuronal precursor cells continued to be produced throughout life in the subependymal layer around the ventricles and migrate into the olfactory bulbs where they differentiate into granule cells. That and subsequent studies overturned the long-held belief (Rakic, 1985; Morshead and van der Kooy, 1992) that the adult brain produces no new neurons. In this chapter we will first examine the origins and hallmarks of the extraordinary plasticity of the olfactory pathways and then will consider the factors that can impact this plasticity. Because of the role that stem cells play in enabling plasticity, the origin and utility of neural and embryonic stem cells will be addressed in Sec. VI of this chapter.

Maruniak

suffice it to say that these basal cells endow the olfactory epithelium with its peerless responses to trauma by providing replacements for lost receptor neurons. Even after seemingly complete destruction of the entire population of receptor neurons, the olfactory epithelium is able to reconstitute itself and restore behavioral function. In animal experiments, receptor neurons are typically destroyed either by cutting their axons or by exposure of the nasal cavity to a toxic chemical such as ZnSO4, TritonX, or methyl bromide (Fig. 1). In most cases acute destruction of receptor neurons leads to a dramatic increase in mitosis of the basal cells (Camara and Harding, 1984; Schwartz-Levy et al., 1991; Carr and Farbman, 1992; Schwob et al., 1992) and restoration of the olfactory epithelium and odor-guided behavior within a month or two (Harding and Wright, 1979; Monti-Graziadei et al., 1980; Matulionis et al., 1982; Samanen and Forbes, 1984; Costanzo, 1985; Yee and Costanzo, 1995; Schwob et al., 1999; Cummings et al., 2000). In humans, a number of diseases may cause loss of receptor neurons, but it is generally believed that viral infection is one of the leading causes (Douek et al., 1975; Doty, 1979) (see Chapter 22). Another major cause of anosmia is injury or severance of the receptor axons during head trauma (Doty et al., 1997) (see Chapter 30). Even though the olfactory epithelium is able to regenerate new receptor neurons, for reasons not yet understood there is often incomplete or no recovery of olfactory acuity after posttraumatic loss. Explanations have centered around the possibility that glial scar formation might inhibit regeneration or block the choanae in the cribiform plate between the epithelia and bulbs (Pasterkamp et al., 1998).

II. PLASTICITY OF THE OLFACTORY EPITHELIUM The incredible plasticity of the olfactory epithelium is largely attributable to its possession of a population of stem cells, the basal cells, which allow turnover of the receptor neurons. Chapter 5 of this book discusses the role of these stem cells in the maintenance of the normal olfactory epithelium. For the purpose of the present review,

Figure 1 A section through the nasal septum showing normal olfactory epithelium on the left and the great loss of receptor neurons from the epithelium on the right after the olfactory nerves on that side were cut. Stained with an antibody to olfactory marker protein. (Scale bar
30 m.)

Plasticity Within the Olfactory Pathways

III. PLASTICITY OF THE OLFACTORY CNS STRUCTURES A.

Olfactory Bulbs

The plasticity of the olfactory CNS pathways, though not as great as that of the olfactory epithelia, surpasses that of other parts of the adult brain. The extraordinary elements of olfactory CNS plasticity can be traced in large part to the olfactory bulbs, which are unique in the scope of their responses to trauma and sensory deprivation throughout life. This unique plasticity is largely endowed by the continual turnover of the granule cells, which are resupplied by precursors migrating from a population of stem cells around the lateral ventricles (Fig. 2). These stem cells will be addressed in Sec. VI of this chapter. Sense-appropriate stimulation during postnatal development is essential for full elaboration of the CNS components of sensory systems. For most senses such stimulation must occur within a circumscribed period of time after birth called the critical period. A critical period is a developmental window of time during which stimulus-driven innervation of CNS sensory structures normally occurs. If an animal is deprived of appropriate stimulation during the critical period, then the CNS fails to develop properly. In most cases this leads to irreversible sensory deficits. In contrast, deprivation outside a critical period typically has little or no permanent effects. Studies from our and other labs have shown that olfaction responds quite differently to deprivation than the other senses. The most striking difference is that there appears to be no critical period. Deprivation at any postnatal time leads to similar negative changes in the anatomy and physiology of the system. Thus, unilateral naris closure for longer than one month causes the deprived-side bulbs of rodents to atrophy by about 25% compared to the open-side bulbs (Fig. 1) (Meisami, 1976; Brunjes and Borror, 1983;

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Benson et al., 1984; Brunjes and Frazier, 1986; Maruniak et al., 1989). This effect is of a much larger magnitude than has been reported for the other senses. Recently, Brunjes’ group showed that the olfactory system is almost completely plastic in being able to recover even from the effects of neonatal naris closure (Cummings et al., 1997). While the resiliency of the olfactory bulbs is great, it is limited because interneurons are the only type of cell that is replaced. Thus, if an insult or degenerative process causes loss of mitral or tufted cells—the output neurons of the bulbs—then the function of the bulb is irretrievably impaired. By contrast, the recovery of the olfactory epithelium can occur after virtually complete destruction because all of its cell types appear to be able to be regenerated (Huard et al., 1998) (see Sec. II) (see also Chapter 5). The dramatic changes that occur in the deprived bulb are manifold and all lamina of the bulbs seem to be affected (Brunjes and Frazier, 1986; Henegar and Maruniak, 1991). The layer that is most markedly affected is the external plexiform layer, which contains mainly the processes of, and synaptic interactions between, the mitral/tufted cells and granule cells (Fig. 3). The granule cell interneurons are by far the most numerous type of neuron in the bulbs and are involved in various sorts of odor processing. In fact, most of the gross anatomical effects of naris closure can be traced to the loss of granule cells. However, in possums, which are born in a very immature state and in which mitral cell formation continues postnatally, neonatal deprivation also decreases the number of mitral cells (Cummings et al., 1997). In adult mice we ascertained that naris closure reduced the number of granule cells by 30% (Henegar and Maruniak, 1991). Using tritiated thymidine autoradiography and quantification of apoptotic cells, we established that these losses were due to decreased proliferation and reduced survival of granule cell precursors within the deprived bulbs (Corotto et al., 1994). 1.

Figure 2 Path of the rostral migratory stream from the proliferative areas around the lateral ventricle into the olfactory bulb.

Olfactory Bulb Neurochemistry

In addition to morphological changes, there are also significant changes in the neurochemistry and metabolism of odor-deprived bulbs. Levels of succinate dehydrogenase and cytochrome oxidase are depressed by neonatal closure (Cullinan and Brunjes, 1987), while NADPH diaphorase (nitric oxide synthase) immunoreactivity is not affected (Croul-Ottman and Brunjes, 1988). The amount of dopamine and tyrosine hydroxylase messenger RNA in deprived bulbs is reduced by at least half (Stone et al., 1990; Wilson and Wood, 1992). To compensate for these reduced dopamine levels in the deprived bulb, dopamine D2 receptors are upregulated by 32% (Guthrie et al.,

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decreases in some bulb enzymes such as tryrosine hydroxylase and neurotransmitters such as dopamine and norepinephrine, while not affecting others (Nadi et al., 1981; Kawano and Margolis, 1982; Baker et al., 1983). B.

Figure 3 Effects of unilateral naris closure of 2 months on the morphology of the olfactory bulbs of a mouse. There is a striking reduction in the overall size of the deprived bulb on the left. The largest decreases are in the granule cell layer (GCL) and the external plexiform layer (EPL). The rostral migratory stream is located in the middle of the bulbs between the arrows within the right bulb. (Scale bar
100 m.)

1991). In contrast, beta-adrenergic receptor levels are significantly reduced in the deprived bulb (Woo and Leon, 1995), possibly in compensation for a transient increase in bulbar norepinephrine (Wilson and Wood, 1992). In bulbs deprived of olfactory stimulation during adulthood, dopamine and tyrosine hydroxylase levels are also markedly decreased while catecholamine and GAD levels are unaffected (Kosaka et al., 1987; Stone et al., 1991; Baker et al., 1993; Philpot et al., 1998). Finally, deprivation appears to rapidly and significantly depress bulbar metabolism (Korol and Brunjes, 1990). Over the long term, metabolic effects may be further exaggerated by a decrease in vascularization that occurs in deprived bulbs (Korol and Brunjes, 1992). Destruction of the receptor neurons by axotomy or chemical lesion causes a greater loss of bulb size than the 25% seen after naris closure. Axotomy leads to a 33% loss of bulb weight after a month (Baker et al., 1984), while ZnSO4 irrigation results in a 40–75% decrease within a month (Margolis et al., 1974; Meisami and Manoochehri, 1977). Some of this excess loss can be attributed to the disappearance of the olfactory nerve layer as a result of destruction of the receptor neurons or their axons. Additional loss may be due to neuronal degeneration within the olfactory bulb following lesion of the olfactory receptor neurons (Pinching and Powell, 1971; Gozzo and Fülöp, 1984). As with deprivation following naris closure, axotomy or chemical lesion of the receptor neurons causes striking

Olfactory Cortex

The most common methods for assessing plasticity of the olfactory cortex are unilateral naris closure, olfactory bulbectomy, and lateral olfactory tract lesions. Of these, olfactory bulbectomy is, by far, the most common (Alberts, 1974; Brunjes, 1992). In this section, we first examine the relatively scant data available on the effects of deprivation on the physiology and anatomy of the olfactory cortex. Next, we discuss the effects of bulbectomy and lateral olfactory tract lesion. 1.

Deprivation

The kinds of striking morphological changes seen in the deprived olfactory bulbs do not extend to higher-order cortical structures. Brunjes’ group found that size and other features of the anterior olfactory nucleus (the second-order structure in the olfactory CNS) were relatively unaffected by neonatal naris closure (Brown and Brunjes, 1990). However, a more detailed study of the anterior piriform cortex revealed that there were some subtle reductions in size and other morphological features (Wilson et al., 2000). 2.

Bulbectomy

In the early postnatal olfactory system, bulbectomy appears to have little or no effect on the morphology of the olfactory cortex (Friedman and Price, 1986). By contrast, bulbectomy at later times causes a number of striking effects on the olfactory cortex and other parts of the brain. For example, in the adult, bulbectomy results in rapid transneuronal degeneration of pyramidal neurons in the piriform cortex (Heimer and Kalil, 1978; Capurso et al., 1997). While ablation of the olfactory bulbs causes loss of olfactory capabilities, it additionally leads to other deficits that cannot be attributed solely to loss of the ability to smell (Alberts, 1974; Edwards, 1974; Miro et al., 1982; Hall and Macrides, 1983; Brunjes, 1992). One of the more interesting of such collateral effects is the finding that the olfactory bulbs play a role in chronobiology. For example, olfactory bulbectomy has been shown to unmask the photoperiodic response (i.e., light-controlled seasonal reproductive changes) in animals that do not normally show photoperiodism (Nelson and Zucker, 1981). Furthermore, removal of the bulbs or transection of the lateral olfactory tracts (the main output pathway of the bulbs) causes an

Plasticity Within the Olfactory Pathways

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The neurotropins (members of the nerve growth factor gene family) play an essential role in the activity-dependent development and survival of neurons in sensory systems (Thoenen, 1995) and appear to play similar roles in the olfactory system throughout life. Some are expressed by olfactory neurons at different times in their life cycles, while others appear to be target-derived factors supplied by target tissues. We will first consider the growth factors that are known to play a role in the olfactory epithelium and then review those that function in the olfactory bulbs.

attributable to problems in the olfactory epithelium, it is important to understand the factors that control neurogenesis, recovery, and maintenance of the epithelium. The basal cells of the olfactory epithelia are like other stem cells in requiring fibroblast growth factor (FGF), epidermal growth factor (EGF), and transforming growth factor (TGF) for survival and differentiation (Reynolds and Weiss, 1992; Richards et al., 1992; Kuhn et al., 1997). Herzog and Otto (1999) assessed the ability of these three growth factors to facilitate recovery of the epithelium after destruction by ZnSO4 irrigation. They found that all three enhanced reinnervation of the bulbs in a dose-dependent manner, with TGF being the most effective. A role for the neurotropins in olfactory epithelial development and plasticity has been inferred from the expression of their receptor subtypes, Trks A, B, and C and nerve growth factor receptor (NGFR), at different stages in development and after various insults. The olfactory receptor neurons hold the distinction of being the only lineage of neurons to sequentially express all of those receptors during its life cycle (Roskams et al., 1996). Destruction of the olfactory epithelium by Triton X upregulates expression of NGFR in the olfactory nerve layer of the bulb, and levels return to pre-lesion values within 16 weeks (Turner and Perez-Polo, 1994) Buckland and Cunningham (1999) compared the expression of several neurotropins before and after bulbectomy and found the following. Glial cell line–derived neurotrophic factor (GDNF) is normally expressed in mature receptor neurons and mitral cells but disappears from receptor neurons after bulbectomy. Ciliary neurotrophic factor (CNTF) immunoreactivity is normally expressed strongly by neurons at all stages but after bulbectomy it is also largely lost. Brainderived neurotrophic factor (BDNF) is seen only in horizontal basal cells and is unaffected by bulbectomy. Two additional compounds appear to play roles in development, neurogenesis, and regeneration of the olfactory epithelium. The first of these, insulin like growth factor-1 (IGF-1), seems to function in the support of neurogenesis in the epithelium (Pixley et al., 1998). The second, nitric oxide (which also functions as a second messenger and neurotransmitter), appears to play a part in the development and regeneration of the olfactory epithelium. In fact, the highest levels of nitric oxide synthase are seen in regenerating receptor neurons (Roskams et al., 1994).

A.

B.

increase in basal levels of gonadotropin secretion (Pieper et al., 1989). Another interesting nonolfactory effect of bulbectomy is the similarity of the animal’s affect and neurochemistry to that seen in depression. In fact, the bulbectomized animal has become accepted as an animal model for depression since many of the affective sequelae of bulbectomy can be ameliorated by antidepressants (Van Riezen et al., 1977). One of the characteristic consequences of bilateral bulbectomy is abnormally high circulating levels of corticosterone, which are typically associated with depression and stress (Steckler et al., 1999). Bulbectomy also causes marked increases in the density of innervation by serotonergic fibers of the frontal cortex (Zhou et al., 1998). In addition, it elevates the expression of NMDA receptors in the prefrontal cortex (Petrie et al., 2000; Webster et al., 2000). Finally, bulbectomy causes long-term increases in neuropeptide Y expression in the piriform cortex and dentate gyrus, suggesting a possible role for this peptide in depression (Holmes et al., 1998). Perhaps all of these effects of bulbectomy are related since humans with seasonal affective disorder (SAD), suffer from seasonal depression. Thus, in those afflicted with SAD, the olfactory bulbs might function abnormally or unusually, causing the individual to become somewhat photoperiodic and experience depression in the wintertime. However, a recent test of olfactory acuity in SAD patients found no significant deficits (Postolache et al., 1999). IV. INFLUENCE OF GROWTH FACTORS ON PLASTICITY OF THE OLFACTORY PATHWAYS

Olfactory Epithelium

While the olfactory epithelium has a robust capacity for recovery from injury, it frequently experiences varying degrees of loss of its sensory neurons following trauma (see Sec. II). Because the loss of olfactory acuity is often

Olfactory Bulbs

It is clear that the neurotropins are also important for the production and survival of neuronal precursors for the olfactory bulbs. For example, infusion of BDNF into the lateral ventricles of adult rats leads to a 100% increase in

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neuronal precursors arriving in the olfactory bulbs (Zigova et al., 1998). In BDNF mutant mice there is excessive cell death in the granule, periglomerular, and subventricular layers of the bulb (Linnarsson et al., 2000). Naris closure reduces bulbar levels of the orphan nuclear receptor NGFIB (Liu and Baker, 1999), which may mediate interactions between NGF and the retinoic acid pathways (Katagiri et al., 2000). While the role of NGF in the bulbs is not clear, destruction of the olfactory epithelium by Triton X causes a dramatic decline in NGFR in the glomeruli (Turner and Perez-Polo, 1994). In keeping with the generally accepted role of EGF as a mitogen, Craig et al. (1996) found that infusion of EGF into the ventricles of mice caused an increase in total number of neuronal precursor cells and enhanced their migration. FGF-1 was found to be selectively present in glomeruli and the external plexiform layer of the adult bulb, perhaps providing trophic support for the innervating receptor neurons (Key et al., 1996). Our study of the effects of unilateral naris closure on the adult olfactory bulb showed that deprivation caused decreased neurogenesis and increased cell death in the rostral migratory stream within the ipsilateral bulb (Corotto et al., 1994). The most obvious explanation for those findings is that normal levels of odor stimulation and consequent electrical activity in the bulbs causes the production of one or more growth factors that support the production and survival of granule cells and their precursors. In fact, some studies suggest that both growth factors and electrical activity must be present for full support of neuronal survival (Ghosh et al., 1994; Meyer-Franke et al., 1995).

V.

INFLUENCE OF THYROID HORMONES ON THE OLFACTORY PATHWAYS

Thyroid hormone is crucial for the growth and development of animals (Meisami, 1984) and particularly for elaboration of the proper biochemistry, physiology, and anatomy of the developing CNS, including the olfactory pathways (Dussault and Ruel, 1987). A number of studies have shown that hypothyroidism drastically affects the development of the peripheral olfactory system, as well (Mackay-Sim and Beard, 1987; Pasternostro and Meisami, 1991)—an alteration that can be reversed by thyroxine treatment (Paternostro and Meisami, 1993). The most important postnatal action of thyroid hormone in the olfactory periphery is in support of neurogenesis and maturation (Paternostro and Meisami, 1994, 1996). In fact, odor preferences of adult mice made experimentally hypothyroid are markedly altered (Beard and Mackay-Sim, 1987), although olfactory sensitivity, per se, may not be (Brosvic et al., 1996). There are

limited data suggesting that dysosmia and hyposmia occur in some hypothyroid humans (McConnell et al., 1975). One possible way in which thyroid hormone may exert its effects is through its ability to increase neurotropin levels in the CNS (Giordano et al., 1992). It also has been shown to synergize with NGF in the olfactory bulbs to support normal growth and maturation (Clos and Legrand, 1990).

VI. THE ROLE OF STEM CELLS IN PLASTICITY OF THE OLFACTORY BULBS The stem cells that supply a continuous stream of granule cell precursors to the adult olfactory bulbs are located around the lateral ventricles (Smart, 1961; Moorshead and van der Kooy, 1992; Morshead et al., 1998). They appear to be a remnant of the stem cells whose progeny formed the forebrain during development (Garcia-Verdugo et al., 1998). In fact, the proliferating progeny still traverse the same pathway, the rostral migratory stream, that furnished all the cells for the development of the olfactory bulbs (Fig. 2). Besides the olfactory bulbs, there is only one other component of the adult mammalian CNS that undergoes turnover of its neurons: the hippocampus. Interestingly, in both structures the class of neurons that undergoes replacement is granule cell interneurons. In the case of the hippocampus the stem cells reside in the dentate gyrus (Gage et al., 1998). There is currently some controversy about the actual location of the stem cells in the lateral ventricles. One camp contends that the ependymal cells that line the ventricles are the true stem cells (Johansson et al., 1999b), while the other believes that it is in the overlying subependymal layer that the stem cells reside (Chiasson et al., 1999). Whatever the case, the rostral migratory stream is believed to be an extension of these proliferative zones that passes through the forebrain and into the center of the olfactory bulbs. Once inside the olfactory bulbs migrating precursors change direction and move radially out of the rostral migratory stream to destinations in the granule cell layer or around the glomeruli (Zigova et al., 1996). The discovery by our lab and others that a functional population of neural stem cells remains around the lateral ventricles of adult rodents stimulated research and brought great hope for their use in repair of the nervous system. The ensuing studies on neural stem cells were performed at the same time that great strides in our knowledge of the power of embryonic stem cells was occuring. These studies have shown that the lines, if any, between the properties of adult neural and embryonic stem cells are quite blurred.

Plasticity Within the Olfactory Pathways

Embryonic stem cells have been found to possess almost miraculous powers because they are completely undifferentiated and appear to be able to become any type of cell. For example, it is now clear that embryonic stem cells can be made to differentiate into virtually any type of cell including neural cells or glia in culture or in vivo (Liu et al., 2000). Similarly, the cells around the ventricles, labeled neural stem cells because they were thought to be capable of giving rise to only neurons or glia, are now known to be also undifferentiated and capable of becoming almost any kind of cell (Flax et al., 1998; Clarke et al., 2000). One of the surprising properties of stem cells is that, when injected into an animal, they seem to mostly differentiate into the correct site-specific type of cell (Lewis, 2000). For example, neural stem cells have been shown to be able to differentiate into blood cells when placed in the bone marrow (Bjornson et al., 1999). Furthermore, Gage’s group has shown that stem cells taken from the hippocampus will differentiate into olfactory bulb neurons when implanted into the rostral migratory stream and allowed to migrate into the bulbs (Suhonen et al., 1996). Thus, it seems that a stem cell is a stem cell is a stem cell, and all are undifferentiated and totipotent. Continuous cultures of both embryonic and neural stem cells have been established. Embryonic stem cell lines have been initiated by collecting cells of the inner cell mass from blastocyst-stage embryos and growing them in culture (Evans and Kaufman, 1981; Martin, 1981). Such stem cells can be induced to differentiate into a neuronal phenotype by retinoic acid treatment (Bain et al., 1995). Alternatively, treatment with bone morphogenic protein (BMP) inhibits differentiation into the neuronal phenotype (Finley et al., 1999). Neural stem cells can be harvested from the lining of the lateral ventricles and grown indefinitely as a cell line in culture (Gage et al., 1995). Such cells have even been derived from adult human tissue (Johansson et al., 1999a; Kukekov et al., 1999). To facilitate detection in experiments, neural and embryonic stem cell lines are usually transfected with a reporter gene that allows them to be easily visualized in host tissues (e.g., by immunohistochemistry or fluorescence). When stem cells from such cultured lines are injected back into the blastocyst stage of an embryo, they are widely incorporated into the resulting animal (Bradley et al., 1984; Gossler et al., 1989). In fact, even stem cells of other species are readily incorporated at this stage and form chimeric adults (Brustle et al., 1998). In the developing brain, injection of stem cells into the ventricles leads to widespread incorporation of their progeny into all parts of the CNS. This procedure has allowed the amelioration of neurological diseases in mutant mice (e.g., Yandava et al., 1999). As a result of the tremendous utility and potential of

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stem cells for research and clinical application, the journal Science designated stem cell research as the breakthrough of the year in 1999. A reasonable question at this point is why one would even bother with adult neural stem cells when cultured embryonic stem cells can seemingly do everything. One reason to use adult cells would be to perform autotransplantation back into an adult host’s body without worrying about rejection. In most situations, removal of host stem cells, expansion in vitro, and then autotransplantation would seem to be safest route. In addition, this strategy would circumvent the ethical issues of creating chimeric humans by implantation of stem cells from another human or even animal. Thus, it would seem to be appropriate to identify and study all potential sources of stem cells, including neural stem cells, in the adult human so that their utility under different circumstances can be ranked. Several sources of viable neural stem cells have already been demonstrated in adult humans. Not surprisingly, the lateral ventricles and the hippocampus have been used to obtain stem cells that can be grown and expanded in culture (Johansson et al., 1999a; Kukekov et al., 1999). In addition, the olfactory bulbs themselves have been shown to be a source of culturable neural stem cells. Recently, stem cells were obtained from the bulbs of patients undergoing neurosurgery, placed in culture, expanded, and differentiated into neurons and glia (Pagano et al., 2000). Thus, while the olfactory bulbs may be able to be used as a source of stem cells in adults, the procedure necessary to harvest such cells would undoubtedly be a risky one. For researchers and clinicians in olfaction there are a number of unanswered questions about the use of stem cells. Experiments still need to be performed addressing questions of the limits of stem cell transplantation in our system. Can they be used to generate mitral or tufted cells in olfactory bulbs where those cell types have been lost? A good research animal in which to answer the latter question might be the PCD mouse, which loses its mitral cells over a fairly short period of time during young adulthood (Greer and Shepherd, 1982). Another important question is whether stem cells can be used to repair the olfactory epithelia in patients who have lost the sense of smell because of peripheral damage. A recent experiment suggests that this approach will be fruitful. Goldstein et al. (1998) found that stem cells harvested from the olfactory epithelium of bulbectomized rats could be used to produce both neuronal and nonneuronal cell replacements when implanted in an olfactory epithelium that had been destroyed by methyl bromide. While these precursors were clearly multipotent, it is not known whether they possess the same kind of totipotency seen in stem cells from the CNS. If they do, they might provide an

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easily accessible and harvestable source of stem cells for culture, expansion, and subsequent reimplantation back into an adult. The future appears to be quite bright for stem cell research and application. Their presence and importance in the extraordinary plasticity of the peripheral and central olfactory pathways should direct much future work to olfaction. VII. THE ORIGIN OF OLFACTORY PLASTICITY I would like to conclude this chapter with a discussion of hypotheses about why olfactory pathways express such a high degree of plasticity. As previously stated, the extraordinary components of olfactory plasticity can be traced to the presence of stem cells, which normally supply replacements for receptor neurons and granule cells. Since other sensory systems lose their ability to replace neurons with maturation, the question becomes why the olfactory system does not. While the rationale for replacement of receptor neurons is not clear, there are two generally accepted contributing factors. First, because the receptor neurons are among the most, if not the most, exposed nerve cells in the body (Nakashima et al., 1984), it is believed that under normal conditions they are so vulnerable that they have to be routinely replaced. Even in their relatively protected location in the recesses of the nasal cavity, they come in contact with environmental pathogens, toxins, and particulate matter and are exposed to wide ranges of temperature and humidity. Second, there is evidence that turnover of receptor neurons allows the epithelium to change its sensitivity to odors. For example, research on rats has found that repeated exposure to an odor can increase the sensitivity of the olfactory epithelium to that odor (Wang et al., 1993). Furthermore, in both humans and rats, repeated exposure to an odor can increase behavioral sensitivity to that odor and even other odors (e.g., Doty et al., 1981; Doty and Ferguson-Segall, 1989; Stevens and O’Connell, 1995; Wysocki et al., 1989; Yee and Wysocki, 2001). It is not known why the olfactory bulbs and hippocampus continue to replace interneurons throughout life while the rest of the brain remains static. Certainly, neither is exposed to any more trauma than other neurons of the brain and so that cannot be a factor. Hypotheses for the hippocampus mainly center around the role that new interneurons could play in learning (Gould et al., 2000). Selective replacement of granule cells may enable the bulbs also to contribute to changes in odor sensitivity (a form of learning, as well). This could act in concert with

Maruniak

the receptor neuron turnover mentioned above to provide a coordinated adjustment of olfactory sensitivity to seasonal changes in odors (e.g., those related to food sources, reproduction, aggression, and parenting). In fact, several lines of evidence suggest that such a mechanism is plausible. First, we have shown that on a global level odor deprivation leads to reduced accumulation of newly produced granule cells and increased death of existing ones (Corotto et al., 1994). Our study suggests that the normal electrical activity of receptor neurons maintains a balance between addition of new granule cells to, and subtraction of existing granule cells from, the olfactory bulbs. Other studies, at a more local level, suggest that an individual odor stimulates a unique and characteristic set of functional columns in the bulbs (Onoda, 1992; Sallaz and Jourdan, 1993; Guthrie et al., 1993). A functional column is an odor-processing unit consisting of glomeruli, which are activated by inputs from the receptor neurons of the nose, the mitral cells that receive this input, and a group of granule cells that modify the responses of the mitral cells (Guthrie et al., 1993). If the effects of global deprivation are extrapolated to the level of a functional column then it seems logical that chronic activity or inactivity within individual columns might determine whether associated granule cells increase or decrease in number. In other words, odor-stimulated columns would tend to add new granule cells and odor-deprived columns would tend to lose granule cells. This would provide a mechanism by which the bulbs could contribute to adjustments in odor sensitivity. VIII.

SUMMARY

The olfactory pathways of adults possess a degree of plasticity that approaches that of the developing nervous system. The olfactory epithelia display an unsurpassed ability to recover from trauma, while the bulbs show an unusually strong response to, and ability to recover from, sensory deprivation. Neurotropins, other growth factors, and some hormones appear to mediate different aspects of these plastic responses. The resiliency of the olfactory pathways arises from their possession of two populations of neurons that turn over in adulthood: the olfactory receptor neurons of the periphery and the granule cells of the brain. Replacements for the receptor neurons are supplied by the basal cells of the epithelium. New granule cells originate as the progeny of stem cells which are located around the lateral ventricles. These precursors migrate into the bulbs via the rostral migratory stream. Much work is currently being directed at determining the utility of using the two types of olfactory stem cells to repair damaged parts of the nervous system.

Plasticity Within the Olfactory Pathways

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30 Head Injury and Olfaction Richard M. Costanzo, Laurence J. DiNardo, and Evan R. Reiter Virginia Commonwealth University, Richmond, Virginia, U.S.A.

I.

and taste centers for evaluation and treatment of their chemosensory dysfunction (Cain, 1989; Ikeda et al., 1999; Kondo et al., 1998). Standardized tests have played an important role in the development of new data on the epidemiology and incidence of this sensory disorder (Deems et al., 1991). For example, Doty and colleagues used the University of Pennsylvania Smell Identification Test (UPSIT) to measure changes in olfactory function in 66 patients with head trauma over periods ranging from 1 month to 13 years (Doty et al., 1997). They found that 36.6% improved, 18% worsened, and 45% had no change. However, UPSIT testing revealed that only 3 patients (5% of the study sample) regained normal olfactory function on retesting. In addition, this study demonstrated that among those patients who believed they had improved, less than half showed a statistical improvement in their UPSIT scrore. Several studies suggest that the likelihood of anosmia is dependent on the severity of the injury (Costanzo and Becker, 1986; Doty et al., 1997; Sumner, 1975). Heywood et al. (1990) matched olfactory test scores with patients’ Glasgow coma scale (GCS) ratings and found a correlation between the severity of head injury and the amount of olfactory disturbance. In mild injury (GCS 13–15), 13% of patients were totally anosmic and 27% showed difficulty with odorant detection or identification. In the moderate head injury group (GCS 9–12), 11% were anosmic while 67% showed some degree of olfactory impairment. Among those with severe head injury (GCS 3–8), 25% were anosmic and 67% had some degree of impairment. Partial or complete unilateral loss of olfactory function can also

INTRODUCTION

The first published report of post-traumatic anosmia appeared in volume 1 of the London Hospital Report (Jackson, 1864). In this article, John Hughlings Jackson describes the case of a 50-year-old patient who lost his sense of smell after being knocked off his horse by a highwayman. The patient, who received a blow to the head, suffered a severe concussion and afterwards never regained his sense of smell. Subsequent reports of post-traumatic anosmia in the late 1800s also involved blows to the head or falls from horses or horse-drawn vehicles (Ferrier, 1876; Legg, 1873; Notta, 1870; Ogle, 1870; Rotch, 1878). Costanzo and Zasler (1991) noted that approximately one of every 400 people in the United States suffers from the sequelae of head injury. Most of these injuries occur among young adult males between the ages of 15 and 24 years. Although the major cause of head injury among this age group is vehicular accidents, in older populations falls and assaults play a prominent role as well. Early studies of posttraumatic olfactory loss suggest a 4–7% incidence of anosmia following head injury (Kraus et al., 1986; Leigh, 1943; Mifka, 1964; Zusho, 1982). However, more recent studies report a higher incidence of anosmia—reaching 60% in cases of severe head injury (Costanzo et al., 1987; Deems et al., 1991; Doty et al., 1997; Ogawa and Rutka, 1999). This might be accounted for in part by increased awareness of olfactory loss in head trauma, the use of standardized tests for the quantitative measurement of olfactory function (Cain et al., 1983), or a selection bias introduced when studying patients who present to smell 629

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occur and is more likely to go unnoticed than complete bilateral anosmia. Parosmia, abnormal odor sensations or the presence of a strange odor in the absence of a stimulus, has been reported in 25–33% of patients with head trauma (Doty et al., 1997; Duncan and Seiden, 1995). Although the mechanisms underlying parosmia are not fully understood, it has been shown that the number of patients experiencing parosmia decreases with time following injury. This improvement in function may reflect a true resolution of olfactory function or an adaptation process in which the patient learns to ignore or tune out these abnormal olfactory sensations. II.

MECHANISMS OF INJURY

Traumatic olfactory dysfunction may be caused by several mechanisms: (1) sinonasal contusions or fractures with or without direct damage to the olfactory apparatus, (2) tearing or shearing of olfactory nerve filaments, or (3) contusion or hemorrhage within the olfactory-related brain regions (Fig. 1). A.

Sinonasal Tract Alterations

Zusho (1982) studied a group of 212 patients with posttraumatic anosmia who came for follow-up evaluation of head and facial injuries. He found that 44.8% had facial or skull fractures and 11.3% had facial contusions with fractures of the nasal bone. Following sinonasal tract alterations, unilateral olfactory loss may be present and hyposmia, rather than anosmia, often results. Potential mechanisms include (1) mucosal hematoma, edema, or avulsion within the olfactory

cleft with direct injury to the olfactory neuroepithelium, (2) scarring with synechia formation or fractures of the nasal skeleton or septum with subsequent airflow alterations that prevent odorants from reaching the olfactory cleft (Fig. 1A), or (3) posttraumatic rhinosinusitis with resultant alterations in nasal airflow or mucus quantity and viscosity. Although this cause of hyposmia is not common, it is important to recognize it since it is potentially treatable. Often reduction of mucosal edema, repair of nasal or septal fractures with relief of airway obstruction, or the treatment of sinusitis can improve olfaction. Resolution of olfactory deficits is more likely to occur when there has been no direct trauma and, consequently, no irreversible scarring or damage to the olfactory cleft. B.

Shear Injuries

The delicate axons of olfactory receptor cells pass through small foramina of the cribriform plate at the base of the skull and synapse directly with cortical cells in the olfactory bulb. Tearing or shearing of these axons may occur with fractures of the naso-orbito-ethmoid region involving the cribriform plate or with rapid translational shifts in the brain secondary to coup or contracoup forces generated by blunt head trauma (Fig. 1B). Fracture of the cribriform plate is actually an uncommon cause of anosmia. Anosmia is more likely to be produced by posterioranterior coup and contra-coup forces (Sumner, 1975; Zusho, 1982). This is due to a shearing effect on the delicate olfactory axon fibers as the brain shifts with respect to the cranial base. This frequently results in complete bilat-

Figure 1 Mechanisms of posttraumatic olfactory dysfunction. (A) Injury to the sinonasal tract with obstruction of airflow to the olfactory cleft. (B) Shearing of the olfactory nerves at the cribriform region. (C) Cortical contusions and brain hemorrhage involving the olfactory cortex.

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eral anosmia. It is of interest to note that nasal endoscopy and the use of biopsy techniques have produced clinical ultrastructural correlates of posttraumatic anosmia that support the notion of shearing of olfactory nerve fibers (Moran et al., 1985). Specifically, the fila olfactoria have been found to be traumatically severed at the cribriform plate. Regenerating axons apparently fail to reach the olfactory bulb due to scar formation and obliteration of the foramina in the cribriform plate. C.

Brain Contusion and Hemorrhage

Head trauma often results in traumatic brain injury in the form of cortical contusion or intraparenchymal hemorrhage (Fig. 1C). Contusion of the olfactory bulbs is one plausible explanation for posttraumatic anosmia. Damage localized to the olfactory bulbs or hemorrhage in the vicinity can occur without involvement of other brain regions (Costanzo and Zasler, 1992). This can lead to direct neuronal injury, or secondary ischemic insult. Inamitsu and coworkers (1990) demonstrated retrograde degeneration of the olfactory neuroepithelium in rats after surgical ablation of the olfactory bulbs. Despite subsequent neuronal regeneration, the number of mature neural elements identified at the olfactory neuroepithelium was diminished. Posttraumatic disorders of odor discrimination are often associated with cortical lesions (Costanzo and Zasler, 1991; Yee and Costanzo, 1998). Higher-order olfactory neurons project mostly to the anterior pyriform cortex, amygdala, and temporal lobe region. Other projections terminate in areas of the frontal lobe. Projections are bilateral and extensively distributed (Yousem et al., 1996). Therefore, it is difficult to attribute complete anosmia to cerebral damage alone. Levin and colleagues (1985) suggest that impaired olfactory recognition without anosmia may result from local or diffuse injury to the orbitofrontal and temporal lobe regions. It is consequently not surprising that behavior disturbances and memory disorders frequently accompany impaired olfactory recognition. III. A.

CLINICAL ASSESSMENT History

Because of the potentially life-threatening nature of head trauma and the frequent co-occurrence of other visceral or orthopedic injuries requiring immediate medical attention, both the patient and his or her treating physicians might not recognize posttraumatic olfactory loss until well after the time of injury. This time lag often encompasses treatments such as neurosurgical procedures, facial fracture

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reduction, nasal intubation, and administration of numerous medications that might also affect olfaction, thus complicating evaluation of patients’ olfactory complaints. A detailed history, therefore, is an important first step. The patient should be asked about pretraumatic olfactory function, and any previous deficits, either transient or prolonged, should be explored in an effort to secure the etiology. Consideration must be given to olfactory loss associated with an upper respiratory tract infection. Even without a history of previous impairment, common causes of olfactory disturbance such as rhinosinusitis, previous head trauma, or nasal obstruction should be ruled out. The patient should be asked about present olfactory function. The severity and nature of the olfactory dysfunction should be documented. Factors such as the completeness of loss, uni- or bilaterality of deficits, and the presence of qualitative changes in olfaction (e.g., alterations in known smells, or dysosmias, or olfactory hallucinations, or phantosmias) should be ascertained. This helps the physician estimate the potential impact of the olfactory loss on the patient. The time course of the olfactory disturbance yields important information about its etiology. As discussed above, most posttraumatic olfactory losses are the result of shear injury to the olfactory nerve fibers. Such injuries typically, but not always, result in immediate olfactory loss. The patient should be asked about clear rhinorrhea or “salty-tasting” postnasal drip, especially without an antecedent history of rhinosinusitis. This might be indicative of a cerebrospinal fluid (CSF) leak resulting from fracture of the anterior cranial base, potentially involving the cribriform. Posttraumatic CSF rhinorrhea does not necessarily indicate a breach in the cribriform plate, because fractures of the adjacent ethmoid sinus roof are often the cause of dural disruptions (Raveh et al., 1988). Gradual, progressive losses are typical of inflammatory disease, as can result following sinonasal trauma. Immediate but transient improvement with topical vasoconstriction further supports an obstructive etiology. The direction and severity of head injury should be ascertained. Zusho (1982) found that the direction of the external force resulting in anosmia at the time of injury was most often posterior-anterior. In a more recent study, Doty and colleagues found that, on average, a single focused impact to the back of the head produced only slightly larger deficits (39.3%, 66 of 168 cases studied) than an impact to the front (36.3%, 61 of 168 cases studied) of the head (Doty et al., 1997). Transverse, superior, and inferior forces were less common. The severity of head injury is also associated with the incidence of posttraumatic anosmia. In particular, the Glasgow coma scale, or perhaps more accurately the duration of posttraumatic amnesia, seems to correlate with the occurrence of posttraumatic anosmia (Levin et al.,

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1985; Sumner, 1975). It is important to note that anosmia may result from even minor head injuries. The physician should thus make every possible effort to reconstruct the mechanism of injury. If the injury resulted in loss of consciousness or amnesia and the patient is unable to provide specific details, any available family members or witnesses should be consulted (with the patient’s permission) to provide additional history. As the physician working up the olfactory complaint is seldom seeing the patient at the time of trauma, the emergency department or inpatient hospital records should be obtained and carefully evaluated for clues. These clues might include mention of the location of head or facial lacerations or ecchymosis, the presence of epistaxis or clear rhinorrhea, or coexisting cranial nerve deficits. Results of any radiographic workup including plain films, magnetic resonance (MR), or computed tomography (CT) scans of the head or maxillofacial area should be reviewed, and whenever possible the films themselves should be reviewed with a neuroradiologist. Subtle findings in the sinonasal and cribriform area might be overlooked in films obtained solely to evaluate intracranial injury. Operative notes from any surgical procedures performed, in particular neurosurgical procedures or maxillofacial fracture reduction, should be reviewed in detail. Lastly, both the inpatient chart and the outpatient office notes from any other treating physicians should be reviewed for mention of olfactory complaints, or denial of the same. This is certainly important in determining the mechanism of a patient’s olfactory loss, but may prove even more critical when evaluating a patient involved in legal proceedings surrounding the traumatic event. Discrepancies in the record might suggest a patient is seeking to magnify their level of olfactory impairment for personal gain. This would indicate a need for more thorough olfactory testing to document malingering. Other sequelae of head injury should also be considered since they may functionally influence olfactory disorders. Gustatory disturbance and cognitive dysfunction are particularly important. Zusho (1982) observed a 16.5% incidence of gustatory complaints in posttraumatic anosmic patients. Formal olfactory and gustatory testing is required to confirm this finding because taste and smell disturbance are easily confused by the patient. Mott and Leopold (1991) suggest obtaining a psychosocial history for all patients with traumatic olfactory loss. Neuropsychological testing should be considered since frontal lobe contusions are frequently cited in posttraumatic anosmia and can cause subtle changes in cognitive function (Zasler et al., 1990). Associations between posttraumatic anosmia and hearing impairment (41%), tinnitus (22.6%), disequilibrium (14.2%), and visual disturbances (2.8%) have also been described (Zusho, 1982).

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B.

Physical Examination

The treatment of any life-threatening injuries obviously takes precedence in the management of head trauma. The details of evaluation and stabilization of the head-injured patient are complex, and will not be reviewed here. Subsequently, the head-injured patient rarely receives an immediate sensory evaluation, with the possible exception of ophthalmological evaluation in cases where screening neurological examination reveals visual compromise. Frequently, either the patient does not recognize an olfactory deficit until some time after injury, or evaluation of such deficits is deferred while other treatment or rehabilitation issues are addressed. Whether occurring at the time of injury or some time later, evaluation of patients with olfactory deficits should include a thorough examination by an otorhinolaryngologist. In addition to evaluation for potential causes of olfactory impairment, the examination should not neglect auditory, vestibular, and other cranial nerve testing, given previously discussed potential associations with olfactory loss. In addition, the chemosensory function of the trigeminal nerve must be considered separately when evaluating olfaction. The examining physician must keep in mind the common pathophysiological mechanisms of posttraumatic olfactory loss. Signs of blunt or severe head trauma might suggest generation of coup-contra-coup forces and a shearing effect on the olfactory fibers. The presence of facial or scalp lacerations, ecchymosis, edema, or tenderness are suggestive of such injuries. Direct injury or even fracture of the cribriform area may be associated with midfacial or nasal fractures. Traumatic telecanthus, described as widening or lateral displacement of the medial canthus, is seen with injuries to the naso-orbito-ethmoid complex that might also involve fracture of the cribriform plate and CSF fistula. Nasal endoscopy is critical in the evaluation of posttraumatic olfactory deficits. A 4.0 mm 30 degree rigid endoscope is typically used, although examination of patients with significant septal deviation or other anatomical narrowing may best be performed with a 2.4 mm endoscope. The intranasal examination is conducted both before and after topical nasal vasoconstriction with either phenylephrine (0.25%) or oxymetazoline (0.025%) to determine the degree of reversible mucosal edema. Routine evaluation should be systematic and include inspection of the inferior, middle, and superior meati as well as the nasopharynx. Purulent discharge from the middle or superior meati is indicative of rhinosinusitis. Specific abnormalities potentially obstructing airflow, such as hematoma, septal deviation, turbinate hypertrophy, neoplasia, or polyposis, should be sought. Inspection of the olfactory cleft itself is critical. Given the typically

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narrow dimensions of this space, which might be further compromised by deflections of the middle turbinate or septum, access can be challenging. This may be facilitated by use of the 2.4 mm endoscope or by gentle lateralization of the middle turbinate after application of topical anesthesia. The injured olfactory cleft may manifest mucosal edema, hematoma or ecchymosis, mucosal lacerations, or CSF leaks. C.

Radiography

High-resolution CT and MR scans are valuable adjuncts in the diagnosis of posttraumatic anosmia. Plain-film radiographs may be of some value in the acute setting to assist in the diagnosis of skull or facial fractures. However, plain films have limited ability to diagnose intracranial abnormalities or fractures of the delicate bones of the cribriform fossa and have thus been supplanted by CT and MR in the workup of posttraumatic olfactory loss. Modern imaging technologies are most useful when properly chosen and performed (Yousem et al., 1996, 1999). CT scanning should utilize thin cuts (1–2 mm) through the skull base in both axial and coronal planes. Abnormalities of the nasal

Figure 2 Magnetic resonance image showing bilateral orbitofrontal contusions in a patient with bilateral anosmia following head trauma.

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cavities producing airflow obstruction, posttraumatic sinusitis, and fractures of the cribriform plate are easily demonstrated without need for intravenous contrast. While fractures of the anterior cranial base may suggest CSF leak, the localization of a suspected leak may be achieved using intrathecal metrizimide, a water-soluble contrast agent. Intracranial soft tissue injuries are best evaluated by MR scanning. Hematoma and contusion are possible intracranial causes of posttraumatic anosmia and may be sought using axial and coronal MRI (Fig. 2). We find the greatest yield in identifying treatable causes of posttraumatic olfactory loss can be achieved by thin-cut CT of the maxillofacial region including the cribriform fossa. This obviates the need for intravenous contrast and the greater expense of MR imaging. MR is reserved for cases in which other neurological findings in addition to anosmia require workup. D.

Olfactory Function Testing

All patients reporting olfactory deficits following head trauma should undergo olfactory testing to confirm and quantify the degree of loss (see Chapters 10 and 22). The development and standardization of olfactory screening tests (OSTs) has improved identification of posttraumatic olfactory losses considerably (Cain, 1989; Costanzo and Zasler, 1991; Doty and Kobal, 1995). Before the use of OSTs, it is likely that most olfactory disturbances resulting from head trauma went unrecognized. There are now several standardized tests available for evaluation of olfactory function. The UPSIT consists of four booklets containing 10 “scratch-and-sniff ” microencapsulated odorants each. The analysis of response data can be of use in the detection of malingerers (Doty et al., 1984). Another test developed at the University of Connecticut employs both odor-detection and odor-identification subtests (Cain et al., 1988). Detection threshold levels have utility in evaluating receptor cell function, and identification scores are useful in uncovering cortical contusions and brain injury (Costanzo and Zasler, 1991). In Japan, testing is often performed using a graded series of odorant concentrations presented on strips of blotting paper to deliver different concentrations of odor stimuli to subjects (Ikeda et al., 1999; Takagi, 1987). Recently, a device that delivers odorants into the nose, termed the “Jet Stream Olfactometer,” has been introduced for evaluating smell function (Ikeda et al., 1999). Using differing concentrations of various odorants, both detection and recognition thresholds may be determined. Although comprehensive olfactory testing is routine at specialized smell and taste centers, this is not the case in most emergency rooms, neurosurgical wards, or head injury rehabilitation facilities. However, screening tests such as the 3-item Pocket

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Smell TestTM and the 12-item Brief Smell Identification TestTM are available commercially (Sensonics, Inc., Haddon Heights, NJ) and are likely to be more suitable for emergency room use or bedside testing. Although olfactory function testing can be extremely valuable in the assessment of the head-injured patient, it may be some time before testing kits gain widespread acceptance and testing becomes routine. E.

Treatment and Prognosis

Many posttraumatic olfactory disturbances are irreversible. Recovery may occur in some cases but is dependent on the mechanism of olfactory injury. The repair of sinonasal obstruction or the resolution of cerebral hemorrhage or contusion may improve olfaction. Sumner (1975) suggested that early recovery from posttraumatic anosmia could result in these instances. As with other neuronal injuries, the prognosis for recovery from posttraumatic anosmia worsens with time. Sumner (1964) noted that 39% of those who recover olfactory function do so within the first 10 weeks after injury. Costanzo and Becker (1986) emphasized that if recovery of olfaction does not occur within 6 months to 1 year following injury, recovery is unlikely. They reported that 33% of all moderately to severely head-injured patients showed some improvement in olfaction, whereas 27% worsened. In a more recent study, Doty et al. (1997) retested 66 patients with olfactory dysfunction several years following head trauma and found that 24 (36%) improved slightly, 30 (45%) had no change, and 12 (18%) worsened. Improvement of anosmia over a long duration may be explained by the regeneration or recovery of damaged olfactory neurons. Costanzo and colleagues have demonstrated recovery of olfaction in hamsters following transection of the olfactory nerve fibers at the cribriform. Regenerating olfactory receptor cell axons have been shown to grow through the cribriform plate and reestablish contact with cells in the olfactory bulb (Costanzo, 1983; Koster and Costanzo, 1996; Yee and Costanzo, 1995). The capacity for olfactory receptor cell recovery in humans has not been established. IV.

SPECIAL CONSIDERATIONS

Although loss of the sense of smell may seem to some like an insignificant functional impairment, such deficits can be disabling and have a negative impact on patients’ quality of life (Miwa et al. 2001). The impact of olfactory dysfunction on an individual’s health, safety, ability to function in the workplace or at home, and the effect on quality of life are all important issues that require consid-

eration (Cain et al., 1987; Cone and Shusterman, 1991; Hoffman et al., 1998). Both the medical and lay communities have historically underestimated the impairment attributable to posttraumatic olfactory deficits. Olfaction has important safety functions such as the early detection of fire, gas leaks, spoiled foods, or dangerous fumes, as well as hedonic functions such as the assessment of the palatability of foods and beverages and the detection of fragrances or aromas. While these functions are less vital to a person’s well-being and functionality than vision or hearing, their loss still negatively impacts a person’s quality of life and potentially the level of disability. The following section provides the clinician with practical information to address the functional ramifications of posttraumatic anosmia. A. Disability Evaluation and Activities of Daily Living The American Medical Association impairment rating system, Guides to the Evaluation of Permanent Impairment, lists total bilateral loss of smell as a 3% impairment of the whole person. Unilateral or partial bilateral loss precludes any permanent impairment rating from being assigned (AMA, 1993). We feel this rating system may not fully represent the functional disability associated with olfactory loss following head trauma and/or brain injury. In order to evaluate the degree of functional disability as well as resultant handicap, the evaluating professional must first consider the particular patient’s activities of daily living (ADLs), vocational status, and avocational pursuits. Such evaluation is, in most cases, difficult if not impossible to accomplish during a clinic visit and may require in-depth questioning and on-site assessment. From a functional standpoint, the main areas that must be considered relative to potential adverse effects of smell loss are safety issues, vocational pursuits, appetite and nutrition, hygiene, homemaking, child care, and hobbies and leisure activities. Personal safety is a major consideration for individuals with impaired olfactory function. The inability to detect smoke, gas leaks, spoiled foods, and chemical vapors in the home or workplace presents a major risk to health and safety. Appropriate provisions must be provided to ensure maximal safety for the patient as well as other persons in the home environment. Patients should be advised concerning the installation and use of smoke detectors and the routine monitoring of gas appliances. Perishable foods should all be dated when purchased. Foods should be stored optimally to minimize the chance for spoilage. Precautions should be exercised in the use and storage of noxious substances used in household cleaning and gardening.

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Patients employed as firefighters, chemists, plumbers, bakers, cosmeticians, florists, food technologists, and cooks might be significantly impaired in their job performance if they could no longer smell. Appropriate job analysis is required prior to making final recommendations regarding whether or not a given patient should attempt vocational reentry. Employee and employer education may be critical, as might job or workplace modifications to increase patient safety. Some individuals may not be able to return to prior jobs due to inherent safety risks not addressable via compensatory strategies. Varney (1988) found a significant negative correlation between the presence of anosmia and successful vocational reintegration although the frequency of vocational dysfunction among anosmic and hyposmic patients in general is unlikely as high as previously reported (Correia et al., 2000). Unfortunately, there are no large-scale well-controlled studies on the effect of anosmia on vocational reentry following traumatic brain injury, it would seem quite apparent that certain vocational pursuits might become more difficult, if not dangerous, with complete anosmia. Anosmic patients may report alterations in the taste of foods rather than a loss of smell. In a study of 750 patients with complaints of abnormal smell or taste perception, Deems and colleagues found that fewer than 4% of those with an olfactory loss and gustatory complaints actually had demonstrable gustatory loss (Deems et al., 1991). Also, patients who complained only of gustatory loss were three times more likely to show objective evidence of olfactory loss than gustatory loss. Given the link between appetite and eating with the aromas of food, it is not surprising that anosmic patients complain of “not enjoying food anymore.” Excess use of salt, seasoning, or sweeteners in an attempt to find missing stimulation is not uncommon. Nutrition as well as medical status can be compromised by anosmic patients who seek out select compensatory food items that may be excessively salty, sweet, or crunchy. The astute clinician can assist the anosmic patient in gaining greater enjoyment from their meals by remembering that the palatability of food is derived from more than just smell; taste, texture, and flavor all play a role. Altering flavor via modification of texture, vision, temperature, or taste may prove a useful compensatory tool for the anosmic or hyposmic patient relative to eating-satisfaction issues (Schiffman, 2000). The anosmic individual may have problems with cooking, child care, or pet care. Patients with anosmia who cook should be instructed to follow recipes and avoid overor underseasoning. Close monitoring of cooking is essential to avoid burning or overcooking. Child care issues are multiple and include education regarding timely diaper changes, hygiene timing, and food preparation to ensure

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the child’s optimal welfare. Pet care may also present a challenge to the anosmic patient. Scheduled litter box and animal pen inspection may prove a useful compensatory technique. Anosmic patients will be unable to detect their own body odor, the body odor of others, the smell of clothing if soiled or musty, or the smell of colognes or perfumes. A schedule should be established for hygiene tasks such as washing clothes and bathing. Appropriate use of body fragrances can be encouraged by training the person to measure out specified quantities of cologne or perfume. Avocational activities may also be adversely affected by loss of smell following head trauma. Activities such as entertaining, gardening, painting, wood burning, leatherwork, and cooking may all lose at least a component of their inherent hedonic value secondary to the inability to perceive the associated smells and odors. From a quality-of-life standpoint, we seldom think what it would be like not to be able to smell the grass, trees, and flowers when we step outside, appreciate the smell of bacon on the griddle and coffee percolating on a weekend morning, smell our children or spouse, or breathe in crisp pine-scented mountain air or salt-laden air at the seashore. Individuals with posttraumatic anosmia may never again appreciate these smells and the associated pleasures that accompany them. Studies have shown a significant relation between olfactory disturbances and clinical depression (Deems et al., 1991) and other psychological effects (Toller, 1999). B.

Medicolegal Considerations

Assessment of olfaction in the head-injured patient may be an important factor in medicolegal matters involving personal injury litigation. Since subjective reports or simple bedside testing may be unreliable in objectively documenting loss of olfactory function and identifying malingerers, special protocols for sensory testing should be employed to validate reproducibility of results (Doty et al., 1995; Heywood and Costanzo, 1986). In addition, a thorough medical and psychiatric history should be obtained to rule out disorders that may affect smell but are unrelated to the history of head trauma, including depression and medication-induced dysosmia (Schiffman, 1983, 1994; Schiffman et al., 1999). Complete evaluation including neurological, otolaryngological, and radiological examinations as described previously should also be performed to support or refute any patient claims of injury. C.

Counseling and Information Resources

In many cases of posttraumatic anosmia there is no medical treatment available to the patient to help restore

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olfactory function. However, the physician can provide important advice and counseling about the risks and hazards associated with anosmia. This includes safety issues such as the inability to detect gas leaks, chemical vapors, and smoke, as well as routine ADLs such as food preparation, child care, personal hygiene, and the proper use of household chemicals such as cleaning agents, bleach, solvents, and pesticides. A list of sample questions for patients with olfactory impairment is given in Table 1. Often patient counseling and reassurance are important in helping patients to cope with the psychological burden of their olfactory loss. By helping patients understand that they have a recognized medical condition, the physician can usually allay patients’ fears or frustrations, even in the absence of definitive treatment. Resources for obtaining additional information on olfactory impairment and brain injury are provided in Table 2.

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Table 2 Resources for Information on Olfactory Impairment and Head Injury 1.

National Institute on Deafness and Other Communication Disorders (NIDCD) Address: Office of Health Communication and Public Liaison, 31 Center Drive, MSC 2320, Bethesda, MD 20892-2320 Voice: (301) 496-7243 FAX: (301) 402-0018 E-mail: [email protected] Internet: http://www.nih.gov/nidcd/health/st.htm

2.

Association for Chemoreception Sciences (AChemS) Address: 744 Duparc Circle, Tallahassee, FL 32312 Voice: (850) 531-0854 FAX: (850) 531-0854 E-mail: [email protected] Internet: http://neuro.fsu.edu/achems/TasteAndSmell.htm

3.

American Academy of Otolaryngology–Head and Neck Surgery (AAO-HNS) Address: One Prince Street, Alexandria, VA 22314 Voice: (703) 519-1589 FAX: (703) 299-1125 E-mail: [email protected] Internet: http://www.entnet.org/smelltaste.html

4.

American Association of Neurological Surgeons 22 South Washington Street, Park Ridge, IL 60068 Voice: (847) 692-9500 FAX: (847) 692-2589 E-mail: [email protected]

SUMMARY

Olfactory loss is a frequent sequela of traumatic head injury. Patients are typically unaware of their loss until some time after injury, and it is often overlooked during initial clinical evaluation. It is important that the physician understand the mechanisms that can cause posttraumatic anosmia and be familiar with current methodologies for clinical assessment. A thorough history and physical exam including otolaryngological evaluation are essential. Modern imaging technologies

Table 1 Sample Questions for Patients with Olfactory Impairment Does anyone in your home smoke cigars or cigarettes? Do you have smoke detectors in your home? How many and where are they located? Do you have gas appliances (stove, furnace, water heater, grill, fireplace)? Do you have a wood-burning fireplace or kerosene heater? Are you responsible for the care of children, pets, or animals? Have you experienced changes in your appetite? Do you add excess salt or sugar to enhance the taste of food? Are you concerned about bad breath or body odor? Do you check and monitor foods for spoilage? Do you use household chemicals such as cleaners, bleach, solvents, and pesticides? Is your sense of smell important in your job or work environment?

Internet:http://www.neurosurgery.org/pubpages/patres/ headinjury.html

can play an important role in the patient work-up. Specific olfactory function testing may be performed by a variety of techniques and is critical in verifying the presence of olfactory loss and determining its severity. Although there is often no therapeutic intervention that can reverse posttraumatic anosmia, spontaneous recovery may occur in some groups of patients, including those whose dysfunction is secondary to hematomas or whose overall function is not severely compromised. Olfactory impairment may increase risk for personal injury due to inability to detect gas leaks, smoke, spoiled foods, and other potentially harmful conditions. It is important for the treating physician to assess for any personal and work-related disabilities, as patients may benefit from counseling or compensatory strategies. Although loss of olfactory function is rarely a life-threatening disability, it nevertheless adversely affects the quality of life and can in some cases lead to changes in mood, appetite, and behavior.

Head Injury and Olfaction

ACKNOWLEDGMENTS This work was supported by Grant DC00165 from the National Institute on Deafness and Other Communication Disorders.

REFERENCES AMA (1993). Guides to the Evaluation of Permanent Impairment. American Medical Association, Chicago. Cain, W. S. (1989). Testing olfaction in a clinical setting. Ear Nose Throat J. 68:316, 322–316, 328. Cain, W. S., Gent, J., Catalanotto, F. A., and Goodspeed, R. B. (1983). Clinical evaluation of olfaction. Am. J. Otolaryngol. 4:252–256. Cain, W. S., Leaderer, B. P., Cannon, L., Tosun, T., and Ismail, H. (1987). Odorization of inert gas for occupational safety: psychophysical considerations. Am. Ind. Hyg. Assoc. J. 48:47–55. Cain, W. S., Gent, J. F., Goodspeed, R. B., and Leonard, G. (1988). Evaluation of olfactory dysfunction in the Connecticut Chemosensory Clinical Research Center. Laryngoscope 98:83–88. Cone, J. E., and Shusterman, D. (1991). Health effects of indoor odorants. Environ. Health Perspect. 95:53–59. Correia, S., Faust, D., and Doty, R. L. (2000). A reexamination of the rate of vocational dysfunction among patients with anosmia and mild/moderate closed head injury. J. Clin. Exp. Neuropsychol. 15:1–12. Costanzo, R. M. (1983). Neural regeneration and functional reconnection following olfactory nerve transection in hamster. Soc. Neurosci. Abstr. 9:699. Costanzo, R. M., and Becker, D. P. (1986). Smell and taste disorders in head injury and neurosurgery patients. In Clinical Measurements of Taste and Smell, H. L. Meiselman and R. S. Rivlin (Eds.). MacMillian Publishing Company, New York, pp. 565–578. Costanzo, R. M., and Zasler, N. O. (1991). Head trauma. In Smell and Taste in Health and Disease, T. V. Getchell, R. L. Doty, L. M. Bartoshuk, and J. B. Snow, Jr. (Eds.). Raven Press, New York, pp. 711–730. Costanzo, R. M., and Zasler, N. D. (1992). Epidemiology and pathophysiology of olfactory and gustatory dysfunction in head trauma. J. Head Trauma Rehabil. 7:15–24. Costanzo, R. M., Heywood, P. G., Ward, J. D., and Young, H. F. (1987). Neurosurgical applications of clinical olfactory assessment. NY Acad. Sci. 510:242–244. Deems, D. A., Doty, R. L., Settle, R. G., Moore Gillon, V., Shaman, P., Mester, A. F., Kimmelman, C. P., Brightman, V. J., and Snow, J. B., Jr. (1991). Smell and taste disorders, a study of 750 patients from the University of Pennsylvania Smell and Taste Center. Arch. Otolaryngol. Head Neck Surg. 117:519–528. Doty, R. L., and Kobal, G. (1995). Current trends in the measurement of olfactory function. In Handbook of Olfaction and Gustation, R. L. Doty (Ed.). Marcel Dekker, Inc., New York, pp. 191–225.

637 Doty, R. L., Shaman, P., and Dann, M. (1984). Development of the University of Pennsylvania Smell Identification Test: a standardized microencapsulated test of olfactory function. Physiol. Behav. 32:489–502. Doty, R. L., McKeown, D. A., Lee, W. W., and Shaman, P. (1995). A study of the test-retest reliability of ten olfactory tests. Chem. Senses 20:645–656. Doty, R. L., Yousem, D. M., Pham, L. T., Kreshak, A. A., Geckle, R., and Lee, W. W. (1997). Olfactory dysfunction in patients with head trauma. Arch. Neurol. 54:1131–1140. Duncan, H. J., and Seiden, A. M. (1995). Long-term follow-up of olfactory loss secondary to head trauma and upper respiratory tract infection. Arch. Otolaryngol. Head Neck Surg. 121:1183–1187. Ferrier, D. (1876). The hemispheres considered physiologically. In The Functions of the Brain. G. P. Putnam’s Sons, New York, pp. 181–211. Heywood, P. G., and Costanzo, R. M. (1986). Identifying normosmics: a comparison of two populations. Am. J. Otolaryngol. 7:194–199. Heywood, P. G., Zasler, N. D., and Costanzo, R. M. (1990). Olfactory screening test for assessment of smell loss following traumatic brain injury. Proceedings of the 14th Annual Conference on Rehabilitation of the Brain Injured Williamsburg, Virginia, 1990. Hoffman, H. J., Ishii, E. K., and Macturk, R. H. (1998). Agerelated changes in the prevalence of smell/taste problems among the United States adult population—results of the 1994 disability supplement to the National Health Interview Survey (NHIS). Ann. NY Acad. Sci. 855:716–722. Ikeda, K., Tabata, K., Oshima, T., Nishikawa, H., Hidaka, H., and Takasaka, T. (1999). Unilateral examination of olfactory threshold using the Jet Stream Olfactometer. Auris Nasus Larynx 26:435–439. Inamitsu, M., Nakashima, T., and Uemura, T. (1990). Immunopathology of olfactory mucosa following injury to the olfactory bulb. J. Laryngol. Otol. 104:959–964. Jackson, J. H. (1864). Illustrations of diseases of the nervous system. London Hospital Report 1:470–471. Kondo, H., Matsuda, T., Hashiba, M., and Baba, S. (1998). A study of the relationship between the T & T olfactometer and the University of Pennsylvania Smell Identification Test in a Japanese population. Am. J. Rhinol. 12:353–358. Koster, N. L., and Costanzo, R. M. (1996). Electrophysiological characterization of the olfactory bulb during recovery from sensory deafferentation. Brain Res. 724:117–120. Kraus, W., Fife, D., and Ramstein, K. (1986). The relationship of family income to the incidence, external causes, and outcomes of serious brain injury, San Diego County, California. Am. J. Public Health Vol 76:1345–1347. Legg, J. W. (1873). A case of anosmia following a blow. Lancet 2:659–660. Leigh, A. D. (1943). Defects in smell after head injury. Lancet 1:38–40. Levin, H. S., High, W. M., and Eisenberg, H. M. (1985). Impairment of olfactory recognition after closed head injury. Brain 108:579–591.

638 Mifka, P. (1964). Post-traumatische anosmie. Wien. Med. Wochenschr. 114:793. Miwa, T., Furukawa, M., Tsukatani, T., Costanzo, R. M., DiNardo L. J. and Reiter E. R. (2001). Impact of olfactory impairment on quality of life and disability. Arch. Otolaryngol. Head Neck Surg. 127:497–503. Moran, D. T., Jafek, B. W., Rowley, J. C. 3d., and Eller, P. M. (1985). Electron microscopy of olfactory epithelia in two patients with anosmia. Arch. Otolaryngol. 111:122–126. Mott, A. E., and Leopold, D. A. (1991). Disorders in taste and smell. Med. Clin. North Am. 75:1321–1353. Notta, A. (1870). Recherches sur la perte de l´odorat. Arch. Gen. Med. 15:385–407. Ogawa, T., and Rutka, J. (1999). Olfactory dysfunction in head injured workers. Acta Otolaryngol. Suppl. 540:50–57. Ogle, W. (1870). Anosmia or cases illustrating the physiology and pathology of the sense of smell. Med. Chir. Trans. 53:263. Raveh, J., Vuillemin, T., and Sutter, F. (1988). Subcranial management of 395 combined frontobasal-midface fractures. Arch. Otolaryngol. Head Neck Surg. 114:1114–1122. Rotch, T. M. (1878). A case of traumatic anosmia and ageusia, with partial loss of hearing and sight. Bost. Med. Surg. J. 99: 130–132. Schiffman, S. S. (1983). Taste and smell in disease. N. Engl. J. Med. 308:1337–1343. Schiffman, S. (1994). Changes in taste and smell: drug interactions and food preferences. Nutr. Rev. 52:S11–S14. Schiffman, S. S. (2000). Intensification of sensory properties of foods for the elderly. J. Nutr. 130:927S–930S.

Costanzo et al. Schiffman, S., S. Zervakis, J., Suggs, M. S., Shaio, E., and Sattely-Miller, E. A. (1999). Effect of medications on taste: example of amitriptyline HCl. Physiol Behav. 66:183–191. Sumner, D. (1964). Post-traumatic anosmia. Brain 87:107–120. Sumner, D. (1975). Disturbance of the senses of smell and taste after head injuries. In Handbook of Clinical Neurology, P. J. Vinken and G. W. Bruyn (Eds.). North-Holland Publishing Co., Amsterdam, pp. 1–25. Takagi, S. F. (1987). A standardized olfactometer in Japan. A review over ten years. Ann. NY Acad. Sci. 510:113–118. Toller, S. V. (1999). Assessing the impact of anosmia: review of a questionnaire’s findings. Chem. Senses 24:705–712. Yee, K. K., and Costanzo, R. M. (1995). Restoration of olfactory mediated behavior after olfactory bulb deafferentation. Physiol. Behav. 58:959–968. Yee, K. K., and Costanzo, R. M. (1998). Changes in odor quality discrimination following recovery from olfactory nerve transection. Chem. Senses 23:513–519. Yousem, D. M., Geckle, R. J., Bilker, W. B., McKeown, D. A., and Doty, R. L. (1996). Posttraumatic olfactory dysfunction: MR and clinical evaluation. Am J. Neuroradiol. 17:1171–1179. Yousem, D. M., Geckle, R. J., Bilker, W. B., Kroger, H., and Doty, R. L. (1999). Posttraumatic smell loss: relationship of psychophysical tests and volumes of the olfactory bulbs and tracts and the temporal lobes. Acad. Radiol. 6: 264–272. Zasler, N. D., Costanzo, R. M., and Heywood, P. G. (1990). Neuroimaging correlates of olfactory dysfunction after traumatic brain injury. Arch. Phys. Med. Rehabil. 71:814. Zusho, H. (1982). Posttraumatic anosmia. Arch. Otolaryngol. 108:90–92.

31 Saliva: Its Role in Taste Function Robert M. Bradley University of Michigan, Ann Arbor, Michigan, U.S.A.

Lloyd M. Beidler Florida State University, Tallahassee, Florida, U.S.A.

I.

INTRODUCTION

particularly important in taste function because they provide the environment for the majority of the tongue’s taste buds (Bradley, 1995). Salivary secretion is under the control of the autonomic nervous system (Garrett and Proctor, 1998). The output of saliva is reflexively controlled and is therefore very dependent on afferent sensory input. Even though there is a baseline flow of saliva, increased flow of saliva requires reflex activation of the autonomic innervation. Normally the total electrolyte content of whole saliva is only about 10% of serum concentrations. However, the osmolality may vary greatly under different physiological conditions and salivary flow rates. The acini of the glands secrete a solution called primary saliva that has about the same Na and K concentrations as blood plasma. However, as the saliva passes through the various ducts and tubules, the primary saliva is modified, Na being reabsorbed from the saliva and K secreted into the saliva. Both the parasympathetic and sympathetic branches of the autonomic nervous system are involved in the control of secretion. The cells of origin of the parasympathetic innervation are located along the medial border of the solitary tract nucleus in the brainstem in the superior and inferior salivatory nuclei, while the sympathetic innervation originates in the intermediolateral nucleus situated in the upper thoracic segments of the spinal cord (Bradley, 1991). The parasympathetic secretomotor fibers travel to the glands with various

Saliva is a complex fluid secreted by glands that drain into the oral cavity. This fluid coats the whole surface of the oral mucosa, including the apical extensions of the taste receptor cells, providing the external milieu of the taste receptor. Thus, chemicals that eventually interact with the taste receptive membranes have to traverse this fluid layer; hence, the initial events in taste transduction that are sometimes referred to as perireceptor events involve saliva (Bradley, 1991; Matsuo 2000; Spielman, 1990). Salivary glands are composed of a large number of identical, blindly ending, tubes. The closed end consists of a cluster of pyramid-shaped acinar cells forming the gland acini. The lumen of the acini are contiguous with a duct system that converge on the main excretory duct that drains saliva (called final saliva) into the oral cavity (Baum, 1993). There are one or two types of acinar cells and four or five cell types in the duct system. The bulk of saliva is secreted by three paired salivary glands: the parotid, submandibular, and sublingual glands. However, a series of smaller salivary glands in the mucosa of the lips (labial), cheeks (buccal), palate (palatine), and tongue (lingual) also contribute significantly to the overall volume of saliva. The lingual salivary glands that drain into the clefts of the circumvallate and foliate papillae on the posterior tongue (von Ebner’s glands) are thought to be 639

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Figure 1 Brainstem and spinal cord autonomic circuits involved in the reflex control of salivary secretion. VII, facial nerve; IX, glossopharyngeal nerve. (From Bradley, 1995).

cranial nerve branches, while the sympathetic innervation distributes with blood vessels. Before synapsing with the basolateral membranes of the gland acini, the parasympathetic fibers first synapse in peripheral autonomic ganglia, and the sympathetic fibers synapse in the superior cervical ganglion (Fig. 1). II.

CHEMICAL COMPOSITION OF SALIVA

The chemical composition of the secretion of each type of salivary gland differs. The concentration of a given substance in whole saliva varies with flow rate, species, sex, physical activity, pharmacological state, and time of day. While 99% of saliva is water, the remaining 1% consists of ions and organic chemicals. A.

Ions

The most important cations of saliva are sodium and potassium, while the predominant anions are chloride and bicarbonate. Other electrolytes also occur in saliva including calcium, phosphate, fluoride, thiocyanate, magnesium, sulfate, and iodide. Salivary water and ions are derived from blood plasma, but their concentrations in final saliva depends on a number of factors, of which the most important is flow rate (Fig. 2) (Schneyer et al., 1972).

B.

Proteins

Recent advances in electrophoresis, DNA, and protein sequencing and identification with labeled antibodies have greatly increased our knowledge of the organic composition of saliva. It is estimated that over 200 different proteins and peptides are contained in the saliva of human and mouse (see Table 1). Many of these, however, may be either isozymes or members of the same protein family. They may range in size from small peptides to large immunoglobins and include highly acidic, as well as basic, proteins. The heterogeneity of the salivary protein population is primarily due to peptide or protein manufacture in the acini with polygenic determinants. This may be followed by further modifications during the transcription and translation process, such as glycosylation or phosphorylation during the passage through the tubules. A variety of other molecules, including steroids and a number of organic molecules, are included in saliva. A given substance or a whole family of proteins may be separated with the aid of binding columns or gels, purified, sequenced, and then computer-compared to other known proteins. A more elegant method of protein identification is to isolate salivary RNA and derive a specific DNA clone whose gene can be sequenced and computer-compared. Many substances in the blood serum are also found in saliva. The salivary concentration of some of these is

Saliva: Its Role in Taste Function

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Figure 2 Electrolyte composition, pH, and osmolality of human parotid gland saliva at resting (0.01 mL/min) and stimulated (1.0 mL/min) flow rates. (Data from Shannon et al., 1974.)

proportional to the serum concentration. Since it is more convenient to obtain a sample of saliva than of blood, the former is often used in routine tests to follow changes in serum concentrations of agents of interest (e.g., chemicals, viruses), such as human immunodeficiency virus (HIV). Salivary tests have been utilized for a number of steroid hormones, for drugs including cocaine and marijuana, and for a measure of cigarette smoking. Small molecules [molecular weight (MW) 100] pass into the saliva from the blood compartment by filtration. The speed of transfer of many larger molecules is related to the lipophilicity or partition coefficient and the pK. The salivary flow rate and pH are also important in determining the drug transfer. Most drugs are not bound to salivary proteins, whereas they appear in both bound and unbound (active) forms in the blood plasma. III. A.

GENERAL FUNCTIONS OF SALIVA Tissue Permeability and Lubrication

Saliva coats the entire tissue area of the oral cavity. The mean total surface area of the adult mouth is about 215 cm2, and the average thickness of the mobile salivary film is 0.07–0.10 mm (Collins and Dawes, 1987). The teeth repre-

sent 21%, the dorsum of the tongue 12%, and the combined ventral surface of the tongue and floor of the mouth 12.3% of the total surface area, or 16% of the oral mucosa. This thin aqueous salivary layer is only a small barrier to passage of water-soluble molecules into the epidermal layer of the oral cavity. The speed of tissue permeation is more dependent on the lipid character of the epidermal layer. However, the thin aqueous layer on the surface of the teeth is little protection for the exposed dental plaque that normally coats various surfaces of the teeth, and substances such as sugars and acids have access since no lipid barrier exists between the plaque and oral cavity. Passage of chemicals through the more highly keratinized tissue of the dorsal tongue surface has been studied with animal models. Penetration was shown to be proportional to the magnitude of the lipid:aqueous partition coefficient (Mistretta, 1971) The high molecular weight glycoproteins such as the mucins adhere to the cell surfaces and contribute to the slippery and tenacious character of the surfaces of the oral cavity. They also give saliva its rather high viscosity. Both viscosity and slipperiness contribute to saliva’s unusual properties, which distinguish it from other aqueous solvents. The water retention expressed by the solvation of the carbohydrate residues of the glyco-

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Table 1 Proteins and Peptides in Mammalian Whole Salivaa

proteins contributes to lubrication under light load and high sliding speed. Lubrication under heavy loads is provided by salivary statherin due to its amphipathic properties. The polar end of statherin adorbs to the hydroxyapatite surface of the tooth, and its less polar tail extends outward to form an oriented film, which increases horizontal sliding (Douglas et al., 1991). Salivary lubrication is also very important in bolus formation, swallowing, and speech production.

starch for enzyme reaction and the length of time the starch remains in the oral cavity before being swallowed. Since the stomach acidity is too high for amylase enzymatic activity, it is thought that little further starch digestion occurs until the food enters the small intestine and is in contact with pancreatic amylase (Rosenblum et al., 1988). However, starch digestion is not totally eliminated in pathological conditions of the pancreas in which pancreatic amylase activity is impaired. Young infants can digest starch in spite of an effective absence of pancreatic activity during the first 6–8 months of life, and since amylase is present in the saliva of term infants it is likely that this nonpancreatic source of amylase is responsible for starch digestion (Lebenthal and Lee, 1984; Murray et al., 1986; Zoppi et al., 1972). Moreover, salivary amylase activity in the stomach is protected by the substrates of amylase as well as the end products of amylase digestion (Rosenblum et al., 1988). Thus, salivary amylase may play a more important role in starch digestion not only in the mouth, but in other parts of the digestive tract as well. The adhering of products of starch digestion to the dental plaque proceeds over a much longer time period than that of the free starch in the saliva of the oral cavity. The longer period of adhesion affords amylase sufficient time to break the starch into its smaller sugar components. These are then metabolized by the bacteria, and acid is released, which attacks the enamel surface. If sticky foods containing mono- or disaccharides are taken into the mouth, acid will also accumulate at the surface of the teeth over a long time period. This is in contrast to the intake of sugar-containing drinks, which are much more rapidly removed from the oral cavity, resulting in a low pH state of short duration and producing less tooth decay. The von Ebner salivary glands secrete lipase. Fat digestion can begin in the mouth and is a very important reaction in those infants having little pancreatic function or those with cystic fibrosis. Furthermore, lingual lipase can function after being swallowed and passed into the stomach, where the pH is low (Hamosh, 1990). Perhaps the most important function of saliva in the digestive process is solubilization of the ingested food. The largest component of saliva is water. Many of the components of food are water-soluble and are released into the oral cavity during chewing. Solubilization also increases dispersion, and both increase the efficiency of enzyme action.

B.

C.

N-Acetyl-D-glucosaminidase Agglutinagen A Agglutinagen B Agglutinagen C Albumin Aldolase Amylase family Angiotensin II Anticomplementary factor Antileukoprotease Antitrypsin Aproerytherin Biopterin Bone marrow colonystimulating factor Calmodulin Carbonic anhydrase Cathepsin Collagenase Cystatin family Elastase Endothelial growthstimulating factor Esterase Esterase B Esteropeptidase Ferritin Fibronectin Fucosyltransferase Gastrin Glucagon Glutamine–glutamic acid protein family Granulocytosis-inducing factor

6-Hydroxymethylpterin IgA Prostaglandins IgG Pterin IgM Renin Kallikrein Lactoferrin Lactoperoxidase Lethal factor Lingual lipase Lipoproteins Lysozyme Mesodermal growth factor Metalloprotease Monopterin Mucins Neopterin Nerve growth factor Neural tube growth factor Parotid glycoprotein family Peroxidase Plasminogen activator Platelet-activating factor Proline-rich proteins Sialomucin Sialotonin Somatostatin Statherin Tissue plasminogen activator Tonin Transferrin VEG protein Vitamin B–binding proteins Wound contraction factor Xanthopterin

aIf individual protein family members and isoenzymes are included separately, the total number approaches 200.

Digestion

Amylase is a major protein component of human saliva. It initiates the breakdown of starch in the oral cavity. The extent of such digestion depends on the availability of the

Wound Healing

Licking of wounds is a common observation in many mammals. This instinctive behavior has been shown to be important in assisting wound healing. Saliva contains a

Saliva: Its Role in Taste Function

number of substances that have a role in wound healing such as epidermal growth factor (EGF), nerve growth factor (NGF), and transforming growth factor–alpha (TGF) (Bodner, 1991; Humphreys-Beher, et al., 1994; Schultz et al., 1991). The tongue, in particular, is constantly exposed to destructive mechanical and chemical trauma, and the EGF of saliva has an important role in promoting wound healing within the oral cavity (Noguchi et al, 1991). More recently, EGF in saliva has been shown to play a role in maintaining taste buds. Removal of the submandibular and sublingual salivary glands results in the loss of taste buds in the fungiform papillae, which can be prevented if EGF is supplied in the drinking water (MorrisWiman et al., 2000), indicating that salivary EGF has an important role in taste bud maintenance. D.

Temperature Control

A number of different mammals, including rats, that do not lose heat by sweating or panting, spread saliva on their body surface, resulting in heat loss by evaporative cooling. In this means of cooling, conversion of body water to water vapor results in a heat loss by the process of the latent heat of evaporation (Mitchell, 1974). The importance of saliva in temperature control is demonstrated in rats that have been desalivated. Rats without saliva can tolerate heat exposure for a much shorter time than normal rats (Hainsworth, 1968). In the rat the submaxillary and sublingual gland provide the most significant contribution to evaporative cooling and the parotid gland provides supplemental secretion during extreme hyperthermia (Hainsworth and Stricker, 1969, 1971). E.

Control of Oral Flora and Dentition

Saliva regulates the bacterial and fungal populations that reside in the oral cavity. Lactoferrin, lactoperoxidase, N-acetyl-D-glucosaminidase, and lysozyme all have antimicrobial action. The mucin glycoproteins have a high viscosity, which helps clear the bacteria from the oral cavity as well as modulate the bacterial colonization on various surfaces. The histidine-rich proteins also help control fungal growth. Excessive growth of bacterial and fungal populations may lead to oral infections. The immunoglobins aid in the control of such infections. In addition, bacterial accumulations on the surface of the tooth localize acid production due to sugar metabolism. The salivary calcium phosphate supersaturated levels help control the hydroxyapatite crystal formation on the surface of the tooth. The cystatins, statherin, and proline-rich proteins help control the level of salivary calcium phosphate. Statherin in particular inhibits

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calcium phosphate precipitation and controls the mineralization of the tooth. F.

Buffering Capacity

The pH of resting (or unstimulated salivary secretion) saliva is about 5.7–6.2. This value is very dependent on salivary flow rate, rising with the magnitude of flow rate to become more alkaline (7.6) (Fig. 2). The major components of the salivary buffering system are bicarbonates and phosphates. The buffering capacity is dependent not only on the concentrations of phosphates and carbonates at any given time, but also on their rate of replacement by changes in flow rate and by the total amount of saliva in the oral cavity at any given time. Small pH electrodes have been used to follow the changes in dental plaque pH after a sucrose rinse. The initial pH of the plaque fell soon after the sucrose rinse and then returned to the control value after 20 minutes as the buffering role of the saliva became effective. The buffering capability of saliva became apparent if the access of saliva to the dental plaque was restricted. The initial pH fell and remained low for over 20 minutes (Englander et al., 1959). G.

Role of Saliva in Taste Function

Saliva plays a dominant role in the initial events in taste transduction. It is the medium in which solid food must dissolve and be transported to the taste bud receptor cell membranes. Since a layer of saliva always coats the tongue surface and fills the taste pores, taste stimuli have to diffuse through this layer to gain access to the receptor sites on the apical portion of the taste bud cells. This process has been modeled by Beidler (1961) and DeSimone and Heck (1980). When calculations based on this model were compared to measures of the latency of the electrophysiological response (Marowitz and Halpern, 1977), diffusion time was not a major component in response latency. Taste buds in the fungiform papillae are bathed in saliva derived from the major salivary glands. However, the largest population of taste buds is located in the circumvallate and foliate papillae. The taste bud density along the papilla walls is very high, amounting to 60% of the total lingual taste buds in rats (Bradley, 1995). The clefts of the circumvallate and foliate papillae are very narrow and deep, isolating them from saliva of the major glands. Thus, slow diffusion cannot account for the rapid entry of tastant-laden saliva, and a pumping action during tissue movement has been suggested as a mechanism. The fluid milieu of the clefts is provided by saliva entering the base of the grooves from the von Ebner salivary glands.

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Electrophysiological recordings from circumvallate taste buds concurrent with stimulation of von Ebner gland secretion has demonstrated that fluid from these glands can effectively rinse away a taste stimulus (Gurkan and Bradley, 1988). Thus, the lack of saliva can greatly impede the sensation of taste by decreasing tastant solubilization as well as tastant transport. In addition to solubilization and transport, saliva has a number of other roles in taste sensation. 1. Saliva as an Adapting Solution Since saliva is a solution containing ions that can be tasted, the taste receptor membranes are constantly exposed to a low concentration of these tastants. Of these ions, sodium, potassium, chloride and bicarbonate can achieve concentrations above their taste-detection level (Fig. 2). Sodium ions have been shown to influence saltiness (Delwiche and O’Mahony, 1996; McBurney and Pfaffmann, 1963) resulting in elevated taste thresholds. Moreover, since the ambient concentrations of sodium and potassium vary with salivary flow rate, their taste detection thresholds may also vary. Thus, when measuring taste thresholds, it is important to rinse the adapting solution away. The significance of saliva as an adapting solution was demonstrated by Matsuo and coworkers, who compared taste responses in anesthetized and unanesthetized animals (Matsuo et al., 1994; Matsuo and Yamamoto 1990, 1992). In acute recordings from the chorda tympani in rats in which the saliva is effectively washed off the tongue, the response to 0.1 M NaC1 was of much greater magnitude than the response to 0.5 M sucrose. In contrast, the responses to these two stimuli were of similar magnitude in unanesthetized animals in which the tongue was not rinsed. Similar results can be obtained in anesthetized animals if the tongue is adapted to saliva rather than distilled water. However, in experiments on the hamster chorda tympani, tongue adaptation to saliva has no effect on the response to sucrose, perhaps reflecting a species difference (Rehnberg et al., 1992). Moreover, the dramatic effect of saliva on sweet responses in the rat are not observed at either the solitary nucleus (Nakamura and Norgren, 1991) or pontine taste relay nuclei (Nishijo and Norgren, 1991). 2. Role of Saliva in Bitter Taste The most studied bitter taste is that of sucrose octaacetate (SOA) in mice. A close linkage of mouse genes for SOA bitter taste and salivary proline-rich proteins (PRP) was found by Azen et al. (1986). This is of interest in the study of bitterness in humans since PRPs represent up to 70% of the total proteins in human parotid saliva.

Bradley and Beidler

The salivary PRPs also chemically interact with many tastants in foods. For example, the taste threshold of tannic acid in mice parallels the ability of PRPs to bind tannic acid (Glendinning, 1992). It is interesting that when tannin-containing foods are ingested by many wild animals, their bodies react physiologically to increase the concentration of PRPs in the saliva and thus bind more tannin, which in turn decreases its salivary concentration and increases the taste threshold. The concentration of salivary PRPs in many animals can be greatly altered by the administration of adrenergic antagonists such as isoproterenol (Bannister et al., 1989; Mehansho et al., 1985). 3.

Role of Saliva in Sour Taste

The saliva’s ambient pH, its ability to maintain it (buffering capacity), and the type of acid introduced into the oral cavity are all important determinants of the magnitude of sourness of acids. A reduction in the perceived sourness of HCl has been shown to occur as a result of the buffering action of salivary bicarbonate (Christensen et al., 1987; Helm et al., 1982; Norris et al., 1984). Since the pH of saliva varies with a number of physiological parameters, sourness and acid detection can vary. Similar results have been reported in electrophysiological recordings from the rat chorda tympani. Responses of the chorda tympani to HCl differ when the tongue is adapted to saliva or water. The response to HCl is less when the tongue is bathed in saliva compared to responses recorded in water adaptation (Matsuo and Yamamoto, 1992). Chorda tympani responses to HCl were 40% lower in awake rats licking 0.01 M HCl than in anesthetized rats in which saliva was absent (Matsuo et al., 1994).

4.

Von Ebner Gland Proteins

A salivary protein was found in human and rat von Ebner salivary glands that belongs to the lipocalin superfamily of lipophilic ligand carrier proteins (Bläker et al., 1993; Schmale et al., 1990). The protein is expressed in the acinar cells of the gland and is secreted into the saliva. It was postulated that this salivary protein binds bitter hydrophobic tastants and thus concentrates them in the saliva adjacent to the taste buds in the clefts of the circumvallate and foliate papillae. The hydrophobic tastant carriers would increase the availability of the bitter substances to the taste receptor microvilli. The role of von Ebner protein in bitter taste has been tested in mice. Because the von Ebner protein is not synthesized in mice, it was possible to introduce the gene from the rat responsible for expressing the protein in

Saliva: Its Role in Taste Function

mice and then perform behavioral tests. The bitter-tasting substance used in the behavioral test was dentatonium benzoate, and tests were performed on transgenic mice expressing the von Ebner protein and on control mice. While both transgenic and nontransgenic mice were aversive to the higher concentrations of dentatonium benzoate, at 0.01 mM the preference ratios were significantly higher in the transgenic animals (Kock et al., 1994). Thus, the control mice appeared to be more sensitive to dentatonium benzoate, throwing some doubt on the role of von Ebner protein in bitter taste. Further doubts about the role of von Ebner protein in bitter taste function comes from its absence in some species despite an almost universal ability to taste bitter compounds, its lack of differential expression when stimulation with the four taste modalities (Spielman et al., 1993), and its identical neuclotide sequence to a protein present in tears known as tear prealbumin (van’t Hof et al., 1997). Lastly, compounds known to be extremely bitter in humans do not bind to the von Ebner protein (Schmale et al., 1993). Thus, the physiological function of the von Ebner protein remains an enigma. It may contribute to the clearance of taste stimuli (Kock et al., 1994) or may play a role in the control of inflammatory processes (van’t Hof et al., 1997). A further protein that is located in the ducts, but not the acini of von Ebner glands, has been described (Li and Snyder, 1995), which also has no known function but serves to reinforce the unique character of the salivary glands associated with the foliate and circumvallate papillae.

5. Gustin An acidic zinc-containing salivary protein named gustin was described in human parotid saliva (Henkin et al., 1975). Gustin has been implicated in taste bud growth functions, and a role in taste bud maintenance was indicated and was decreased in concentration in patients with taste disorders (Shatzman and Henkin, 1981). Because gustin contained zinc, it was suggested that zinc is an important ion in normal taste function (Henkin, 1984). Moreover, loss of taste function was thought to be related to zinc deficiency, and zinc supplement was suggested for those patients who complained of loss or alteration of taste function. However, the role of zinc in taste disorders has been questioned since in a double-blind study the use of zinc was effectively equivalent to a placebo in the treatment of taste disorders (Anonymous 1979; Henkin et al., 1976). Moreover, it has recently been shown that gustin is actually carbonic anhydrase VI and is therefore not a protein uniquely associated with taste buds (Thatcher et al., 1998).

645 Table 2 Electrochemical Seriesa Gold Mercury Palladium Silver Tin Aluminum

 1.7 volts  0.91 volt  0.83 volt  0.80 volt  0.14 volt  1.7 volts

a

Values as reduction potentials. Source: From Hunsberger, 1974.

H.

Saliva and Galvanic Currents

A source of electrical current is formed when two dissimilar metals are placed in an ionic medium such as a body fluid. Galvani utilized such an arrangement to stimulate nerve and muscles in his studies of electricity (Herness, 1985), and Volta studied the effect of metals placed into the oral cavity which stimulated the taste buds (Piccolino, 1997). Today it is common for adults to have dental amalgam fillings or gold crowns, which, under certain conditions, may act as a source of electrical current (Hugoson, 1986). There may be two consequences. First, the currents may stimulate sensory nerves and initiate sensations of taste, pain, or other unusual sensory qualities. Second, electrolysis at the interface of the metal and ionic saliva may release metallic ions injurious to the surrounding tissue and cause corrosive damage. Recently, the release of mercury from amalgam fillings has led to public debate concerning their safety. The magnitudes of galvanic currents are dependent on the ionic content of saliva, including its pH, and the nature of the metals. The saliva can vary with many conditions, including the salivary flow rate. However, the magnitude of the voltages produced is mostly influenced by the choice of the two metals involved, the magnitude being proportional to the difference in their reduction potentials (see Table 2). Note that a large potential difference may be generated when an individual with a gold crown takes a piece of aluminum foil into the mouth. Since the path of current may pass down the root canal and enter the nerve, the generated pain is both immediate and strong! The return current path is by way of the bony fluid and the saliva in contact with the foil (Schreiver and Diamond, 1952). Note also that a gold crown should never be in direct contact with an amalgam filling! IV. SJÖGREN’S SYNDROME AND SALIVARY FUNCTION Sjögren’s syndrome (SS) is an autoimmune disease characterized by exocrine gland dysfunction. One of the symptoms

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of this disorder is decreased function of the salivary glands, reducing salivary flow and resulting in a dry mouth or xerostomia (Sreebny and Broich, 1987; Van der Reijden et al., 1999). SS is more frequent in females than males (9:1) and is most often found in individuals over 40 years of age. Since saliva plays such an important role in taste function, it is not surprising to find that SS patients often complain of impaired taste function. Patients with SS were found to have a decreased taste acuity (Henkin, 1972). However, individuals with chronic and complete absence of the salivary glands have normal taste function when tested, indicating that the taste system itself is normal (Weiffenbach, 1986). Moreover, in a more recent investigation of SS patients with complaints of impaired taste function, judgments of taste intensity were normal despite a decreased threshold sensitivity (Weiffenbach et al., 1995). Thus, the complaints probably do not reflect changes in the peripheral taste system but may be due to the problems with taste stimulus solubilization and delivery to the taste receptors. In addition, since taste transduction involves apical ion channels on the taste receptor membranes, the absence of a source of permeating ions may impair taste transduction.

V. EFFECTS OF DRUGS ON SALIVARY SECRETION Although SS is often related to salivary dysfunction, a large number of other diseases may also be involved, including often recognized diseases such as hypertension, rheumatoid diseases, diabetes, and hyperlipidemia. Those patients with cystic fibrosis, sarcoidosis, Bell’s palsy, pancreatitis, and thyroiditis also have symptoms, as do those with adrenocortical diseases. Since many of these diseases are observed in the elderly population and since many of the associated medications also have an effect on saliva production, complaints of dry mouth are common in the elderly. Nearly one in five older adults has xerostomia (Rhodus and Brown, 1990). Over 400 drugs have been identified as producing xerostomic side effects (Sreebny and Schwartz, 1986). The drugs that produce dry mouth can be categorized according to therapeutic function (Table 3). The number of people aged 65 years or older in the United States is about 50 million. Many of these are treated for several conditions requiring several drugs to be taken daily. Dry mouth is a common complaint. Fortunately, most patients with dry mouth have no irreversible damage to the major salivary glands. Furthermore, many such patients take four or more drugs daily. The physician should reconsider the number of drugs given and their dose and should

Bradley and Beidler Table 3 Drugs that Effect Salivary Secretion Category

Number of drugs

Antihistamine Anticholinergic Antihypertensive Anorexic Diuretic Antidepressant Antipsychotic Analgesic Antinauseant Antiparkinsonism Psychotherapeutic Cough and cold preparation Antidiarrheal Anti-inflammatory

56 48 45 44 33 25 24 15 11 11 11 9 8 7

suggest substitutes. Since dryness is particularly noted at night, the drug schedule should be such that maximum dosage does not occur at that time. VI. REMEDIES FOR LOW SALIVARY FLOW RATES A.

Changing Patient Habits

Very slow salivary flow rates greatly decrease the buffering capacity of the fluid in the mouth, its ability to remove the starch or sugars from the surface of the tongue, and the amount of antibacteriosides, antibodies, growth factors, enamel-supporting agents, and numerous other beneficial proteins that normally pour into the oral cavity. No longer can the patient depend on saliva to maintain a healthy oral environment. Mastication initiates the salivary response. Human consumption of a liquid diet (Metrecal) rather than normal solid food for 1 week was shown to decrease parotid flow by 30% (Hall et al., 1967). A similar 2-week experiment with rats showed a 39–52% decrease in parotid gland weight accompanied by atrophy (Hall and Schneyer, 1964). Such experiments suggest that persons with low salivary flow rate should change their diet to increase the magnitude and length of time of chewing. Crisp vegetables and fruits should replace mashed potatoes and other soft foods, and nuts and hard pretzels should be taken as snacks for those with moderate but not severe dry mouth. Between meals a noncaloric object can be held in the mouth. It is interesting to note that American Indians were reported to place a pebble in the mouth to stimulate salivary flow when water was not immediately available.

Saliva: Its Role in Taste Function

The patient should be advised to gargle with a water rinse immediately following a meal or snack to slow the progression of tooth decay. Chewing gum for 30 minutes or more after meals enhances remineralization of carieslike lesions in human enamel (Manning and Edgar, 1992). In addition, it is important to rinse the mouth with a suitable buffer before retiring or taking a nap. If a tablet is used instead of a liquid, it is important not to concentrate the tablet in any given area of the oral cavity or else damage may occur. It is preferable to follow the tablet with liquid a few minutes after the remains of the tablet disappear. The use of commercial mouthwashes that contain high concentrations of alcohol is to be discouraged. Since the concentration of those agents in the oral fluid that normally aid in guarding against tissue damage is greatly decreased, the patient should have his dentist check the mouth frequently. A continuous dialogue between medical doctor and dentist is warranted. Continued use of tobacco in any form as well as of alcohol should be discouraged. Use of the two together is even more deadly since many of the injurious chemicals associated with tobacco are soluble in alcohol, which easily penetrates the barrier of the outer cell layer of the oral tissue and interacts with the dividing basal cells beneath. B.

Saliva Substitutes

One of the most important aspects of saliva is mouthfeel and associated bolus movement. Water alone is not a good substitute except for temporary relief (Gans et al., 1990). Considering the large number of different chemicals found in whole mouth saliva, one would expect a good saliva substitute to be rather complex. The high molecular weight mucins are the primary contributors to the saliva’s viscosity. A substitute of about 1% carboxymethylcellulose (CMC) is often used in commercially available salivas to produce an acceptable viscosity. However, lubrication of hard surfaces is poor with such a simple fluid. Replacement of CMC with mucin enhances consumer acceptability. The proline-rich glycoproteins of saliva serve to lubricate hard surfaces, and the addition of serum albumin enhances the effect (Hatton et al., 1987). Because of their formulation simplicities, most artificial salivas do not produce a long-lasting lubricating effect (Olsson and Axell, 1991). Recently, polymer-based saliva substitutes have been developed as an artificial saliva with some success (van der Reijden et al., 1996). Most saliva substitutes highlight viscosity but do not adequately address the problem of remineralization. The substrates are usually buffered and contain mineral salts including fluorides. However, there is no substitution for statherin and the anionic proline-rich proteins that are the

647

normal salivary components involved in remineralization (Douglas, 1991). The antimicrobial function of normal saliva is very important and is served by secretory IgA, histidine-rich proteins, lactoferrin, and lactoperoxidase. Artificial salivas do not adequately address this issue. Since both oral bacterial and fungal infections are not uncommon and acquired immune deficiency is of increasing importance, Levine et al. (1987) suggested that separate artificial salivas with appropriate oral medications be developed to serve people with these ailments.

C.

Salivary Stimulation

Many patients with salivary deficits still have some salivary tissue functioning. The flow rate is normally increased by mechanical and gustatory stimulation (Kjeilen et al., 1987). The myoepithelial cells respond to chewing and initiate the reflex leading to salivary flow. The latter is directly related to the number of teeth involved. The gustatory-salivary reflex is best initiated with sour (citric acid) and sweet stimuli. Thus, chewing sweet-sour gum or lozenges is helpful for many with xerostomia. Unfortunately, maximum stimulation is usually shortlived. De Rossi et al. (1987) reported the parotid glands’ resting flow rate as 0.08 mL/min for men aged 41–60, which rose to 0.59 mL/min after they chewed fruit gum and 1.7 mL/min after ingestion of lemon juice. Sreebny and Broich (1987) indicate that unstimulated flow of whole saliva of normal people is about 0.3–0.5 mL/min; most of this is of submandibular/sublingual origin. After 2% citric acid stimulation the whole saliva flow rate rises to about 1–3 mL/min, with the parotid and submandibular/sublingual glands contributing a large fraction of the total saliva, the former slightly more than the latter. The stimulated saliva is very important in eating and in speech. However, the feeling of oral dryness may be more correlated with unstimulated saliva that is primarily mucus in character. Thus, most commercial artificial salivas, which do not contain mucin-like substances, are not very effective in relieving the dry mouth sensation (Sreebny and Broich, 1987). Salivary gland secretion can be stimulated directly by parasympathetic drugs. Fox et al. (1991) used 5-mg capsules of pilocarpine hydrochloride given three times a day. Two thirds of the patients with salivary hypofunction increased their saliva production with only mild and tolerable side effects. These trials lasted 5 months. If parasympathetic agonists can increase salivary gland output, then one would expect that antagonists would decrease salivary gland output. Many dry mouth complaints of the elderly are due to side effects of their medication.

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ACKNOWLEDGMENTS The preparation of this chapter was supported in part by NIH grants from the National Institute of Deafness and other Communication Disdorders numbers DC00288 and DC04198 to RMB. REFERENCES Anonymous (1979). Ineffectiveness of zinc in treating ordinary taste and smell dysfunctions. Nutr. Rev. 37:283–285. Azen, E., Lash, I., and Taylor, B. (1986). Close linkage of mouse genes for salivary proline-rich proteins (PRPs) and taste. Trends Genet. 2:199–200. Bannister, A. J., Divecha, N., Ashmore, M., and McDonald, C. J. (1989). Basic proline-rich proteins of murine parotid glands: induction of mRNA by isoprenaline and post-secretion processing. Eur. J. Biochem. 181:371–379. Baum, B. J. (1993). Principles of saliva secretion. Ann. NY Acad. Sci. 694:17–23. Beidler, L. M. (1961). Taste receptor stimulation. In Progress in Biophysics and Biophysical Chemistry, XII, J. A. V. Butler, H. E. Huxley, and R. E. Zirkle (Eds.). Pergamon Press, New York, pp. 107–151. Bläker, M., K. Kock, C. Ahlers, F. Buck, and Schmale, H. (1993). Molecular cloning of human von Ebner’s gland protein, a member of the lipocalin superfamily highly expressed in lingual salivary glands. Biochim. Biophys. Acta Gene Struct. Expression 1172:131–137. Bodner, L. (1991). Effect of parotid submandibular and sublingual saliva on wound healing in rats. Comp. Biochem. Physiol. [A] 100A:887–890. Bradley, R. M. (1991) Salivary secretion. In Smell and Taste in Health and Disease, T. V. Getchell, R. L. Doty, L. M. Bartoshuk, and J. B. J. Snow (Eds.). New York: Raven Press, pp. 127–144. Bradley, R. M. (1995) Essentials of Oral Physiology. Mosby, St. Louis. Christensen C. M., Brand J. G., and Malamud D. (1987). Salivary changes in solution pH: a source of individual differences in sour taste perception. Physiol. Behav. 40:221–227. Collins, L., and Dawes, C. (1987). The surface area of the adult human mouth and thickness of the salivary film covering the teeth and oral mucosa. J. Dent. Res. 66:1300–1302. Delwiche, J., and O’Mahony, M. (1996). Changes in secreted salivary sodium are sufficient to alter salt taste sensitivity: Use of signal detection measures with continuous monitoring of the oral environment. Physiol. Behav. 59:605–611. De Rossi, G., Focacci, C., and Campioni, P. (1987). Salivary gland physiology and radioisotope uptake. In Radioisotope Study of Salivary Glands, G. De Rossi (Ed.). CRC Press, Boca Raton, FL, pp. 2–21. DeSimone, J. A., and Heck, G. L. (1980). An analysis of the effects of stimulus transport and membrane charge on the salt, acid and water-response of mammals. Chem. Senses 5:295–316.

Bradley and Beidler Douglas, W., Reeb, E., Ramasubbu, N., Raj, P., Bhandary, K., and Levine, M. (1991). Statherin: a major boundary lubricant of human saliva. Biochem. Biophys. Res. Commun. 180: 91–97. Englander, H. R., I. L. Shklair, and Fosdick, L. S. (1959). The effects of saliva on the pH and lactate concentration in dental plaques I. Caries-rampant individuals. J. Dent. Res. 38:848–853. Fox, P., Atkinson, J., Macynski, A., Wolff, A., Kung, D., Valdez, I., Jackson, W., Delapenha, R., Shiroky, J., and Baum, B. (1991). Pilocarpine treatment of salivary gland hypofunction and dry mouth (xerostomia). Arch. Intern Med. 151: 1149–1152. Gans, R., Watson, G., and Tabak, L. (1990). A new assessment in vitro of human salivary lubrication using a compliant substrate. Arch. Oral Biol. 35:487–492. Garrett, J. R., and Proctor, G. B. (1998). Control of salivation. Front. Oral Biol. 9:135–155. Glendinning, J. (1992). Effect of salivary proline-rich proteins on ingestive responses to tannic acid in mice. Chem. Senses 17:1–12. Gurkan, S., and Bradley, R. M. (1988). Secretions of von Ebner’s glands influence responses from taste buds in rat circumvallate papilla. Chem. Senses 13:655–661. Hainsworth, F. R. (1968) Evaporative water loss from rats in the heat. Am. J. Physiol. 214:979–982. Hainsworth, F. R., and Stricker, E. M. (1969) Evaporative cooling in the rat: effects of partial salivation. Am. J. Physiol. 217:494–497. Hainsworth, F. R., and Stricker, E. M. (1971). Evaporative cooling in the rat: differences between salivatory glands as thermoregulatory effectors. Can. J. Physiol. Pharmacol. 49: 573–580. Hall, H., and Schneyer, C. (1964). Salivary gland atrophy in rat induced by liquid diet. Proc. Soc. Exp. Biol. Med. 117: 789–793. Hall, M., Merig, J., and Schneyer, C. (1967). Metrecal-induced changes in human saliva. Proc. Soc. Exp. Biol. Med. 124: 532–536. Hamosh, M. (1990). Lingual lipase. In Lingual and Gastric Lipases: Their Role in Fat Digestion, M. Hamosh (Ed.). CRC Press, Boca Raton, FL, pp. 35–106. Hatton, M., Levine, M., Margarone, J., and Aguirre, A. (1987). Lubrication and viscosity features of human saliva and commercially available saliva substitutes. J. Oral Maxillofae. Surg. 45:496–499. Helm, J. F., Dodds, W. J., Hogan, W. J., Soergel K. H., Egide, M. S., and Wood, C. M. (1982). Acid neutralizing capacity of human saliva. Gastroenterology 83:69–74. Henkin, R. I. (1984). Zinc in taste function. A critical review. Biol. Tr. Elem. Res. 6:263–280. Henkin, R. I., Talal, N., Larson, A. L., and Mattern, C. F. T. (1972). Abnormalities of taste and smell in Sjorgren’s syndrome. Ann. Intern. Med. 76:375–383. Henkin, R. I., Lippoldt, R., Bilstad, J., and Edelhoch, H. (1975). A zinc protein isolated from human parotid saliva. Proc. Natl. Acad. Sci. USA 72:488–492. Henkin, R. I., Schechter, P. J., Friedewald, W. T., Demets, D. L., and Raff, M. (1976). A double blind study of the effects of

Saliva: Its Role in Taste Function zinc sulfate on taste and smell dysfunction. Am. J. Med. Sci. 272:285–299. Herness, M. S. (1985). Neurophysiological and biophysical evidence on the mechanism of electric taste. J. Gen. Physiol. 86:59–87. Humphreys-Beher, M. G., Macauley, S. P., Chegini, N., van Setten, G., Purushotham, K., Stewart, C., Wheeler, T. T., and Schultz, G. S. (1994). Characterization of the synthesis and secretion of transforming growth factor- from salivary glands and saliva. Endocrinology 134:963–970. Hugoson, A. (1986). Results obtained from patients referred for the investigation of complaints related to oral galvanism. Swed. Dent. J. 10:15–28. Kjeilen, J., Brodin, P., Aars, H., and Berg, T. (1987). Parotid salivary flow in response to mechanical and gustatory stimulation in man. Acta Physiol. Scand. 131:169–175. Kock, K., Morley, S. D., Mullins, J. J., and Schmale, H. (1994). Denatonium bitter tasting among transgenic mice expressing rat von Ebner’s gland protein. Physiol. Behav. 56:1173–1177. Lebenthal, E., and Lee, P. C. (1984). Alternative pathways for digestion and adsorption in early infancy. J. Pediatr. Gastroenterol. Nutr. 3:1–3. Levine, M., Aguirre, A., Hatton, M., and Tabak, A. (1987). Artificial salivas: present and future. J. Dent. Res. 66: 693–698. Li, X.-J., and Snyder, S. H. (1995). Molecular cloning of Ebnerin, a von Ebner’s gland protein associated with taste buds. J. Biol. Chem. 270:17674–17679. Manning, R., and Edgar, W. (1992). Salivary stimulation by chewing gum and its role in the remineralization of caries-like lesions in human enamel in situ. J. Clin. Dent. 3:71–74. Marowitz, L. A., and Halpern, B. P. (1977). Gustatory neural response of the chorda tympani to lick-duration stimuli. Chem. Senses Flav. 2:457–485. Matsuo, R. (2000). Role of saliva in maintaining taste sensitivity. Crit. Rev. Oral Biol. Med. 11:216–229. Matsuo, R., and Yamamoto, T. (1990). Taste nerve responses during licking behavior in rats: Importance of saliva in responses to sweeteners. Neurosci. Lett. 108:121–126. Matsuo, R., and Yamamoto, T. (1992). Effects of inorganic constituents of saliva on taste responses of the rat chorda tympani nerve. Brain Res. 583:71–80. Matsuo, R., Yamamoto, T., Ikehara, A., and Nakamura, O. (1994). Effect of salivation on neural taste responses in freely moving rats: Analyses of salivary secretion and taste responses of the chorda tympani nerve. Brain Res. 649:136–146. McBurney, D. H., and Pfaffmann, C. (1963). Gustatory adaptation to saliva and sodium chloride. J. Exp. Psychol. 65:523–529. Mehansho, H., Clements, S., Sheares, B. T., Smith, C., and Carlson, D. M. (1985). Induction of proline-rich glycoprotein synthesis in mouse salivary glands by isoproterenol and by tannins. J. Biol. Chem. 260:4418–4423. Mistretta, C. (1971). Permeability of tongue epithelium and its relation to taste. Am. J. Physiol. 8:73–78. Mitchell, D. (1974) Physical basis of thermoregulation. In Environmental Physiology, D. Robertshaw (Ed.). Butterworth, London, pp. 1–32.

649 Morris-Wiman, J., Sego, R., Brinkley, L., and Dolce, C. (2000). The effects of sialoadenectomy and exogenous EGF on taste bud morphology and maintenance. Chem. Senses 25:9–19. Murray, R. D., Kerzner, B., Sloan, H. R., Juhling, M. H., Gilbert, M., and Ailabouni, A. (1986). The contribution of salivary amylase to glucose polymer hydrolysis in premature infants. Pediatr. Res. 20:186–191. Nakamura, K., and Norgren, R. (1991). Gustatory responses of neurons in the nucleus of the solitary tract of behaving rats. J. Neurophysiol. 66:1232–1248. Nishijo, H., and Norgren, R. (1991). Parabrachial gustatory neural activity during licking by rats. J. Neurophysiol. 66:974–984. Noguchi, S., Ohba, Y., and Oka, T. (1991). Effect of salivary epidermal growth factor on wound healing of tongue in mice. Am. J. Physiol. 260:E620–E625. Norris, M. B., Noble, A. C., and Pangborn, R. M. (1984). Human saliva and taste responses to acids varying in anions titratable acidity and pH. Physiol. Behav. 32:237–244. Olsson, H., and Axell, T. (1991). Objective and subjective efficacy of saliva substitutes containing mucin and carboxymethylcellulose. Scand. J. Dent. Res. 99:316–319. Piccolino, M. (1997) Luigi Galvani and animal electricity: Two centuries after the foundation of electrophysiology. Trends Neurosci. 20:443–448. Rehnberg, B. G., Hettinger, T. P., and Frank, M. E. (1992) Salivary ions and neural taste responses in the hamster. Chem. Senses 17:179–190. Rhodus, N., and Brown, J. (1990). The association of xerostomia and inadequate intake in older adults. J. Am. Diet. Assoc. 90:1688–1692. Rosenblum, J. L., Irwin, C. L., and Alpers, D. H. (1988). Starch and glucose oligosaccharides protect salivary-type amylase activity at acid pH. Am. J. Physiol. 254:G775–G780. Schmale, H., Holtgreve-Grez, H., and Christiansen, H. (1990). Possible role for salivary gland protein in taste reception indicated by homology to lipophilic-ligand carrier proteins. Nature 343:366–368. Schmale, H., Ahlers, C., Bläker, M., Koch, A., and Spielman, A. I. (1993). Perireceptor events in taste. In The Molecular Basis of Smell and Taste Disorders, D. Chadwick, J. Marsh, and J. Goode (Eds.). John Wiley, Chichester, pp. 167–185. Schneyer, L. H., Young, J. A., and Schneyer, C. A. (1972). Salivary secretion of electrolytes. Physiol. Rev. 52:720–777. Schreiver, W., and Diamond, L. (1952). Electromotive forces and electric currents caused by metallic dental fillings. J. Dent. Res. 31:205–229. Schultz, G., Rotatori, D. S., and Clark, W. (1991). EGF and TGF in wound healing and repair. J. Cell. Biochem. 45:346–352. Shannon, I. L., Suddick, R. P. and Dowd, F. J. (1974). Saliva: composition and secretion. In Monographs in Oral Science, Vol. 2. S. Karger, Basel, pp. 1–103. Shatzman, A. R., and Henkin, R. I. (1981). Gustin concentration changes relative to salivary zinc and taste in humans. Proc. Natl. Acad. Sci. USA 78:3867–3871. Spielman, A. I. (1990) Interaction of saliva and taste. J. Dent. Res. 69:838–843.

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Bradley and Beidler Van der Reijden, W. A., Vissink, A., Veerman, E. C. I., and Nieuw Amerongen, A. V. (1999). Treatment of oral dryness and related complaints (xerostomia) in Sjögren’s syndrome. Ann. Rheum. Dis. 58:465–473. Van’t Hof, W., Blankenvoorde, M. F. J., Veerman, E. C. I., and Nieuw Amerongen, A. V. (1997). The salivary lipocalin Von Ebner’s gland protein is a cysteine proteinase inhibitor. J. Biol. Chem. 272:1837–1841. Weiffenbach, J. M., Fox, P. C., and Baum, B. J. (1986). Taste and salivary function. Proc. Natl. Acad. Sci. USA 83:6103–6106. Weiffenbach, J. M., Schwartz, L. K., Atkinson, J. C., and Fox, P. C. (1995). Taste performance in Sjögren’s syndrome. Physiol. Behav. 57:89–96. Zopi, G., Andreotti, G., Pajno-Ferrara, F., Njai, D. M., and Gaburro, D. (1972). Exocrine pancreatic function in premature and full term neonates. Pediatr. Res. 6:880–889.

32 Morphology of the Peripheral Taste System Martin Witt University of Technology Dresden, Dresden, Germany

Klaus Reutter University of Tübingen, Tübingen, Germany

Inglis J. Miller, Jr. Wake Forest University School of Medicine, Winston-Salem, North Carolina, U.S.A.

I.

perception. One aim of this chapter is to provide sufficient knowledge of peripheral gustatory anatomy as a basis for understanding other chapters of this book. Some structural details about the human peripheral taste system are well known, but it is also worthwhile to provide comparative anatomical information to fill in the gaps or understand and establish basic principles. The fundamental question of how tastants are perceived has been addressed for more than two millennia, and the concepts, theories, and experimental “proofs” proposed have been discarded in the past that our present-day concepts can be incorporated. Therefore, this chapter also includes important historical paradigms in taste research which may serve as caveats for sculpting the discipline as contemporary concepts emerge. Aristotle (384–322 B.C.), applying Platonic concepts, argued that taste sensation was carried from the tongue via the blood to the liver or heart, which was the common seat of the soul and all sense perception (Siegel, 1970). Galen’s (Claudius Galenus, 129–201 A.D.) anatomical analyses challenged this notion: his detailed studies on the innervation of the tongue describe correctly the different functions of the three principal nerves supplying the tongue (lingual, glossopharyngeal, and hypoglossal nerves) and demonstrate their origin at the base of the brain. Galen posited that the lingual nerve communicated gustatory sensations, a concept yet resonant in contemporary

INTRODUCTION Many papillae are evident, I might say, innumerable, and the appearance is so elegant that they catch the view and thoughts of the observer, and control him for a long time and not without enjoyment....All this delights the curious mind, when observing with an engyscope; and if anyone asks to what they are similar, I am unsure whether I should compare this huge number of papillae first with grapes or fruits of the bay, or innumerable mushrooms emerging between fine, densely standing blades of grass...

These are the first detailed and enthusiastic words on papillae and their “membranes” in human tongues by Lorenzo Bellini (1665) (Fig. 1), who knew that Marcello Malpighi (1628–1694) had reported on lingual papillae the year before. Malpighi discovered mucosal elevations associated with nerve fibers, naming these elevations “papillae,” as the morphological substrates for gustatory sensation (Fig. 2). The peripheral taste apparatus includes the gustatory sensory organs or taste buds and their innervation, and the specific papillae within which taste buds are assembled. What one needs to know about taste buds in relation to how one perceives tastants depends on the approach to taste 651

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Figure 1 Cover sheet of Lorenzo Bellini’s book summarizing examination of the anatomy of the kidney and taste organs. (Langerack, Leiden, 1711.)

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neurobiology. One strand of nerve fibers corresponding to cranial nerve IX (CN IX) (rediscovered in humans by Panizza in 1834) was already known to Galen as the principal gustatory nerve of the tongue. CN IX also carries some motor fibers to the pharynx. Galen also noted excretory ducts of the lingual and submandibular glands (in ox). The particular structure of the tongue surface was described initially by Casserius (1609) and later by Malpighi (1664; cited: Malpighi, 1687) and Bellini (1665) (reviewed by Jurisch, 1922). Further evidence of the significance of the papillary epithelium and its cells came from observations in taste organs of the frog (Waller, 1847, 1849; Fixsen, 1857). Taste buds were identified initially on the barbels and skin of fishes by Leydig (1851) and described as becherförmige Organe (goblet-shaped organs), whose function he associated with tactile sensitivity. Schulze (1863) subsequently suggested they were chemosensory structures. Similar organs in mammals were described as Schmeckbecher (taste goblets) (Lovén, 1868) and Geschmacksknospen (taste buds) or Geschmackszwiebeln (taste onions) (Schwalbe, 1868). Herrick (1904) translated Geschmacksknospen as “taste buds.” Their location within lingual papillae, the latter already associated with the loci of taste perception, lent credence to their identity as taste sensor organs. Nineteenth-century studies focused on cytological features, nerve supply (Figs. 3, 4), and the development of taste buds. While the nerve-dependent nature of taste sensation was known since Galen’s time, its significance for sensory organ physiology blossomed in the mid-1800s. With development of the neuron doctrine (see Koelliker, 1844), previously described “ganglion globules” (Ganglienkugeln) (Ehrenberg, 1833) could now be acknowledged as part of a specialized cellular system (cell theory of Schleiden and Schwann) (Schwann, 1839). Two major prerequisites favored the expansion of scientific

Figure 2 Depiction of a bovine tongue by Marcello Malpighi (1686) showing “patches” where papillae were observed. Note the concentration of fungiform papillae (dots) on the tip of the tongue. The drawing of vallate and foliate papillae is still rather vague.

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Figure 3 These illustrations from Bourgery and Jacob (1839) show the human tongue including nerve and blood supply, as well as the lingual muscle system. In spite of precise macroscopical observations on the innervation of glands and muscles, it still represents indirectly the (nowadays revised) morphological concept of gustation in the early nineteenth century, according to which the principal taste nerves are the lingual (p in A and B) and glossopharyngeal (t in B) nerves. The chorda tympani, although depicted near the submandibular ganglion (q in A), does not reach the lingual dorsum. Ebner’s glands are not known to the authors. (A) D, Submandibular gland with q, chorda tympani and submandibular ganglion and p, lingual nerve; s, hypoglossal nerve; E, sublingual gland; F, Nuhn’s gland. (B) C, Styloid process; b, stylopharyngeus muscle, the leading muscle for t, glossopharyngeal nerve; k, lingual artery; p, lingual nerve, interrupted to show the glossopharyngeal nerve; q, part of the chorda tympani nerve; s, hypoglossal nerve; X, trunk of the facial nerve. (See color insert.)

knowledge in the nineteenth century: (1) replacement of the often speculative natural philosophy by the experimentally based natural sciences, mainly represented by Francois Magendie (1783–1855) in France and Johannes

Müller (1801–1858) in Germany, and (2) technical advances, which included improved microscopes (K. Zeiss, 1816–1888, together with Ernst Abbé, 1840–1905; and E. Leitz, 1843–1920), as well as use of histological

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per contiguitatem.” This first observation of secondary sensory cells in a taste organ that was clearly different from primary sensory cells, as had already been described in the olfactory epithelium, albeit without the understanding of the “contact” nature of taste bud cells and their innervating afferents as later described ultrastructurally, was opposed by Retzius (1892). Using the Golgi silver impregnation method, Retzius attributed a sensory function only to free nerve endings. Later, Krause (1911) clearly demonstrated that the finest nerve fibers enter the taste bud and ascend to the taste pore but do not merge with taste bud cells whose basal processes end near the basal cells.

Figure 4 The first precise depiction of a human tongue by Samuel Soemmering (1806). (Above) The anterior part (left side) of the tongue shows numerous fungiform papillae. Behind the V-like arrangement of vallate papillae (right side), the lingual tonsils and the entrance to larynx with the epiglottis are visible. (Below) Lateral view of the tongue shows the left lingual artery and its ramifications into gustatory papillae, which appear as red dots after injection of the artery with a red dye (observation of Soemmering).

techniques, e.g., introduction of chromic acid for histological examination of neural tissue (Hannover, 1840), allowing for the distinction between cells and fibers. Further, gold chloride staining facilitated discriminating finely ramifying nerve fibers (Gerlach, 1858). Helmholtz (1842) observed a direct continuity between nerve cells (globuli gangliosi) and their fibers in evertebrates. The demand for the neuron doctrine in vertebrates was established by Koelliker (1844). Introduction of the methylene blue staining method (Ehrlich, 1886) facilitated observations that fine nerve fibers in the frog taste disc widened with varicosities, penetrated the gustatory epithelium and approached the sensory cells “with extremely sharp small knobs, [which]...connect taste cells not continuously, but

II.

LINGUAL PAPILLAE AND TASTE BUD DISTRIBUTION

A.

Nongustatory Papillae

Approximately 60 years before taste buds were identified as gustatory organs, an illustration of the human tongue by Soemmering (1806) (Fig. 4) accurately showed the regional distribution of lingual papillae. A line called the sulcus terminalis (an ontogenetic remnant, see below), which is located posterior to the vallate papillae, separates the body of the tongue from the lingual root. It may be seen that the sulcus terminalis extends laterally to the pharyngeal wall from the foramen caecum (also an ontogenetic remnant, see below) near the midline (see Figs. 4, 12). The root of the tongue is covered by a papilla-free smooth epithelium, and beneath this epithelium lie mucous glands and a reticular connective tissue filled with lymphatic follicles, which led to the designation of “lingual tonsil.” Ducts of intralingual salivary glands (Ebner, 1873) empty into the troughs of vallate and foliate papillae (see below). The dorsal surface of the tongue is covered with filiform and conical papillae from the sulcus terminalis to the tongue tip. Filiform papillae are the most prevalent type, while the number of conical papillae may vary. Both types of papillae are sparse along the lingual margin and abundant in the middle regions. Conical papillae have a cylindrical base and taper to a sharp point at their apex. Filiform papillae (L. filum
thread) have a pyramidal shape and a narrow tail of cornified cells extending from their apical tips as a pennant. The fila are part of the fibrous mat on the tongue’s surface in the hypertrophic condition called “hairy tongue.” B.

Gustatory Papillae

Taste buds occur in distinct papillae of the tongue, the epithelium of the palate, oropharynx, larynx (epiglottis),

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and the upper esophagus. Taste buds of most vertebrates are bulb-shaped structures composed of about 50–120 bipolar cells (see Figs. 5, 9, 11). With the exception of basal cells, the slender taste bud cells arise from an interrupted basal membrane and converge with their apical protrusions, the microvilli, into the mucus-filled taste pit. Together, these cells form the organ’s sensory epithelium. The nuclei of the cells are located in the lower third of the taste bud, which is approximately the region where most afferent nerve fiber terminals are distributed. Sensory cells possess transmembrane receptors and/or ion channels for specific taste stimuli at apical and lateral portions of the cell membrane. Taste buds are demarcated from surrounding nongustatory epithelial cells by specialized epithelial cells (marginal cells). 1.

Distribution of Lingual Taste Buds

The pattern of taste bud distribution over the tongue surface is similar among humans and other mammals. Lingual taste buds are found exclusively within gustatory papillae, i.e., those bearing taste buds. Similar types of gustatory papillae are located on homologous regions of adult mammalian tongues. The gustatory papillae include the vallate, foliate, and fungiform papillae. As the term suggests, typical fungiform papillae are mushroom-shaped, with a slender neck and an enlarged head (Figs. 6–8). But the majority of fungiform papillae vary in form, and the filiform papillae are intermingled among them. Shortly after the published discovery of taste buds in humans (Schwalbe, 1868; Lovén, 1868), the first systematic investigations on the distribution of taste buds within the oral cavity in humans were carried out by the medical student, Hoffmann (1875). He emphasized that taste buds are more

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sparse within foliate papillae and the soft palate, including the uvula. Hoffmann concluded that the development of taste perception is dependent on the number of taste buds on a particular location. Of the approximately 4600 total taste buds in all three lingual fields in humans, vallate buds comprise about 48% (2200), foliates about 28% (1280), and fungiforms 24% (1120). However, taste bud numbers vary greatly among individuals (Miller and Reedy, 1990a), with some adults possessing a total of only 500 taste buds (Linden, 1993). The taste bud density of foliate papillae seems to be constant in life (Hou-Jensen, 1933), but age-related differences have been reported for vallate papillae, which are more numerous and containing more taste buds in younger individuals (Jurisch, 1922). There are also more marginal fungiform papillae during the late fetal and newborn period, but they usually lack taste buds and are referred to as “sucking papillae” (Habermehl, 1952; Yamasaki and Takahashi, 1982). 2.

Vallate papillae, comprehensively described by Haller (1766) and Soemmering (1806), lie directly anterior to the sulcus terminalis and extend in a V-shaped line across the root of the tongue (Fig. 4). They are round and measure between 2 and 8 mm in diameter. The pores of taste buds open into the trenches around the bases of each vallate papilla (Fig. 6). The papillae are innervated by an enormously large nerve fiber plexus originating from the glossopharyngeal nerve [cranial nerve, IX (CN IX) see below] compared to foliate or fungiform papillae. The number of vallate papillae per human tongue varies between 4 and 18 (n
2264 tongues), with an average of 9.2  1.8 papillae (Munch, 1896). Ninety-eight percent of all tongues have a central median papilla (Fig. 4). The presence of three or four lateral papillae on each tongue half was observed most often (20%). Atrophic changes were observed in papillae of some men 40 years old and some women 55–60 years old, though Jurisch (1922) reported that the number of vallate papillae did not appear to change systematically as a function of age. The average numbers of taste buds per papilla are summarized in Table 1. 3.

Figure 5 Two taste buds of a rabbit foliate papilla. Some cells, as well as some nerve fibers, are reactive for NSE.

Vallate Papillae

Foliate Papillae

Foliate papillae in humans were first reported by Albinus (1754) and histologically described by Hönigschmied (1873) but did not become the focus of scientific attention in humans until the twentieth century. These papillae, located bilaterally along the posterolateral margins of the tongue surface, consist of parallel rows of ridges (folia) and valleys, which lie adjacent to the lower molar teeth.

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Figure 6 Schematic drawings of taste bud–bearing mammalian lingual papillae (longitudinal sections). (A) Fungiform papilla (papilla fungiformis) with two apically situated taste buds and their innervation. On the right side, a (taste bud–free) filiform papilla. (B) Foliate papillae (pp. foliatae). (C) Vallate papilla (p. vallata). In B and C, the taste buds are directed to the lateral trenches of the papillae. Serous Ebner glands drain to the trenches. Dermal connective tissue is rich in nerve fibers; below the taste buds they form subgemmal plexus.

Ducts located between the folia transmit secretions from mostly serous lingual glands within the root of the tongue. Scanning electron microscopy of human foliate papillae and transmission electron microscopy of their resident taste buds was reported by Svejda and Janota (1974) and Azzali et al. (1996). The number of taste buds in human foliate papillae was reported by Hou-Jensen (1933) and Mochizuki (1939) (see Table 1). Confusion in finding taste buds within the folds of human foliate papillae reflects papilla structure. As many as 20 parallel ridges and furrows are found on the posterolateral margin of the human

tongue. The rostralmost furrows (lateral rugae) contain no glandular ducts or taste buds, and their epithelia are more cornified than that between foliate papillae (Hou-Jensen, 1933). Fungiform papillae can be found on the tops of these lateral rugae. Although human foliate papillae were thought to be “rudiments” (in comparison to the well developed rabbit foliate papillae), Mochizuki (1939) calculated an average of 1300 taste buds per tongue, which exceeds the number of fungiform taste buds [800 buds: Braus (1940); 1000 buds: Miller and Bartoshuk (1991)]. Indeed, contiguous taste buds within the same cleft may

Table 1 Distribution of Taste Bud Numbers in or on Human Oral or Lingual Structures Vallate papillae – 400 252  151a 234  114a 240  125a

Fungiform papillae 20–30 (fetal)

1000 (total) 800 (total)

Foliate papillae ?

Palate

Larynx

15–20 per papilla.

1279 – 708–1328  2500 (neonate)

585 1120 a

per papilla.

 25 (senile)

Ref. Hoffmann, 1875 Wyss, 1870 Arey et al., 1935 Mochizuki, 1939 Miller and Bartoshuk, 1991 Braus, 1940 Hou-Jensen, 1933 Lalonde and Eglitis, 1961 Jowett and Shrestha, 1998 Cheng and Robinson, 1991 Miller and Reedy, 1990b

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Figure 8 Human fungiform papilla during development, week 15. One taste pore is visible (arrow). Scale bar
50 m.

Figure 7 Fungiform papillae during development (12th postovulatory week). (Above) Anterior part of the tongue. Paramedian sagittal section. (Below) Detail of the anterior part at the tongue’s tip. Only a few papillae contain taste pores. Filiform papillae are not developed yet. M, mandible. Scale bar
1 mm (above), 250 m (below).

form a more functional unit, since they share access to a common taste stimulus pool. Foliate papillae are innervated by branches of the glossopharyngeal nerve (CN IX), but the more anterior portion also receives nerve fibers from the chorda tympani (Oakley, 1970; Pritchard, 1991). 4. Fungiform Papillae Due to their morphological heterogeneity, fungiform papillae have been variously described as papillae clavatae, capitatae, lenticulares, obtusae, majores, mediae (Haller, 1766; Soemmering, 1806). These papillae can be easily identified as pink elevations about 0.5 mm in diameter on the anterior portion of the living human tongue. Notwithstanding its convenient location, the fungiform taste bud population has been difficult to quantify since fungiform papillae vary in appearance and are distributed

over a large area of tongue surface. The anterior portion of the tongue extends from the line of vallate papillae to the tongue tip (Figs. 4, 7, 8). This region contains about 30 cm2 of surface area, depending on the size of the person, and the fungiform papillae are spread unevenly over it. The number of taste buds differs among fungiform papillae, and there are large variations among human subjects in fungiform taste bud distribution. Most papillae on the 5 mm margin of the tongue tip are shorter than those on more posterior regions. Following the surface in a posterior direction from the midline of the tip toward the back of the tongue, fungiform papillae become progressively larger in size. They also are larger in more posterior lingual regions. Small, rounded papillae are present on the margin. Some of them contain taste buds, while others are comparable in size to filiform papillae but lack fila. Some papillae on the margin of the tip are elongated like conical papillae, and these, generally, lack taste buds. Fungiform papillae vary in size and shape: some are short and cuboidal, and others are tall with expanded heads like mushrooms. Among papillae on the margin of shorter height and smaller diameter ( 0.5 mm), the distinction between filiform and fungiform papillae becomes obscure. Fungiform papillae occasionally have projections on their apices like small fila on filiform papillae.

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Taste buds have been quantified in terms of tongue surface areas, referred to as “taste bud density,” or number of taste buds per cm2 of tongue surface. There are about 145 gustatory (fungiform) papillae per lateral half of the tongue, with about 30 papillae per cm2 on the tip, but only about 3 papillae per cm2 on the posterolateral area. There are about 30 large fungiform papillae in the posteromedial region, for an estimated total of about 320 fungiform papillae per tongue. On 320 fungiform (gustatory) papillae, we estimate an average of about 3.5 taste buds per papilla, for a total of 1120 fungiform taste buds (Miller and Reedy, 1990a, 1990b) (Table 1). Most investigators who study human fungiform papillae report the existence of papillae without taste buds, which rarely occurs in other mammals (Mistretta and Baum, 1984). Studies in humans show that fungiform papillae lacking taste pores comprise 12.8–67% of the fungiform population per subject (Arvidson and Friberg, 1980; Miller and Reedy, 1990a, b; Cheng and Robinson, 1991). This wide range may reflect how investigators decide which papillae are “fungiform.”

III.

EXTRALINGUAL TASTE BUDS

There are “extralingual” taste buds in regions of the oral, pharyngeal, and laryngeal cavities. Interestingly, Magendie (1820) and Carus (1849) erroneously associated the teeth with taste perception. Verson (1868) described the first “goblet-like organs” within the dorsal epithelium of the epiglottis, and Davis (1877) and Wilson (1980) observed taste buds in and the elicitation of taste perception from the human larynx. Lalonde and Eglitis (1961) counted more than 2500 taste buds on the epiglottis, soft palate, laryngeal pharynx, and oral pharynx of one human neonate. Taste buds are evident in the epiglottis of one neonatal specimen (Rabl, 1895) and esophagus in human fetuses (Ponzo, 1907) and adults (Schinkele, 1942; Burkl, 1954). Taste buds are also found near the openings of sublingual salivary gland ducts in some other primates (Hofer et al., 1979) and near the ducts of the molar glands in rodents (Iida et al., 1983). In chickens and quails, taste buds in nonlingual parts of the oral cavity are almost always associated with salivary gland ducts (Ganchrow and Ganchrow, 1987). Miller and Smith (1984) estimate that about 25% of the hamster’s total taste buds are extralingual, and Mistretta and Baum (1984) accounted for a similar proportion of extralingual taste buds in the rat. It is not known whether extralingual taste buds are functionally different from those on the tongue. Taste buds of the epiglottis and/or uvula could be involved in initiation of upper airway reflexes (Bradley et al., 1983) and in the

pharyngolaryngeal water response, possibly mediated by receptors signaling the absence of chloride ions (reviewed by Lindemann, 1996). Similarly, taste buds of the larynx seem not to play a role in gustation but detect chemicals that are not saline-like in composition (Bradley, 2000). IV.

SALIVARY GLANDS OF THE TONGUE

“Lingua sicca non gustat (A dry tongue does not taste)”: This declaration of Haller (1766) refers to the dependence of taste ability on solutions, within which tastants are dissolved and transported to the taste bud. Extralingual saliva is secreted by small, mostly mucous glands embedded in the epithelium of the cheek and palate. More saliva is produced by the serous parotid gland and the muco-serous sublingual and submandibular glands (Fig. 3), whose secretory ducts open at the tongue frenulum just underneath its tip. Intralingual saliva originates from the mucoserous anterior lingual glands (glands of Blandin and Nuhn) (Tandler et al., 1994) and deep posterior serous salivary glands (Ebner) located in the submucous connective tissue below the foliate and vallate papillae of the tongue (Ebner, 1873; Riva et al., 1999). Their excretory ducts lead to the deepest sites of the papillar furrows (Fig. 6). The gland lobules lie deeply in large patches of connective tissue which, in turn, are separated from each other by muscle bundles. In addition, adjacent to Ebner glands in vallate papillae lie mucous (Weber’s) glands (Nagato et al., 1997), which in humans open into the crypts of the lingual tonsils (Zimmermann, 1927). Neither Weber’s nor Blandin-Nuhn glands lie in close proximity to taste buds, and their particular significance for taste perception is unknown because of the difficulties in collecting saliva from these glands (Tandler et al., 1994). There is biochemical and histochemical evidence that the saliva of Ebner’s glands, as well as that of other nonlingual salivary glands, has more functions than that of a serous “washing solution.” Binding proteins such as Ebnerin (Li and Snyder, 1995) are supposed to modulate sensations. Schmale et al. (1990) isolated a protein from rat Ebner’s glands that is structurally similar to odorant binding proteins in Bowman’s glands of the olfactory mucosa. The gland is under autonomic control (Gurkan and Bradley, 1987). For reports on specific ligand-receptor interaction with taste qualities, see Azen et al., 1990; Spielman, 1990; Schmale et al., 1993; Toto et al., 1993. V.

BLOOD SUPPLY TO GUSTATORY PAPILLAE

The mammalian tongue receives its blood supply from the lingual artery, which is usually a branch of the external

Morphology of the Peripheral Taste System

carotid artery (Fig. 3). Study of the tongue’s vascular system historically parallels that of the lingual papillae. For example, Albinus (1754), Soemmering (1806), and Arnold (1839) performed intravascular injections in order to visualize the papillary surface. More recently, distribution of the blood supply to different regions of the tongue and different types of lingual papillae has been described by Hellekant (1976). Each type of gustatory papilla is supplied by a characteristic capillary configuration (rat: Ohshima et al., 1990; cat, rabbit: Ojima et al., 1997a,b,c), and fine capillary networks are found adjacent to taste buds. The capillary loops of larger papillae in rats and dogs often show a constriction, maybe sphincter-like structures, but rarely arteriovenous anastomoses (Selliseth and Selvig, 1993; Hu et al., 1996). Taste stimuli injected systemically elicit responses in gustatory nerves as the bolus passes through the tongue (Bradley, 1973).

VI.

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buds, involved in finding food; in rocklings (Gaidropsarus) they are assumed to be important for predator avoidance (Kotrschal, 1996). Since the arginine-like receptor in catfish taste buds also occurs in solitary chemosensory cells, Finger (1997) suggests that taste buds might include solitary chemosensory cells within them. During development of fish, solitary chemosensory cells seem to precede the development of taste buds. In mammals, however, solitary chemosensory cell–like cells have been observed only transiently, during development. In newborn rats, single gustducin-immunopositive cells are seen in locations where later-developing vallate papillae will appear (Sbarbati et al., 1999). Individual slender cells, immunopositive for cytokeratin 20 (Witt et al., 1999), an intermediate filament protein that is exclusively present in taste bud and epidermal Merkel cells (Moll, 1993; Zhang and Oakley 1996), are seen occasionally during early ontogenesis of the human tongue. However, gustatory epithelia of adult mammals have not yet been reported to possess solitary chemosensory cells.

SOLITARY CHEMOSENSORY CELLS

In addition to taste buds and free nerve endings, the solitary chemosensory cells comprise another chemosensory system in vertebrates. They are not assembled in clusters, but are dispersed across the surface of the animal. Solitary chemosensory cells are related to taste bud cells in the sense that the former are secondary sensory cells with a slender, bipolar phenotype (Finger, 1997). “Classical” solitary chemosensory cells have been studied mostly in teleosts (Whitear, 1992). The evolutionary benefits of these cells are still in question: in sea robins (Trigon), they are, beside taste

VII. CELL TYPES OF VERTEBRATE TASTE BUDS Peripheral taste organs differ in number, size, and shape in different vertebrate taxa, according to their importance for the particular species. For the sake of brevity, the following overview is restricted to some functionally wellcharacterized vertebrate species. Details are available in the review by Reutter and Witt (1993). Figure 9 shows a scheme representing the organization of cells in fish, frog, and mammalian taste buds.

Figure 9 Longitudinal sections of taste organs from representatives of three different vertebrate classes. (A) Fish (bullhead, Teleostei); (B) amphibian (frog, Anura); (C) mammal. In this schematic drawing, each sensory epithelial cell type is represented once with a distinct grey step. The organs lie in squamous epithelium of different height, on top of dermal papillae, which are also of different height. Each dermal papilla contains nerve fibers and a capillary vessel.

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Taste Buds of Lower Vertebrates — Cell Types

1. Fish In fish, and especially in some teleosts that are well-adapted to the dark, the taste organ is significantly more important for food intake than in amphibians and mammals. Thus, these fish, like the Siluridae, possess many more taste buds than representative of the latter classes (Atema, 1971; Miller and Bartoshuk, 1991; Finger et al., 1996). The fish taste bud is generally pear-shaped and similar to that of mammals (Fig. 9). Electron microscopic studies of teleost taste buds (for reviews see Reutter, 1986; Jakubowski and Whitear, 1990; Reutter and Witt, 1993; Boudriot and Reutter, 2001) have attempted, somewhat incompletely, to relate the ultrastructure and functions of taste bud cell types. This is also apparent in the nonuniform usage of nomenclatures. Most authors refer to elongated “light” and “dark” taste bud cells, as well as “intermediate” and “degenerative” cells (Desgranges, 1965; Welsh and Storch, 1969; Reutter, 1971, 1978; Connes et al., 1988). Generally, light cells are supposed to be “sensory” (receptor) cells, while the dark cells are regarded as “supporting” cells (e.g., Desgranges, 1965; Hirata, 1966; Whitear, 1970). However, Reutter (1992) considers the dark cells also as sensory because they exhibit synaptic contacts with nerve profiles or with basal cells. These observations, however, are not supported by the work of Grover Johnson and Farbman (1976) and Jakubowski and Whitear (1986). According to these authors the differentiation “light” and “dark” in combination with functional terms such as “supporting cells” and “sensory (receptor) cells” are misleading and should be avoided. According to Merkel (1880), Hirata (1966), and Reutter (1973), the taste bud may also have mechanoreceptive functions, particularly in view of the morphology of basal cells (Reutter, 1971, 1986). While the existence of synaptic contacts of light and dark taste bud cells has not yet been finally proven, the different lengths of their microvilli perhaps suggest functional differences: The light taste bud cells have a single long microvillus reaching far into the mucous layer of the taste bud surface, the receptor area. By penetrating this layer, longer microvilli may be exposed to quite different “perireceptor events” (Getchell et al., 1984) than the small microvilli of dark taste bud cells that do not penetrate the mucous layer (Witt and Reutter, 1990). Ultrastructural investigations in nonteleostean fish reveal clearly that taste buds differ within the main vertebrate taxa (Reutter and Witt, 1993). Further, in different systematic groups of fish, the taste buds do not follow only one structural design. Thus, taxon-specific taste buds or cell types do not exist. Similar differences among mammalian taste buds point to a similar, inevitable conclusion

regarding particular phenotypes: There are only speciesspecific taste bud types, and a general “model” seems difficult to find among vertebrates. This underlines the thesis that morphological phenotypes and the structural organization of taste bud cells do not necessarily reveal a general blueprint but, rather, reflect specific environmental conditions and/or feeding behaviors (Reutter and Witt, 1999). The influence of environmental differences has been studied in two closely related teleosts, one of which is sighted (Astyanax mexicanus), and the other of which is a blind cave fish (Astyanax jordani). Whereas taste bud morphology is rather similar, the cave fish compensates for blindness by significantly more gustatory axon profiles (Boudriot and Reutter, 2001) and an expanded expression of Prox 1 gene in developing taste buds (Jeffery et al., 2000). 2.

Amphibians

The taste organs of postmetamorphotic Salientia (
Anura), unlike piscine and mammalian taste bud bulblike formations, are relatively large disk-like epithelial differentiations of the dorsal lingual and palatal mucosa (Fig. 9). Waller (1847, 1849) and Engelmann (1872) called these structures Geschmacksscheibe or “taste discs.” In contrast to Salientia, the taste buds of the urodeles [
Caudata, e.g., mudpuppy (Necturus), newt (Triturus), or Axolotl (Ambystoma)] have a bulb-like shape (Fährmann, 1967; Farbman and Yonkers, 1971; Cummings et al., 1987; Toyoshima et al., 1987; Toyoshima and Shimamura, 1987; Delay and Roper, 1988). The cellular elements of the taste disc, or Endscheibe (Merkel, 1880), of the frog were subject to numerous investigations and received various designations: After Waller (1847, 1849) had first distinguished between papillae conicae (
P. filiformes) and papillae fungiformes, Fixsen (1857) described two different cell types, the so-called “cellulae cylindricae” and “cellulae fusiformes,” the processes of which pass through the whole sensory epithelium to reach the connective tissue core of the papilla. Engelmann (1872) and Merkel (1880) further developed the terminology: Merkel distinguished between “cylindrical cells” (Cylinderzellen) situated on the epithelial surface and surrounding “wing cells” (Flügelzellen), the nuclei of which lie deeper in the epithelium. After Graziadei and DeHan (1971) had described only two cell types (“associate cells” and “sensory cells”) in electron microscopy, the close relationship between “rod cells” (Stäbchenzellen) and cylindrical cells (Merkel, 1880) was recently reintroduced by von Düring and Andres (1976). In addition, the latter authors first described the basal cells and Merkel cells of the frog taste disc, which are the only cells that do not contact the epithelial surface.

Morphology of the Peripheral Taste System

Tadpoles possess so-called premetamorphic papillae, which bear bud-like taste organs at their tops. During metamorphosis, these structures wholly disappear and are replaced by fungiform papillae with large taste disks (Nomura et al., 1979; Zuwala and Jakubowski, 1991; Zuwata, 1997). Tadpole taste discs consist of sensory and supporting cells, and basal cells are lacking. The taste disc in adult frogs contains up to 8 cell types (Reutter and Witt, 1993): mucus cells, wing cells, two types of sensory cells (cylindrical and rod-like type), two types of basal cells [stem and Merkel cell–like basal cells (Zancanaro et al., 1995)], and marginal cells and ciliated cells (Toyoshima et al., 1999) (Fig. 9). The cell types and the history of the nomenclature have been described in detail (Jaeger, 1976; Reutter and Witt, 1993; Witt, 1993; Osculati and Sbarbati, 1995). Taste buds of the mudpuppy (Necturus, Urodela) are similar to those of fishes. They are composed of dark and light cells and possess serotonergic Merkel cell–like basal cells, which are synaptically connected with either nerve fibers or dark and light cells (Delay and Roper, 1988; Delay et al., 1993, 1994). 3.

Reptiles and Birds

Lingual taste buds in reptiles have been described in turtles (Korte, 1980; Iwasaki et al., 1996), tortoises (Pevzner and Tikhonova, 1979), and some lizards (Uchida, 1980). Lizards (Gekkonidae or Anguidae), as well as snakes, have virtually no taste buds on the tongue, but rather on the buccal floor and oral epithelia of the mandible and maxilla (Toubeau et al., 1994). In shape, reptilian taste buds resemble those of mammals. There are up to five different types of taste bud cells, classified into light and dark cells and as types 1, 2, 3, A, B or as types I, II, II and basal cells (Reutter and Witt, 1993). Reptiles and birds belong to the same superclass, and one might expect a similar organization of avian taste buds. However, the few examples of bird taste buds show great variability among species. Buds in grain-eating birds appear in the posterior part of the tongue, near the pharynx, and in the distal palatal mucosa (Saito, 1966; Ganchrow and Ganchrow, 1987; Ganchrow et al., 1991; Sprissler, 1994). Unlike mammals, avian taste buds do not reside within lingual bud-bearing papillae. In addition, taste buds contain tubulus-like channels circumscribed by elongated cells grouped in a rosette configuration, with the channel lumen continuous apically with the taste pore (Berkhoudt, 1985; Ganchrow et al., 1993). Taste buds are richly innervated, and synapses are seen between all cell types (light cells and dark cells) and nerve fibers (Reutter and Witt, 1993; Sprissler and Reutter, 1993; Sprissler, 1994).

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B.

Mammalian Taste Buds — Cell Types

Early histological investigators of mammalian taste buds described two types of elongated, fusiform cells in taste buds of human vallate papillae (Schwalbe, 1868): “supporting” and “taste cells,” the latter divided into Stiftchenzellen (pin cells) and Stabzellen (rod cells) with differences in contrast and brightness (Fig. 10). Classification into “light” and “dark” taste bud cells was also used in early electron microscopic analyses (Engström and Rytzner, 1956). Farbman (1965) considered dark, fusiform taste bud cells of human fungiform papillae as sensory cells (type I), whereas other investigators (Paran et al., 1975) described a type II cell that contains numerous vacuoles and mitochondria, especially in apical areas. This latter cell is not believed to be sensory. Cottler Fox et al. (1987) speculated that the different electron densities are due to irreproducible fixation artifacts. In general, ultrastructural and immunohistochemical criteria are considered more important for the classification of cells than the evaluation of the electron density of the cytoplasm. Moreover, present understanding in mammalian taste bud cytology leads to using a rather heterogeneous nomenclature, based on morphological and functional differences across species. The basis for the current nomenclature was established by the Murrays and collegues (Murray and Murray, 1967, 1971; Murray et al., 1969; Murray, 1986) in taste bud cells of the rabbit foliate papillae. However, these cell types are different in some respect from recent classifications obtained in rodent taste buds (Kinnamon et al., 1985, 1993, 1994; Pumplin et al., 1997). Royer and Kinnamon (1988) observed considerable deviations in the cytoarchitecture of murine foliate taste buds compared to that of other mammals. For example, they did not find type III cells, and all bud cells had synaptic connections with nerve fibers. There are only a few ultrastructural studies of human taste buds. Paran et al. (1975) and Azzali (1997) concluded that human taste bud cells are essentially similar in rabbit and dog (Kanazawa, 1993). An example for a more generalized taste bud is depicted in Figure 11. The classification in this chapter follows the description of rabbit taste buds (Murray, 1986; Royer and Kinnamon, 1991; Reutter and Witt, 1993). 1.

Type I Cells

These cells are the most frequent. They are spindle-shaped and have a basal process that envelops the axons in a Schwann cell–like manner. Type I cells protrude with brush-like, long microvilli (1–2 m) into the taste pit. These cells ensheath type II and III cells with cellular protrusions and may insulate them. Apically, they contain

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Figure 10 Schwalbe’s (1868) taste bud description (from vallate papilla of the swine). Minor bundles and fibrils of nerves are lost in the “interior of taste goblets.” Note the incorrect depiction of basal cells (left side). Isolated cells of the human taste goblets, one of which (4) shows a large microvillus. 1
Supporting cell (Deckzelle, mostly at the margin); 2, 4
pin cell (Stiftchenzelle); 3
rod cell (Stabzelle). According to Schwalbe, both cell types might mediate different taste sensations.

large granules, 100–400 nm in diameter (Murray, 1986). The nuclei of type I cells are irregularly shaped. According to Murray and Murray (1971) and Murray (1986), these cells have a secretory (supporting cells) and possibly phagocytotic function and probably produce the amorphous material of the taste pit (e.g., Farbman, 1965; Menco, 1989; Ohmura et al., 1989; Witt, 1996). 2. Type II Cells Most of these cells are located in the periphery of the taste bud. They are fusiform, but do not possess enveloping processes, or granules. Their cytoplasm is moderately electron-dense, and nuclei are round to oval. Synapses are not observed. Toyoshima and Tandler (1987) describe a modified endoplasmic reticulum with specialized subsurface cisterns adjacent nerve profiles. The function of these cells may be secretory or chemosensory, but synapses in monkeys are not evident (Farbman et al., 1985, 1987). In mice, vallate and foliate “light” (type II) and “dark” (type I and type II) taste bud cells exhibit synaptic contacts with nerve fibers, thus suggesting a gustatory function. However, type I and type II cells do not form synapses with the same

nerve fibers (Kinnamon et al., 1985, 1988; Royer and Kinnamon, 1988). 3.

Type III Cells

Taste buds contain only 5–7% of type III cells. They have unbranched basal and apical processes. Their apical portion protrudes with a single, large microvillus into the taste pit and may reach the taste pore. Type III cells are presumably the only cells that have synaptic contacts with intragemmal nerve fibers. Near the cell nucleus there are numerous serotonin-containing, dense-cored vesicles (80–140 nm in diameter) (Nada and Hirata, 1977; Fujimoto et al., 1987). Proteins [VIP (Herness, 1989), spot 35 protein, and calbindin (Yoshie et al., 1991; Johnson et al., 1992)] were also demonstrated in these cells. Therefore, these cells are considered by most investigators as gustatory sensory cells. 4.

Type IV Cells

Type IV cells (Murray et al., 1969; Murray, 1973) include basal cells (Nemetschek-Ganssler and Ferner, 1964) or

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Figure 11 Mammalian taste bud in longitudinal section, idealized schematic drawing according to electron microscopic findings. Each cell type is depicted once. Following Murray’s nomenclature, cells of type I, II, and III are elongated and form the buds sensory epithelium proper. Apically, these cells end with different types of microvilli within the taste pit and may reach the taste pore. Type IV cells are basal cells, type V marginal cells. Synapses are found especially at the bases of type III cells. Nerve fibers within the short dermal papilla are slightly myelinated, and within the taste bud they form an unmyelinated plexus. Note the basal lamina between dermis (which contains a capillary) and the epithelium.

“pregustatory cells” (Scalzi, 1967). These are relatively small undifferentiated cells that lie at the taste bud’s base, which do not form processes that reach the pore. They contain numerous bundles of intermediate filaments (Royer and Kinnamon, 1991) and differ from Merkel cell–like basal cells of fishes and amphibians. Type IV cells are considered to be undifferentiated stem cells of their bud cell progeny (Murray, 1973, 1986; Roper, 1989). Some authors report on “intermediate cells” (e.g., Kinnamon et al., 1985). It has been pointed out by Farbman et al. (1985) that differences in the electron density of the cytoplasm could also reflect different stages in the maturation of the same cell type indicating different states of function. 5.

Type V Cells

“Marginal cells” (also “perigemmal cells” and, in extension of Murray’s nomenclature, “type V cells”) have been described (Beidler and Smallman, 1965; Farbman, 1980; Gurkan and Bradley, 1987; Reutter and Witt, 1993). However, they have nothing in common with the secretory marginal cells of taste organs in fish and frog

and may possibly be taste bud stem cells (Beidler and Smallman, 1965; Farbman, 1980) which express particular non–taste receptor proteins, e.g., CD44 isoforms (Witt and Kasper, 1998), during human taste bud ontogenesis. C.

Molecular Markers of Taste Bud Cells — Basis for a New Classification?

One of the most intriguing challenges for suggesting possible functional properties of taste bud cells is to identify subsets of these cells by morphological features as well as molecular properties, many of which can be traced even in improved primary taste bud cell cultures (Kishi et al., 2001). Histochemical evidence on the neurochemical nature of taste cells [see the concept of paraneuron by Fujita (1994)] have identified the panneuronal markers, neuron-specific enolase (NSE) (see Fig. 5), and protein gene product 9.5 (PGP 9.5) (Yoshie et al., 1988; Montavon et al., 1996; Astbäck et al., 1997), carbohydrate-binding proteins, the lectins (Witt and Reutter, 1988; Witt and

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Miller, 1992), and members of the intermediate filament family, e.g., cytokeratins 18–20 and vimentin (Zeng et al., 1995; Zhang et al., 1995; Zhang and Oakley, 1996; Witt et al., 1999; Witt and Kasper, 1999), as well as microfilaments (Höfer and Drenckhahn, 1999). However, the relation between molecular marker and taste bud cell type has not been firmly established ultrastructurally. Immunoelectron microscopic studies have tried to match functional parameters with those of conventional electron density. For example, cell adhesion molecules (Nolte and Martini, 1992; Smith et al., 1993, 1994) and several blood group antigens (Pumplin et al., 1997, 1999; Smith et al., 1999) characterize subsets of taste bud cells. Dark cells of rat vallate taste buds have been associated with H blood group antigen (2B8 epitope) expression, and light cells with the Lewis b antigen (Pumplin et al., 1997). These authors conclude that light cells in rat vallate taste buds have at least two molecular phenotypes, namely cells with Lewis b and those without. A subset of the light cell phenotype contains partly the G protein gustducin (Ruiz-Avila et al., 1995; Menco et al., 1997), which is involved in the perception of sweet and bitter taste (Wong et al., 1996). Choline acetyl transferase, an enzyme involved in the synthesis of the neurotransmitter acetylcholine, has been identified in rat type II cells (Menco et al., 1997). Whereas the putative neurotransmitter serotonin is confined to basal cells of fish taste buds (Reutter, 1971) and Merkel cell–like basal cells of amphibian taste organs (Toyoshima and Shimamura, 1987; Delay et al., 1997; Hamasaki et al., 1998), serotonin in mammals has been described in type III cells in the rabbit (Fujimoto et al., 1987; Kim and Roper, 1995) and human taste buds (Azzali, 1997). This led to the hypothesis that these cell types were equivalent in both taxa (Kim and Roper, 1995). Lindemann (1996) suggests the term “serotonergic cells” instead of type III cells (10% of all cells). Generally, neuropeptides are located in intragemmal nerve fibers rather than in particular bud cells. An exception is vasointestinal peptide (VIP) that has been detected in a subset of rat taste bud cells (Herness, 1989) and in light taste bud cells in the carp by electron microscopy (Witt, 1995). Though most of these markers are expressed only in differentiated cells and are not evident after nerve dissection (Smith et al., 1993; Whitehead et al., 1998), their functional correlation with taste perception data is mostly unknown. To avoid the present Babylonian confusion of tongues with regard to taste bud cell nomenclature, future research directions should try to associate electrophysiologically characterized, isolated taste bud cells with a particular cell type based on its specific protein expression. Modern cell

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biological approaches, e.g., introduction of green fluorescent protein chimeras in vitro (Landin and Chaudhari, 2000) or calcium imaging after application of specific neuropeptidergic stimuli (Lu et al., 2000) might contribute to a solution of this problem. Several authors report on morphological and immunohistochemical differences between vallate/foliate and fungiform taste buds within the same species. For example, mouse taste bud cells of fungiform papillae contain more synapses and presynaptic vesicles than those of vallate papillae (Kinnamon et al., 1993), and the number of taste bud cells containing group H blood antigen and gustducin is three times higher in vallate than in fungiform papillae (Smith et al., 1993). In rabbit, lectin carbohydrate profiles of both taste bud populations differ as well (Witt and Miller, 1992). The reasons for and significance of these differences between fungiform and vallate/foliate taste cells are not clear, but factors determining their varying phenotype could include a different local saliva composition (Shatzman and Henkin, 1981; Schmale et al., 1990; Schmale and Bamberger, 1997) or morphogenetic conditions of local epithelium (Smith et al., 1999). Evidence for communication between taste cells includes the presence of cell adhesion molecules (Nolte and Martini, 1992; Smith et al., 1993), heparin-binding proteins (Wakisaka et al., 1998), and membrane receptors that influence the intracellular signal transduction cascades. For example, the hyaluronan receptor, CD44, was identified in a subset of human fetal taste bud cells (marginal cells) and most of adult human taste bud cells (Witt and Kasper, 1998). This transmembrane protein is linked to a series of actin-associated microfilaments, e.g., ezrin and ankyrin, which are located in microvilli of type I cells and might influence the function of ion-translocating membrane proteins (Höfer and Drenckhahn, 1999). In light of efferent neural control, taste bud cell communication may be mediated via local axon reflexes between sensory cells (Caicedo et al., 2000). The leaf-like enclosing morphology of type I (dark) cells suggests an insulating, glia-like function.

VIII. DEVELOPMENT OF THE HUMAN PERIPHERAL TASTE SYSTEM Morphogenesis of the mouth cavity is characterized by the development of the tongue anlage, which appears prior to, and is a prerequisite of, the formation of gustatory papillae. At the embryonic age of 4 weeks, the first structure of the tongue anlage to appear is the tuberculum impar, which is situated between the first (mandibular) and second (hyoid) branchial arches (Fig. 12). Then, anterolateral to

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Figure 12 Development of the human tongue, 5th postovulatory week. The schematic drawing was done by compiling Hinrichsen’s (1990) and our own data. By horizontal section the floor of the forecoming buccal cavity of a human embryo is removed and viewed from dorsally. The floor relief is derived from the branchial arches I (mandibular arch), II (hyoidal arch), III (3rd pharyngeal arch), and IV (4th pharyngeal arch), and by their derivatives which are: 1, the paired lingual swellings; 2, the impar tubercle; and 4, the hypobranchial eminence. These three structures form the tongue anlage. Between 2 and 4, the anlage of the thyroid gland invaginates, and as its remnant the 3 (foramen caecum) is left; 5 is the anlage of the epiglottis. The branchial arches, as well as the tongue anlage, are innervated by the cranial nerves CN V3 (mandibular nerve), CN VII (facial nerve), CN IX (glossopharyngeal nerve), and CN X (vagal nerve). CN XII (hypoglossal nerve) invades the tongue anlage as well and innervates its muscular system. Later, the lingual nerve (from CN V3) and the chorda tympani (running with CN VII) join each other and supply the anterior two thirds of the tongue with somatosensory and gustatory nerve fibers, whereas CN IX and CN X carry taste fibers for the posterior third of the tongue, the epiglottis, and the pharynx.

the tuberculum impar, the paired lingual swellings (which derive from the medial parts of the mandibular arches) fuse with the tuberculum impar. The tongue’s base is formed by the hypobranchial eminence (copula of His) forming within the third and fourth branchial arches. The border between the caudal part and the body of the tongue is demarcated by a V-shaped rim, the sulcus terminalis (Bradley, 1972; Witt and Reutter, 1997). The innervation pattern of cranial nerves, which later supplies particular lingual regions, reflects the early innervation of branchial arches (see Fig. 12). The pretrematic nerve of the first branchial arch is the lingual nerve (from cranial nerve V3); that of the second arch, the chorda tympani (from the intermedio-facial nerve); and that of the third arch constitutes later lingual rami of the glossopharyngeal nerve (for details, see textbooks on anatomy and embryology; e.g., Williams et al., 1989; Hinrichsen, 1990). The first detailed developmental studies on the surface appearance of the tongue were carried out by Froriep (1828) and continued histologically by Tuckerman (1889), Gråberg (1898), and Hellman (1922). Hermann (1885) described the stages of karyokinesis in developing taste

buds. It was unclear to Hermann if supporting, or neuroepithelial, cells were being replaced. Vallate papillae start to develop earlier than fungiform papillae, and begin with the appearance of a central midline papilla just behind the foramen caecum around the 6th postovulatory week. From week 7 on, there develop many hillock-like epithelial elevations on the tongue’s dorsum, as seen with scanning electron microscopy (Fig. 13). Some of these elevations are precursors of fungiform papillae and are especially densely distributed near the midline and the lateral ridges of the tongue (Habermehl, 1952; Hersch and Ganchrow, 1980; Witt and Reutter, 1997). Analysis of serial sections of the tongue encompassing this critical developmental age (weeks 6–8) demonstrates that not every dermal elevation will be the target of nerve fibers. First, around week 7–8, nerve fibers, migrating towards the periphery, form a large intragemmal plexus. Our own studies show that there are no taste bud anlagen without approaching nerve fibers (Witt and Reutter, 1996; Witt and Kasper, 1998). Also, taste bud primordia without dermal papillae are evident, as well as individual bipolar epithelial cells resembling

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At their apical ends, taste bud cells cannot be distinguished by their electron density (week 15) (Fig. 15). There are cells with long, slender microvilli, but, in contrast to adult taste buds (Azzali, 1997), there are no type I cells with typical dark granules believed to secrete the mucous material that fills the taste pit (Witt and Reutter, 1996).

IX. INNERVATION OF THE HUMAN TONGUE AND TASTE BUDS

Figure 13 Scanning electron micrograph of a human embryonic tongue, 7th postovulatory week. Fine dots in the dorsal surface demarcate later fungiform papillae. Anlagen of vallate papillae (arrows) lie in front of the sulcus terminalis. Short arrow indicates the median vallate papillae, which originates first. Scale bar
0.5 mm.

solitary chemosensory cells. These individual cells are immunopositive for cytokeratin 20, a marker for lingual taste bud cells (Zhang and Oakley, 1996; Witt and Kasper, 1999) (see above). Temporal correlation, which would suggest dependence of taste bud development on nerve ingrowth, has not as yet been seen. Lingual taste bud primordia first occur around the 7th and 8th postovulatory weeks (Bradley and Stern, 1967; Bradley, 1972). Taste pores, commonly acknowledged as a sign of taste bud maturity, appear between the 10th and 14th weeks. The presence of a taste pore is not always associated with a fully mature taste bud because the bottoms of early taste pits may be covered by flat epithelial cells (Witt and Reutter, 1997). However, transmission electron microscopic studies show that early taste bud primordia (week 8) synaptically contact nerve fibers, suggesting that the potential for neurotransmission precedes the exposure of sapid molecules to the apical surface of the taste bud cell. At its base, the developing human taste bud (weeks 12–15) contains processes of dark and light cells, as well as processes resembling type III cells (exhibiting synapses with nerve fibers) (Fig. 14).

The tongue is innervated by (1) motor nerve fibers, constituting the hypoglossal nerve (CN XII), which supplies the inner (intrinsic) and the hyoidal tongue muscles; (2) somatosensory nerve fibers, composed of divisions of the trigeminal (CN V3) and glossopharnygeal (CN IX) nerves; (3) autonomic nerve fibers, which stem from the intermedio-facial nerve (CN VII), the glossopharyngeal nerve, and the vagus nerve (CN X); and (4) sensory (gustatory) nerve fibers that transmit taste information centrally from taste buds, namely (1) the chorda tympani, a branch of the intermediate nerve (generally considered a part of the facial nerve; CN VII), (2) glossopharyngeal nerve (CN IX), and (3) vagus nerve (CN X) (summarized in Fig. 16). Classical research papers of the nineteenth century explored the clinical consequences in taste perception associated with diagnostic features of the cranial nerves. These papers have formed the bases for the neurological examination. Two prominent themes were explored: (1) Which cranial nerves are associated with functional attributes of the taste system? (2) How is taste perception affected by neurological diseases? In physiological experiments, Magendie (1820) dissected the lingual nerve of living, unanaesthetized animals and observed loss of taste, but intact movement and sensation of palate, gingiva, and buccal mucosa. Panizza (1834) performed dissection of the glossopharyngeal nerve, resulting in loss of taste. Alcock (1836) described the role of the chorda tympani and the sphenopalatine ganglion. Lussana (1869, 1872) traced the target tissue of the chorda tympani nerve to the anterior two thirds of the tongue. The dependence of taste buds on nerve supply was experimentally shown by von Vintschgau and Hönigschmied (1877): 40 days after dissection of the glossopharyngeal nerve, the number of taste buds dramatically decreased. In similar experiments, Ranvier (1888) observed that taste bud sensory cells degenerate, and supporting cells pushed through the pore to the tongue surface. During this process, he observed cellules migratrices [phagocytic fibroblasts? (Suzuki et al., 1996)] loaded with fed particles, which were “probably responsible for removal of old materia”.

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Figure 14 Basal portion of a developing human taste bud, week 15. Light cells (L), nerve fiber profiles (N), and dark cells (D) are the prominent structures at the base. Type III-like cells contain dense-cored vesicles (arrows). BL
Basal lamina; S
Schwann cell; scale bar
1 m. (From Witt and Reutter, 1996.)

Soon it became evident that experiments based on vivisection were not only painful for the animal (mostly dogs or cats), but also unreliable in their results (Alcock, 1836; Wagner, 1837). Nevertheless, the overall conclusions derived from these nineteenth-century nerve dissection experiments (reviewed by Parker, 1922; Jägel, 1991) cannot deny their import for current knowledge of cranial nerve supply and taste sensitivity. An elegant review of the background of peripheral taste pathways in humans was written by Lewis and Dandy (1930). They examined both the neurological and neuroanatomical literature on gustatory pathways. The sensory distribution of the facial nerve and its clinical importance was described by Hunt (1915). He disentangled the overlapping sensory fields of the facial nerve (including the chorda tympani) from the trigeminal nerve by documenting the distribution of herpes zoster inflammation. The herpetic eruptions outlined the sensory fields of the geniculate ganglion on the tongue, soft palate, and ear. Another basis for evaluating the involvement of the chorda tympani nerve with lingual taste buds came from patients who had undergone middle ear surgery (Bull, 1965; Borg et al., 1967). Contemporary reviews of human (Norgren, 1990) and primate (Pritchard, 1991) taste pathways have incorporated

observations from the second half of the century, particularly those derived from electrophysiological studies. 1. Chorda tympani and greater petrosal nerve: Distal to the intermediate nerve branch of the facial nerve, peripheral axons of some geniculate ganglion somata (the chorda tympani nerve) take a recurrent course within the facial canal in the petrosal part of the temporal bone, pass through the middle ear, and exit the skull via the petrotympanic fissure to join the lingual division of the trigeminal nerve, the lingual nerve. Both intermedio-facial (gustatory) and trigeminal (somatosensory) fibers run in the lingual nerve and distribute to the fungiform papillae on the anterior two thirds of the tongue and may reach also the anterior portion of the foliate papillae. Taste buds on the soft palate are innervated by the greater petrosal branch of the intermedio-facial nerve, whose somata also lie within the geniculate ganglion (Harris, 1952; Miller and Spangler, 1982). Some chorda tympani fibers are reported to anastomose with the greater petrosal nerve via the otic ganglion (Schwartz and Weddell, 1938; Pritchard, 1991). Both the greater petrosal and chorda tympani nerves also carry parasympathetic fibers to their associated salivary glands: the greater petrosal

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Figure 15 Transmission electron micrograph of a human fungiform taste bud during development (week 15). The taste pore (TP) is already open, and some elongated cells stick into the taste pit. However, differences between cell types in the apical portion of the taste bud cannot be made yet. There is no mucus in the taste pit. Approximately two thirds of the taste bud are filled with ramifications of nerve fibers (N). A part of the basal portion is outlined with a rectangle (see Fig. 14). MC
marginal cell; scale bar
10 m. (From Witt and Reutter, 1996, with permission of John Wiley and Sons.)

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Figure 16 Innervation of the human tongue and the taste bud–bearing epithelia (hatched regions), compiled by figures from Feneis (1985) and Sobotta (1993). The cranial nerves CN VII (which includes the intermediate nerve with its branches, greater petrosal nerve and chorda tympani, black), CN IX, and CN X contain sensory gustatory fibers. CN V is the trigeminal nerve with its divisions V1, ophthalmic, V2, maxillary, and V3, mandibular nerves. CN XII, hypoglossal nerve, is the motor nerve of the tongue. gG, Geniculate ganglion; pG, pterygopalatine ganglion; iG, inferior ganglion of the glossopharyngeal (CN IX) and vagal (CN X) nerves, respectively; sG, submandibular ganglion with postganglionic autonomic nerve fibers of the chorda tympani to supply the submandibular and sublingual glands.

2.

nerve serves the palatine glands, while the chorda tympani innervates the submandibular and sublingual glands via the submandibular ganglion. Glossopharyngeal nerve: Axons of the glossopharyngeal nerve originate from ganglion cells mainly in the inferior (petrosal) glossopharyngeal ganglion. These peripheral axons supply both taste buds and general sensory innervation to the vallate and foliate papillae. Salivary glands (Ebner) are supplied by parasympathetic fibers via an intrinsic ganglion (Remak, 1852).

3.

4.

Probably, the glossopharyngeal nerve also supplies taste buds in the pharynx. Vagus nerve: Taste buds on the laryngeal surface of the epiglottis, larynx, and proximal portion of the esophagus are innervated by the superior laryngeal branch of the vagus nerve, which has the perikarya of its chemosensory neurons in the inferior (nodose) vagal ganglion. Trigeminal nerve: The possible role of trigeminal nerve fibers in taste perception has been discussed for

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two millenia, and there is no end in sight. Via the lingual nerve, this nerve conveys somatosensory information from the tongue to the trigeminal ganglion (Gasseri). In fact, most of the nerve fibers entering the fungiform papillae are trigeminal, while a few fibers originate from the chorda tympani (25% in rat) (Farbman and Hellekant, 1978). Palatal trigeminal fibers may respond to sapid stimuli, as revealed by trigeminal transection experiments (Berridge and Fentress, 1985) and electrophysiology (Harada and Smith, 1992). Finally, taste qualities may be influenced by nonsapid stimuli, e.g., temperature: approximately one-half of the nerve fibers involved in taste transduction respond to temperature (Cruz and Green, 2000). The interaction of both gustatory and somatosensory qualities may be as tight-knit as their anatomical proximity. Katz et al. (2000) suggest that gustation should be thought of as an integral part of a distributed, interacting multimodal system. The observation that taste buds degenerate after dissection of their sensory innervation and, subsequently, reappear after regeneration of their peripheral nerves has been a major focus of research in the peripheral taste system. Nineteenth- and early twentieth-century literature on taste bud degeneration, regeneration, and development was reviewed comprehensively by Parker (1922). Olmsted (1920) proposed that trophic maintenance of fish taste buds depended on the transmission of a putative trophic material from nerve to epithelium. Cross reinnervation of the glossopharyngeal nerve to fungiform taste buds (which are normally supplied by the chorda tympani) had no effect on the usual immunohistochemical properties of fungiform versus vallate taste buds (Smith et al., 1999). As a consequence, these authors believe that the protein expression in and subsequent function of taste buds depend on the epithelium from which the cells arise, and not the buds specific nerve supply. Meanwhile, Nosrat and Olson (1995) and Nosrat et al. (2000) detected mRNA of brainderived neurotrophic factor (BDNF) and neurotrophins in developing anterior tongue epithelium, before nerve fibers were observed. This argues for the hypothesis that trophic factors act as target-derived chemoattractants for the early nerve fibers. These, in turn, initiate the formation of taste buds. BDNF-null mutant mice failed to develop taste buds (Oakley et al., 1998). Sensory ganglia involved in taste bud innervation (see above) are reduced by 40% in volume compared to about 20% of trigeminal ganglion under the same condition (Mistretta et al., 1999). Taste buds do not develop after injection of -bungarotoxin into the amniotic fluid in fetal mice. This neurotoxin abolishes motor and sensory nerve development (Morris-Wiman et al., 1999).

Thus, nerve fibers are required to maintain taste buds once the latter are formed and start to function (e.g., Hosley et al., 1987), but controversy exists whether nerves are necessary to initiate taste bud development. At present, there are two main doctrines concerning this issue: (1) the presence of nerve fibers, if not the synaptic contact to local epithelial cells, is a prerequisite for the neurosensory transformation of epithelial cells (Witt and Reutter, 1996; Oakley, 1998a,b; Oakley et al., 1998); (2) initial taste bud development is nerve-independent, suggested by a series of studies in salamanders (Stone, 1940; Barlow et al., 1996; Barlow and Northcutt, 1998a,b). Taste buds seem to develop from local epithelium and not from neurogenic ectoderm [axolotl: Barlow and Northcutt (1997), mouse: Stone et al. (1995)]. It may be that mechanisms of differentiation of the same receptor organ vary among vertebrate taxa. Growth factors other than BDNF may contribute to the maintenance of gustatory papillae, e.g., epidermal growth factor (EGF) supplied by salivary glands (MorrisWiman et al., 2000). More detailed studies on developmental aspects of the peripheral gustatory system including whether taste buds may develop without the stimulation of nerves are described later in this book (see Chapter 36). ACKNOWLEDGMENTS The authors are indebted to Mihnea Nicolescu, who provided the schematic drawings. Drs. Judith and Donald Ganchrow helped with critical reading of the manuscript and contributed thereby to avoid mistakes, redundancies, and senseless phrases. REFERENCES Albinus. (1754). Academicarum annotationum liber 1, Tab.I. Lugdunum Bataviensis (Leiden). Alcock, B. (1836). Determination of the question, which are the nerves of taste. Dublin J. Med. Chem. Sci. 10:256–279. Arey, L., Tremaine, M., and Monzingo, F. (1935). The numerical and topographical relation of taste buds to human circumvallate papillae throughout the life span. Anat. Rec. 64:9–25. Arnold, F. (1839). Tabulae Anatomicae. Icones organum sensuum. Organon gustus. Turici, Zürich. Arvidson, K., and Friberg, U. (1980). Human taste: response and taste bud number in fungiform papillae. Science 209:807–808. Astbäck, J., Arvidson, K., and Johansson, O. (1997). An immunohistochemical screening of neurochemical markers in fungiform papillae and taste buds of the anterior rat tongue. Arch. Oral Biol. 42:137–147. Atema, J. (1971). Structures and functions of the sense of taste in the catfish (Ictalurus natalis). Brain Behav. Evol. 4:273–294.

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33 Central Taste Anatomy and Neurophysiology Edmund T. Rolls University of Oxford, Oxford, United Kingdom

Thomas R. Scott San Diego State University, San Diego, California, U.S.A.

1984). Second-order taste neurons project through the central tegmental tract to terminate in the parvicellular division of the ventroposteromedial thalamic nucleus (VPMpc) (Beckstead et al., 1980; Norgren, 1984; Pritchard et al., 1989). A remarkable difference from the taste system of rodents is this direct projection from the NTS to gustatory thalamus. In rodents there is an obligatory relay from the NTS to the pontine parabrachial taste nuclei, which in turn project to the thalamus (Norgren, 1984; Norgren and Leonard, 1973). The pontine taste nuclei also project to the hypothalamus and amygdala in rodents (Norgren, 1976), providing direct access in rodents to these subcortical structures important in motivational behavior (e.g., feeding) and learning (Rolls 1990). In contrast, in primates there appears to be no such direct pathway from the brainstem taste areas to the hypothalamus and amygdala (Norgren, 1984). This fundamental difference in the anatomy of the rodent and primate taste pathways shows that even in a phylogenetically old system such as taste, the way in which the system functions and processes information may be different across mammalian orders. This may result from the great development of the cerebral cortex in primates and the advantage of using extensive cortical processing from each sensory modality before the representations are integrated in multimodal regions, as suggested below. The thalamic taste area, VPMpc, projects to the primary taste cortex, which forms the rostral part of the frontal

The aims of this chapter are to describe the anatomy and physiology of the central taste system, with reference to the rodent and to the primate to make the findings relevant to taste processing and its disorders in humans. Neuroimaging studies in humans are also described. We also address the convergence of gustatory input with olfactory, somatosensory, and visual afferents, and how the central representation of the sensory properties of food is made relevant to the control of the appetite for a food and food intake. Much of the research in rodents has been carried out at subcortical levels, whereas in primates more research has been carried out on the cortical processing of taste. The evidence suggests that not only the central connections, but also the principles of processing in the taste pathways, may be different in rodents and primates.

I.

GUSTATORY ANATOMY

Diagrams of the taste pathways in primates are shown in Figures 1 and 2. As described in detail in the preceding chapter, three cranial nerves (CN) carry taste information centrally: CN VII (chorda tympani and greater superficial petrosal branches), CN IX (lingual branch), and CN X (superior laryngeal branch). Gustatory axons terminate in the rostral part of the nucleus of the solitary tract (NTS) in the medulla (Beckstead and Norgren, 1979; Norgren, 679

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Figure 1 Drawing of the human brain, with the presumed afferent limb of the taste system superimposed. nst, nucleus of the solitary tract; pbn, parabrachial nucleus; VPMpc, ventroposteromedial nucleus of the thalamus, pars parvocellularis; ins, insula; op, operculum. (Drawing by Mr. Birck Cox, used with permission.)

operculum and adjoining insula (Figs. 1–3) (Pritchard et al., 1986). In macaques, this is at the anterior end of the Sylvian fissure. Cells in the insular-opercular taste cortex (IO) then project anteriorly to a part of the caudolateral orbitofrontal cortex (OFC), in which taste-responsive neurons are found (Baylis et al., 1994; Rolls et al., 1990). This OFC area does not receive inputs from VPMpc, but instead receives projections from the mediodorsal nucleus of the thalamus, which projects to the prefrontal cortex. Thus the caudolateral orbitofrontal taste cortex contains a secondary taste cortical area, which receives gustatory inputs from the primary frontal opercular and insular taste cortices. Afferents also reach the caudolateral orbitofrontal taste cortex from the more ventral part of the rostral insular cortex, the amygdala, the substantia innominata, the rhinal sulcus, and the surrounding orbitofrontal cortex. Through some of these pathways visceral information may reach the caudolateral orbitofrontal taste cortex. Injections of horseradish peroxidase more anteriorly in the orbitofrontal cortex have been shown to label neurons in the secondary taste cortex, but not in the primary taste cortex, providing evidence for tertiary cortical taste regions in more anterior parts of the primate orbitofrontal cortex in which taste-responsive neurons are found (Baylis et al., 1994; Rolls, 1997; Rolls and Baylis, 1994; Thorpe et al., 1983).

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The orbitofrontal cortex receives inputs from other sensory systems, as shown schematically in Figure 2. There are direct connections from the primary olfactory cortex (pyriform cortex) to area 13a of the posterior orbitofrontal cortex, which in turn has onward projections to a middle part of the orbitofrontal cortex (area 11) (Barbas, 1993; Morecraft et al., 1992; Carmichael et al., 1994) (see Fig. 2). Visual inputs reach the orbitofrontal cortex directly from the inferior temporal cortex in which representations of objects are found (Booth and Rolls, 1998; Rolls, 2000c), the cortex in the anterior part of the superior temporal sulcus in which face-responsive neurons are found (Hasselmo et al., 1989a,b; Wallis and Rolls, 1997), and the temporal pole (see Barbas, 1988, 1993, 1995; Barbas and Pandya, 1989; Carmichael and Price, 1995; Morecraft et al., 1992; Seltzer and Pandya, 1989). There are corresponding auditory inputs from the superior temporal cortex (Barbas, 1988, 1993), and somatosensory inputs from somatosensory cortical areas 1, 2 and SII in the frontal and pericentral operculum, and from the insula (Barbas, 1988; Carmichael and Price, 1995). The caudal orbitofrontal cortex receives strong inputs from the amygdala (see Chapter 8) (Price, 2001). The orbitofrontal cortex also receives inputs via the mediodorsal nucleus of the thalamus, pars magnocellularis, which itself receives afferents from temporal lobe structures such as the prepyriform (olfactory) cortex, amygdala, and inferior temporal cortex (see Price et al., 1996). The orbitofrontal cortex projects back to temporal lobe areas such as the inferior temporal cortex and, in addition, to the entorhinal cortex (or “gateway to the hippocampus”) and cingulate cortex (Insausti et al., 1987). The orbitofrontal cortex also projects to the preoptic region and lateral hypothalamus, to the ventral tegmental area (Johnson et al., 1968; Nauta, 1964), and to the head of the caudate nucleus (Kemp and Powell, 1970). Reviews of the cytoarchitecture and connections of the orbitofrontal cortex are provided by Petrides and Pandya (1994), Pandya and Yeterian (1996), Carmichael and Price (1994, 1995), and Barbas (1995). The anatomy of taste pathways beyond these is not known in detail. In primates, taste neurons are found in the hypothalamus to which OFC projects (Burton et al., 1976; Rolls et al., 1986), and in the amygdala (Rolls, 2000; Sanghera et al., 1979; Scott et al., 1993), which receives inputs from the insular cortex (Mesulam and Mufson, 1982b) and from the OFC (Amaral et al., 1992). II.

TASTE PROCESSING

A.

Nucleus of the Solitary Tract

1.

Primates

Taste neurons have been found and their responses analyzed in the rostral part of the nucleus of the solitary tract of

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Figure 2 Schematic diagram of the taste pathways in primates (center) showing how they converge with olfactory, visual, and somatosensory pathways. The gate functions shown refer to the finding that the responses of taste neurons in the orbitofrontal cortex and the lateral hypothalamus are modulated by hunger. VPMpc: Ventroposteromedial thalamic nucleus; V1, V2, V4: visual cortical areas.

macaque monkeys (Scott et al., 1986a). Different neurons were found that responded best to glucose, NaCl, HCl (sour), and quinine HCl (bitter), but the tuning of the neurons was in most cases broad; for example, 84% of the neurons had at least some response to 3 or more of these 4 prototypical taste stimuli. The mean value of the breadth of tuning metric of Smith and Travers (1979) (see Chapter 34), calculated from the responses to the four prototypical stimuli for 52 neurons analyzed in the nucleus of the solitary tract (NTS), was 0.87. Thus, neurons in this part of the primate taste system have relatively broad tuning to differ-

ent stimuli. This distributed encoding may provide an efficient mechanism for information transmission in a relatively small number of neurons (see Erickson, 1985; Hinton et al., 1986; Rolls and Treves, 1998). In macaques, feeding to satiety with glucose has no impact on the responsiveness of NTS cells to the taste of the glucose (Yaxley et al., 1985). 2.

Rodents

Evidence about gustatory coding in the taste pathways of rodents is described in Chapter 35. In rodents the NTS is

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thought to manage a basic level of taste discrimination, to control somatic reflexes of acceptance or rejection, and to regulate autonomic reflexes that anticipate digestive processes. Neurons here may also modulate feeding behavior according to short-term satiety signals. a. Basic Discriminations. Lesions in the gustatory division of the NTS impair a rat’s capacity to discriminate sapid stimuli from water (Shimura et al., 1997a). The concentration-response function is flattened for all basic taste qualities, but the effect is most severe for sucrose and NaCl. The deficit is greater with damage to the NTS than to other gustatory relays. Conversely, rats with only the hindbrain intact show typical acceptance-rejection reflexes (see below), implying that the taste quality that drives the reflex is being properly assessed. b. Acceptance-Rejection Reflexes. Clusters of cells in the ventral regions of gustatory NTS send their projections ventrolaterally to salivatory and pharyngeal areas of the reticular formation, to the hypoglossal nucleus, and toward the facial and ambiguus nuclei, through which acceptancerejection reflexes could be orchestrated. These are fully integrated within the hindbrain, in both rodents (Grill and Norgren, 1978) and humans (Steiner, 1979), and are stereotypical to each of the basic taste qualities.

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c. Autonomic Reflexes. A second projection from ventral NTS goes caudally, to ramify through viscerosensory NTS and the dorsal motor nucleus of the vagus (DMNX), through which it may influence autonomic reflexes. There are several cephalic phase pancreatic and gastrointestinal reflexes associated with digestion, of which the best delineated is the cephalic phase insulin release (Powley et al., 1987). Thousands of  cells in the rats’ islets of Langerhans are stimulated to release insulin by fewer than 200 fibers coursing through the gastric and hepatic branches of the vagus nerve. These originate in the rostromedial division of the DMNX, which is overlain by the gustatory NTS. Neurons in DMNX send apical dendrites into the NTS, functionally fusing the two structures and offering gustatory input direct control over autonomic reflexes (Powley and Berthoud, 1991). Accordingly, a sweet taste elicits insulin release, and this effect is blocked by vagotomy (Louis-Sylvestre et al., 1983). The insulin release is also blocked if the sweet stimulus is paired with gastrointestinal distress to create a conditioned taste aversion, implying that this profound learning experience alters the gustatory code for sweetness. d. Satiety. The acceptance-rejection reflexes mentioned above may be modified by short-term satiety signals in decerebrate rats (Grill and Kaplan, 1990). If satiated, or if blood glucose levels are raised, acceptance reflexes to a

Figure 3 Coronal sections to show the locations of the primary taste cortices in the macaque in the frontal operculum and rostral insula and of the secondary taste cortex in the caudolateral orbitofrontal cortex. The coordinates are in mm anterior (A) or posterior (P) to sphenoid. (From Aggleton and Passingham, 1981.)

Central Taste Anatomy and Neurophysiology

sweet stimulus are lost, as they are in an intact rat, implying that hindbrain cells are sensitive to these immediate signals of satiety. Similarly, when glucose is made systemically more available to neurons by means of intravenous glucose infusions (Giza and Scott, 1983), mild hyperinsulinemia (Giza and Scott, 1987a), or glucagon administration (Giza et al., 1993), gustatory responses to glucose in the NTS show some decline, implying a reduction in the appetitive nature of the taste signal (Giza et al., 1992). The decrease of responsiveness occurs within five minutes, and is reversible. Moreover, the effects are differential, depending on the cell type. Giza et al. (1997a,b) found that neurons that responded specifically to sugars were suppressed by 74% in their response to glucose following hyperglycemia.

B.

Parabrachial Nucleus

In rodents, gustatory afferents terminate in the medial parabrachial nucleus (PBN), whereas afferents communicating visceral information synapse more laterally. However, the gustatory and visceral modalities converge on single cells within the PBN, suggesting that while it is involved in gustatory processing, the primary role of the PBN may be to mediate the interactions between physiological condition and taste. The integrity of the PBN is crucial to the formation and retention of a conditioned taste aversion and for the expression of a sodium appetite. 1.

Processing Taste Information

Neurons in the medial PBN of the rat respond robustly to taste stimuli (Perrotto and Scott, 1976), and the evoked activity is essentially a linear transformation of that evoked from NTS cells by the same stimuli (Scott and Perrotto, 1980). Studies of c-fos expression suggest that discrete subareas of the PBN are activated by visceral stimulation and by tastes of various qualities (Yamamoto et al., 1994). Destruction of PBN neurons, however, has considerably less impact on taste discrimination than do corresponding lesions in the NTS (Flynn et al., 1991a,b). 2.

Responses to Physiological Condition

a. Satiety. Lesions of the PBN block the increase in feeding that is otherwise induced by the fructose analog 2,5-anhydro-D-mannitol (Grill et al., 1995). Since 2,5-AM works only peripherally, this implies vagal mediation. Conversely, when a rat has a satiating load of intralipid administered into the duodenum, taste responsiveness in the PBN declines. The decline is selective, with the greatest effect on the taste activity evoked by sucrose. Among

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subtypes of taste cells, the responsiveness of sucrose-specialized neurons to sucrose were suppressed by 77%. This is nearly the same as the reduction in response of sugar cells to glucose in the NTS (74%) after hyperglycemia had been induced (Giza et al., 1997a,b). The result implies that a reduced response among cells that would otherwise provide a powerful signal for reward may be one mechanism by which satiety is manifested. b. Conditioned Taste Aversions. A conditioned taste aversion (CTA) is formed to a novel taste when it is paired with gastrointestinal malaise. PBN is central to this process. Rats with either electrolytic (Reilly et al., 1993) or ibotenic acid lesions of the PBN could learn neither a conditioned taste (Reilly, 1998) nor odor aversion (Grigson et al., 1998), though their ability to develop a conditioned taste preference was unimpaired. The failure resulted neither from a loss of taste nor from visceral sensitivity, but rather from an inability to form an association between them. There are conflicting data regarding the disruptive effects of PBN lesions on existing CTAs (Grigson et al., 1997; Sakai et al., 1998). Both electrophysiological and c-fos studies have been employed to document the effects of a CTA on activity in the PBN. Creation of a CTA to the taste of NaCl resulted in greater responses to NaCl thereafter (Shimura et al., 1997b), though the impact of that increase on the code for NaCl could not be determined. The same result had been reported using a saccharin-conditioned stimulus in the NTS (Chang and Scott, 1984). The application of immunocytochemical techniques has permitted topographic analysis of the effects of a CTA. Yamamoto and colleagues report that, when a formerly appetitive taste is used as the CS in a CTA paradigm, the c-fos expression it elicits shifts from a PBN subnucleus associated with positive hedonics to one normally activated by aversive tastes (Yamamoto, 1993). Separate subnuclei of the PBN may send discrete projections to forebrain areas that analyze taste and hedonics. If such a CTA-induced reorganization of the topographic representation of taste is confirmed, it would provide a basis for the shift in hedonic value of the CS, established in PBN, but manifested more rostrally. c. Sodium Appetite. Lesions of the PBN blocked the expression of a sodium appetite in rats (Flynn et al., 1991a,b). The implication that PBN is involved in sodium appetite is strengthened by electrophysiological results, showing that gustatory responsiveness to NaCl declines when rats are depleted, just as has been shown in the CT nerve (Contreras and Frank, 1979) and NTS (Jacobs et

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al., 1988). The nature of that involvement has yet to be clarified. C.

Thalamic Taste Area

1. Primates Pritchard et al. (1989) were able to confirm that the parvicellular division of the VPMpc is the thalamic taste relay nucleus in primates by showing that single neurons in it responded to taste stimuli in macaque monkeys. The neurons were relatively broadly tuned, with a mean breadth of tuning of 0.73 (measured across responses to sucrose, NaCl, HCl, and quinine HCl). Responses to sweet and salt were most common, with 56% responding best to sucrose, and 24% responding best to NaCl. Relatively few neurons had best responses to HCl or QHCl (14%), and most of the responses to HCl and QHCl were described as side-band responses in NaCl-best neurons. In addition to gustatory neurons, some neurons in the nucleus responded to tactile stimuli. Rather surprisingly, given the effects of cortical lesions on food preferences described below, Reilly and Pritchard (1995) reported that VPMpc lesions in macaques caused only minor changes in acceptance behavior. 2. Rodents In rats, cells in the parvicellular division of the VPMpc represent an obligatory synapse between gustatory parabrachial nucleus and gustatory cortex. Therefore, these neurons were assumed to play a major role in taste perception. Electrophysiological studies indicate that taste cells in VPMpc have the sensitivities to support such a role. Thalamic taste cells show monotonically increasing responses with stimulus concentration and differential activity to a full range of taste qualities (Scott and Erickson, 1971). Moreover, many taste cells in the rat VPMpc are multimodal for touch and thermal stimulation as well, implying the early phases of the integration of different sensory modalities (Verhagen et al., 1999). Consistent with this evidence, early lesion studies reported that destruction of VPMpc resulted in elevated detection thresholds, blunting of preference-aversion functions, and loss of the capacity to generate conditioned taste aversions or to express a sodium appetite (Wolf and DiCara, 1974). It now appears that these studies were flawed, victims of the persistent technical shortcomings of early lesion experiments: lesions that were misplaced, were inordinately large, or that destroyed fibers of passage that underlay functions unrelated to those of the target site. In recent experiments, lesion sites have been identified with the aid of electrophysiological guidance to ensure proper placement. Neurons are confirmed as being taste

responsive before the lesion is made. With the location assured, the region of destruction can be sharply limited. Finally, fibers of passage are spared by the use of cytotoxic chemicals that destroy only cell bodies. Under these conditions, the presumed functions of VPMpc were not confirmed. Rats with lesions in VPMpc showed only slightly impaired preference-aversion functions across taste qualities and concentrations in both 24-hour, two-bottle and 15-minute, single-bottle tests (Reilly and Pritchard, 1996a). They were willing to expend a normal amount of effort for a gustatory reinforcer (Reilly and Trifunovic, 1999). They were capable of forming normal conditioned taste aversions and preferences and conditioned odor aversions (Grigson et al., 2000; Reilly and Pritchard, 1996b). They were able to express a sodium appetite, with the only abnormality being that the appetite did not increase in strength with multiple sodium depletions (Scalera et al., 1997). In sum, the thalamic taste relay does not appear necessary in rodents for nearly normal perception of and response to taste stimuli, nor for taste-guided behaviors that rely on integrating taste information with physiological condition, including malaise (CTA), nutritional repletion (CTP), or sodium depletion (sodium appetite). Where thalamic-lesioned rats are deficient is in tasks that require a gustatory memory not associated with physiological condition. They are unable to show anticipatory contrast, i.e., the reduction in consumption of a mildly preferred solution in anticipation of one that is highly preferred (Riley and Trifunovic, 1999). Intact rats show suppressed drinking of weak saccharin relative to controls if they have been trained to expect a strong sucrose solution immediately afterward. Rats with VPMpc lesions show no such suppression despite repeated training sessions, implying that they are not capable of forming a gustatory memory. The difference between this training and conditioning based on physiological changes (CTA, CTP, sodium appetite) is that the latter has been shown to involve actual changes in the responsiveness of the taste system (Chang and Scott, 1984; Contreras and Frank, 1979; Giza et al., 1993; Jacobs et al., 1988; McCaughey and Scott, 2000). Thus, the effect may be established elsewhere, and the resulting altered responsiveness of the taste system may sustain the aversion (CTA) or preference (CTP, sodium appetite) response. The implication is that the thalamic taste relay of rodents is concerned not so much with the consummatory aspects of taste-related behavior as with providing a route necessary for the formation of the gustatory memories that guide the search for food (Reilly, 1998). Finally, discrepant reports about the involvement of VPMpc in CTA formation may be reconciled by a close study of the anatomical connections from the parabrachial

Central Taste Anatomy and Neurophysiology

nucleus to thalamus (Bester et al., 1999). The more lateral regions of PBN (internal lateral and ventral lateral subnuclei) project to the midline and intralaminar thalamic complex, which has been shown to be second in importance only to the PBN itself in creating a CTA. From the thalamus, fibers project to the basolateral amygdala, whose integrity is also required for normal CTA formation. In contrast, the more medial divisions of PBN project to VPMpc, carrying taste information not subject to involvement in the CTA. The magnitude of lesions performed in earlier studies apparently conflated these two pathways. D.

Insular-Opercular (Primary) Taste Cortex

As one proceeds beyond the thalamic taste area, about which little is known, and on to the cortical taste areas, the wealth of the data are from primates. In the following analysis, we focus on results gained from recordings in alert macaques. 1.

Anatomical Connections

Afferents to the insular-opercular cortex (IO) include those from the thalamic taste relay in VPMpc, but also connections from primary somatosensory cortex, the (auditory) cortex in the superior temporal sulcus, entorhinal cortex, and the basolateral amygdala. Efferents from this area project to the second somatosensory cortical area, the cortex in the superior temporal sulcus, several parts of the frontal cortex including the OFC, and the lateral, central, and corticomedial nuclei of the amygdala (Augustine, 1996). 2.

Representation of Taste in the Insular-Opercular Cortex

a. Topographic Organization. There are indications of a crude topographic arrangement of taste qualities in the nucleus of the solitary tract (NTS) of the macaque (Scott et al., 1986) that conform to the organization of sensitivities in the human mouth (Collings, 1974). Moreover, a preservation of anatomical organization from NTS to VPMpc has been reported (Beckstead et al., 1980). However, this is apparently not retained in IO (Pritchard et al., 1986). Accordingly, a plot of the location of each taste cell in IO as a function of its most effective stimulus offers no evidence for topographic organization by taste quality. Apparently, the gross physical location of a neuron is not a salient criterion for coding taste quality, i.e., there is no apparent region devoted to perceiving, say, saltiness. This conclusion does not eliminate the possibility of fine-grained topographic organization that would not have been detected by these studies, e.g., cortical columns

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composed of functionally similar neurons. Nor does it imply that gustatory processing areas that are more concerned with the hedonic components of chemicals (e.g., orbitofrontal cortex) may not reintroduce topographic organization. b. Coding of Taste Quality. Coding strategies are discussed in Chapter 15. There, Smith and Scott rely primarily on data from rodents to conclude that there are classes of taste cells and that taste quality is represented by patterns of activity read across them. Analysis of responses from some 800 taste cells from IO leads to the same result in the insular and opercular primary taste cortices (Scott and Plata-Salaman, 1999; Scott et al., 1986b; Yaxley et al., 1990). Figure 4 shows the relationship among response profiles of 399 taste cells in primate IO in the form of a dendrogram (whose organization is described in the figure legend) to four prototypical tastants (Scott and PlataSalaman, 1999). It reveals rather sharp distinctions between the clusters of 153 glucose-oriented cells to the left, 137 NaCl-oriented cells in the center, and 89 quinineoriented cells to the right, from which a small group of 20 HCl-oriented cells extends. Each group is statistically distinct from the others. Therefore, taste neurons may be segregated into statistically independent types, but within them the constituent neurons do not have identical sensitivities. It also appears that the primate taste system devotes ~73% of its neural resources (290 of 399 cells) to the detection of stimuli that humans describe as sweet or salty, i.e., those that dominate the cuisines of the world. Neurons tuned primarily to quinine constitute ~22% of cortical cells, while only ~5% are oriented toward the detection of acids. Taste quality may be represented by the similarity between patterns of activity generated across all taste cells. By this index, cells in IO most accurately represent taste quality. The relationship between the correlations generated from macaque neurophysiological data and from the descriptions of the same stimulus pairs by humans is closest in IO. This is borne out by a quantitative comparison between macaque neurophysiology and human psychophysics (Fig. 5). Humans judged the similarity between each of 21 pairs of stimuli, and these judgments were plotted against the correlation between patterns elicited by the same two stimuli in macaque cortex. The only major discrepancy involved NaCl versus the bitter salts, CaCl2 and MgCl2, a relationship that was closer in the macaque than in humans. Even with this anomaly, the correlation between the two plots is 0.91; without it, 0.97. Therefore, the most accurate analysis of perceived taste quality is likely to take place in primary taste cortex.

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Figure 4 A dendrogram indicating the distribution of similarity among 399 taste cells in the insular-opercular cortex. The basic stimulus that elicited the dominant response from each group of neurons is listed at the bottom, while the degree of similarity among the profiles of the neurons is shown on the ordinate. Heavy lines indicate the levels at which the groups reach statistical independence. (From Scott and Plata-Salamán, 1999.)

3. Other Inputs into the Insular-Opercular Cortex A relatively low proportion of neurons in the primary taste cortex are apparently activated by taste (Scott and PlataSalaman, 1999; Scott et al., 1986b; Yaxley et al., 1990). For example, across seven studies 6% of insular-opercular neurons responded to taste, 24% responded to movements of the jaw, 4% to tactile stimulation of the mouth, 1% to tongue extension, and 1% to the sight of food (Scott and PlataSalaman, 1999). Some of these effects may be produced by inputs from the primary somatosensory cortex, and there is a whole set of somatosensory inputs to parts of the insula just posterior to the primary taste cortex (Mufson and Mesulam, 1982; Robinson and Burton, 1980; Schneider et al., 1993), which is at the anterior (rostral) end of the insular-orbital cortex (Pritchard et al., 1986). Oral and facial somatosensory fields do appear to be represented in the anterior insula, at least close to taste areas (Schneider et al., 1993).

Although neuroimaging and evoked potential studies do not have sufficient spatial resolution to separate activations produced in the anterior insula from more posterior insular areas with known somatosensory and other inputs, activation in this general region can be produced by stimulation of visceral regions of the esophagus (Aziz et al., 1995; Weusten et al., 1994) and of the throat (Roper et al., 1993), as well as by general visceral arousal induced by phobic anxiety (Rauch et al., 1995). Further, activation of this general region has been produced in positron emission tomography (PET) studies when subjects moved their mouths and tongue and during speech, implying involvement in oromotor functions (Raichle, 1991). Fiol et al. (1988) reported that seizures that began in this general region elicited vomiting, and electrical stimulation of this region in experimental animals does the same (Oppenheimer et al., 1992). Also, activation in this general area can be produced by the sight of face expressions of

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Figure 5 Relationship between response profiles of stimulus quality generated from human psychophysical responses (---) and from neural responses in the macaque (—). The correlation between each stimulus pair is nearly identical by these two approaches, with the exception of those between bitter and salty salts (N: 0.3 M NaCl vs. Ca: 0.3 M CaCl2 and Mg: 0.3 M MgCl2). F: 1 M fructose; K: 0.3 M KCl; S: 1 M sucrose. (Human psychophysical data are from Kuznicki and Ashbaugh, 1979.)

disgust (literally bad taste) (Phillips et al., 1997). Further research is needed to investigate the extent to which individual neurons with taste responses in the primary taste cortex at the rostral end of the insular and adjoining frontal operculum actually show convergence of inputs from these other sources. E. Gustatory Responses in the Caudolateral Orbitofrontal Secondary Cortical Taste Area In a study of the role of the orbitofrontal cortex in learning, Thorpe et al. (1983) found a small proportion of neurons (8%) with gustatory responses in the main part of the orbitofrontal cortex. In some cases these neurons were very selective for particular gustatory stimuli. Therefore, when they set out to search for a secondary taste cortical area, Rolls et al. (1990) started recording at the anterior boundary of the opercular and insular cortical taste areas and worked forward towards the orbitofrontal area investigated

by Thorpe et al. (1983). In recordings made from 3120 single neurons, Rolls et al. (1990) found a secondary cortical taste area in the caudolateral part of the orbitofrontal cortex of the macaque. The area is part of the dysgranular field of the orbitofrontal cortex, OFdg (see Figs. 2 and 3), and is situated anterior to the primary taste cortical area in the frontal opercular and adjoining insular cortices. The responses of 49 single neurons with gustatory responses in the caudolateral orbitofrontal taste cortex were analyzed using the taste stimuli glucose, NaCl, HCl, quinine HCl, water, and black currant juice. Examples of the responses of one orbitofrontal cortex neuron to the stimuli are shown in Figure 6, and quite sharp tuning is evident, in that the neuron responded primarily to the taste of glucose. The mean breadth of tuning coefficient (calculated over the responses to the four prototypical stimuli) for 49 cells in the caudolateral orbitofrontal cortex was 0.39. This tuning is much finer than that of neurons in the nucleus of the solitary tract of the monkey, and finer than that of neurons in the primary

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F.

Figure 6 Examples of the responses recorded from one caudolateral orbitofrontal taste cortex neuron to the six taste stimuli, water, 20% black currant juice (BJ), 1.0 M glucose, 1.0 M NaCl, 0.01 M HCl, and 0.001 M quinine HCl (QHCl).

frontal opercular and the insular taste cortices. A cluster analysis showed that at least seven different groups of neurons were present, the mean profiles of which are shown in Figure 7. For each of the stimuli glucose, black currant juice, NaCl, and water, there was one group of neurons that responded much more to that tastant than to the other tastants. The other groups of neurons responded to two or more of these tastants, such as glucose and black currant juice. In this particular region neurons were not found with large responses to HCl or quinine HCl (Rolls et al., 1990). In more recent studies, the area of orbitofrontal cortex from which recordings have been made has been extended, and neurons with large responses to other stimuli such as HCl, quinine, and monosodium glutamate have been found (Baylis and Rolls, 1991; Critchley and Rolls, 1996c; Rolls and Baylis, 1994; Rolls et al., 1994, 1996a,b, 1999). In these studies of further regions of the orbitofrontal cortex, Rolls and colleagues are finding further areas with tasteresponsive neurons, some of which are broadly tuned, but as in the earlier studies (Rolls et al., 1990; Thorpe et al., 1983), some of the neurons are very finely tuned. Moreover, as described below in Sec. IV, some of these neurons are multimodal.

Amygdala

The amygdala receives projections from both the primary (IO) and secondary (OFC) taste cortices. Sanghera et al. (1979) described taste responses in some amygdala neurons in macaques, and Wilson and Rolls (1993, 2000) found more. Nishijo et al. (1988a,b) found ingestion-related amygdala neurons, at least some of which were probably taste-related. Taste-responsive cells composed about 7% of neurons in another study in the primate amygdala (Scott et al., 1993). As opposed to the precision with which stimulus concentration and quality are represented in IO, the amygdaloid representation was found to be rather crude. Inhibitory responses are nearly as prevalent as excitatory ones, such that the net concentration-response functions are nearly flat, with increasing inhibition offsetting greater excitation. When absolute response rates are calculated, concentration-response functions are monotonically increasing to most basic stimuli, but are not steep enough to match human psychophysical functions. The basic taste qualities are less distinctly separated from one another than at lower neural levels. Glucose is clearly discriminable from others; NaCl and MSG are less distinct; HCl and quinine HCl are not separable. Therefore, taste-evoked activity in the amygdala does not appear to provide an adequate neural basis for the discriminative capacity of humans or monkeys with regard to either stimulus concentration or quality. Rather, the role of the amygdala may be to impart hedonic value to the taste experience (see below). G.

Hypothalamus

The lateral hypothalamic area receives projections from OFC and from the amygdala and has been reported to possess taste-responsive cells in rats (Norgren, 1970). In macaques some neurons are found that respond to taste, and these neurons are implicated in responses to food in that they are modulated in a sensory-specific manner by satiety (see below) (Burton et al., 1976; Rolls et al., 1986). These neurons are likely to be involved in autonomic responses to foods and may also be involved in the reward value that food has when the organism is hungry (Rolls, 1999; Rolls et al., 1980). Oomura et al. (1991) and Karádi et al. (1992) have also shown that lateral hypothalamic neurons that are glucose-sensitive (as shown by microelectrophoresis) are more likely to respond to taste and olfactory stimuli than are glucose-insensitive neurons. H.

Basal Ganglia

The ventral striatum, including the nucleus accumbens and the olfactory tubercle, receives afferents from the OFC and

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Figure 7 The mean response profiles (  SD) of each of the main clusters of neurons in the secondary taste cortex. The numbers of the cells included in each cluster are annotated. (From Rolls et al., 1990.)

amygdala and contains some taste-responsive neurons (Williams et al., 1993). Some 19% of the neurons in globus pallidus are reported to respond to taste stimulation, while 16% are sensitive to olfactory stimulation (Karádi et al., 1995), but their response characteristics are not yet well defined. I.

Protein Taste

An important food taste that appears to be different from that produced by sweet, salt, bitter, or sour is the taste of protein. At least part of this taste is captured by the

Japanese word umami, which is a taste common to a diversity of food sources including fish, meats, mushrooms, cheese, and some vegetables. Within these food sources, glutamates and 5-nucleotides—sometimes in a synergistic combination—create the umami taste (Ikeda, 1909; Kawamura and Kare, 1987; Yamaguchi, 1967; Yamaguchi and Kimizuka, 1979). Monosodium L-glutamate (MSG), guanosine-5-monophosphate (GMP), and inosine-5-monophosphate (IMP) are examples of umami stimuli. These findings raise the question of whether umami taste operates through channels in the primate taste system

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which are separable from those for the “prototypical” tastes sweet, salt, bitter, and sour. (Although the concept of four prototypical tastes has been used by tradition, there is increasing discussion about the utility of the concept, and increasing evidence that the taste system is more diverse than this; see, e.g., Kawamura and Kare, 1987.) To investigate the neural encoding of glutamate in the primate, Baylis and Rolls (1991) made recordings from 190 tasteresponsive neurons in the primary taste cortex and adjoining orbitofrontal cortex taste area in macaques. Single neurons were found that were tuned to respond best to monosodium glutamate (umami taste), just as other cells were found with best responses to glucose (sweet), sodium chloride (salty), HCl (sour), and quinine HCl (bitter). Across the population of neurons, the responsiveness to glutamate was poorly correlated with the responsiveness to NaCl, so that the representation of glutamate was clearly different from that of NaCl. Further, the representation of glutamate was shown to be approximately as different from each of the other four tastants as they are from each other, as shown by multidimensional scaling and cluster analysis. Moreover, it was found that glutamate is approximately as well represented in terms of mean evoked neural activity and the number of cells with best responses to it as the other four stimuli, glucose, NaCl, HCl, and quinine. It was concluded that in primate taste cortical areas, glutamate, which produces umami taste in humans, is approximately as well represented as are the tastes produced by glucose (sweet), NaCl (salty), HCl (sour), and quinine HCl (bitter) (Baylis and Rolls, 1991). In a further investigation, these findings have been extended beyond the sodium salt of glutamate to other umami tastants that have the glutamate ion but do not introduce the sodium ion into the experiment and to a nucleotide umami tastant (Rolls et al., 1994). In recordings made mainly from neurons in the orbitofrontal cortex taste area, it was shown that single neurons that had their best responses to sodium glutamate also had good responses to glutamic acid. The correlation between the responses to these two tastants was higher than between any other pair, which included in addition a prototypical set including glucose, sodium chloride, HCl, and quinine HCl. Moreover, the responsiveness to glutamic acid clustered with the response to monosodium glutamate in a cluster analysis with this set of stimuli, and glutamic acid was close to sodium glutamate in a space created by multidimensional scaling. It was also shown that the responses of these neurons to the nucleotide umami tastant inosine5-monophosphate were more correlated with their responses to monosodium glutamate than to any prototypical tastant (Rolls et al., 1994). A psychophysical convergence was also found between umami taste and

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corresponding odors, in that humans rated the intensity of umami flavor higher if the taste of glutamate was combined with the odor of methyl furyl disulfide (garlic) (Rolls, 2000c; Rolls et al., 1998). Thus, neurophysiological evidence in primates does indicate that there is a representation of umami flavor in the cortical areas which is separable from that to the four prototypical taste qualities sweet, salt, bitter, and sour (see also Rolls, 2000c; Rolls et al., 1998). This representation is probably important in the taste produced by proteins. These findings of cortical neurons tuned to respond to protein (umami) taste are supported by the more recent discovery of glutamate taste receptors on the tongue (Chaudhari et al., 2000). In addition, to the extent that certain amino acids taste sweet, salty, or sour, they evoke neuronal activity in the primary taste cortex, which is similar to that evoked by glucose, NaCl, or HCl, respectively (Plata-Salaman et al., 1992).

III. EFFECTS OF HUNGER AND SATIETY ON TASTE PROCESSING AT DIFFERENT STAGES OF THE TASTE PATHWAY Humans report that the hedonic value of tastes declines with satiety (Cabanac 1971), but that the intensity of the taste shows little if any decrease (Cabanac, 1971; Rolls and Rolls, 1977, 1982; Rolls et al., 1981a,b, 1982; Thompson et al., 1976). These findings show that pleasantness of the taste of food decreases to zero when the food is eaten to satiety, but that we can still identify the food, and taste it perfectly well, when we are satiated. In the primate brain, the evidence described below indicates that these two processes are clearly separated in gustatory information processing, with the nature of the taste (which taste it is, and its intensity) represented in the primary taste cortex, and the pleasantness of taste represented only after this processing, in the secondary taste cortex in the orbitofrontal cortex, in the lateral hypothalamus, and to some extent in the amygdala. In contrast, in rodents the separation between the identity of a taste and its hedonic value seems much less distinct, with modulation of taste processing by hunger early on in the taste pathways. It is important to examine the effects of satiety on taste processing, because alteration of the reward value of taste by hunger versus satiety is one of the main ways in which food intake is controlled (Rolls, 1975, 1999). A.

Rodents

Physical and chemical signals of satiety have been shown to have impact on taste responsiveness in the hindbrain of the rat. Gastric distension by air or with 0.3 M NaCl sup-

Central Taste Anatomy and Neurophysiology

presses responses in the NTS, with the greatest effect on glucose (Glenn and Erickson, 1976). Intravenous infusions of 0.5 g/kg glucose (Giza and Scott, 1983), 0.5 U/kg insulin (Giza and Scott, 1987a), and 40 mg/kg glucagon (Giza et al., 1993) all cause reductions in taste responsiveness to glucose in the NTS. The intraduodenal infusion of lipids cause a decline in taste responsiveness in the PBN, with the bulk of the suppression borne by glucose cells (Hajnal et al., 1999). The loss of signal that would otherwise be evoked by hedonically positive tastes implies that the pleasure that sustains feeding is reduced, making termination of a meal more likely (Giza et al., 1992). Conversely, the intracerebroventricular administration of neuropeptide Y—a peptide that induces feeding—causes an increase in appetitive behavioral reflexes to sucrose (Baird et al., 2000). If taste activity in NTS is affected by the rat’s nutritional state, then intensity judgments in rats should change with satiety. There is evidence that they do. Rats with conditioned aversions to 1.0 M glucose show decreasing acceptance of glucose solutions as their concentrations approach 1.0 M. This acceptance gradient can be compared between euglycemic rats and those made hyperglycemic through intravenous injections (Scott and Giza, 1987). Hyperglycemic rats showed greater acceptance at all concentrations from 0.6 to 2.0 M glucose, indicating that they perceived these stimuli to be less intense than did conditioned rats with no glucose load (Giza and Scott, 1987b). B.

Primates

In order to provide evidence as to where hunger controls taste processing in primates, the responses of single neurons at different stages of the taste system have been analyzed while macaques are fed to satiety, usually with a 20% glucose solution. To ensure that the results were relevant to the normal control of feeding (and were not due, for example, to abnormally high levels of artificially administered putative satiety signals such as gastric distension or plasma glucose), Rolls and colleagues allowed the monkeys to feed until they were satiated and determined whether this normal and physiological induction of satiety influenced the responsiveness of neurons in the taste system, which were recorded throughout the feeding until satiety was reached. The recordings were made in the monkey in order to make the results as relevant as possible to our understanding of sensory processing and the control of feeding, and its disorders, in humans. It was found that this modulation of taste-evoked signals by motivation is not a property found in early stages of the primate gustatory system. The responsiveness of taste neurons in the nucleus of the solitary tract is not

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attenuated by feeding to satiety (Yaxley et al., 1985). Thus, taste processing at this early stage of the taste system does not appear to be modulated by satiety, the signals for which include gastric distension (Gibbs et al., 1981) as well as other signals (see Rolls, 1999). It was also found that in the primary taste cortex, both in the frontal opercular part (Rolls et al., 1988) and in the insular part (Yaxley et al., 1988), hunger does not modulate the responsiveness of single neurons to gustatory stimuli. In contrast, in the secondary taste cortex, in the caudolateral part of the orbitofrontal cortex, it was found that the responses of neurons to the taste of glucose decreased to zero while the monkey ate it to satiety, during the course of which its behavior turned from avid acceptance to active rejection (Rolls et al., 1989). This modulation of responsiveness of the gustatory responses of the orbitofrontal cortex neurons by satiety could not have been due to peripheral adaptation in the gustatory system or to altered efficacy of gustatory stimulation after satiety was reached, because modulation of neuronal responsiveness by satiety was not seen at the earlier stages of the gustatory system, including the nucleus of the solitary tract, the frontal opercular taste cortex, and the insular taste cortex. Evidence was obtained that gustatory processing involved in thirst also becomes interfaced to motivation in the caudolateral orbitofrontal cortex taste projection area, in that neuronal responses here to water were decreased to zero while water was drunk until satiety was produced (Rolls et al., 1989). In the secondary taste cortex, it was also found that the decreases in the responsiveness of the neurons were relatively specific to the food with which the monkey had been fed to satiety. For example, in 7 experiments in which the monkey was fed glucose solution, neuronal responsiveness decreased to the taste of the glucose but not to the taste of black currant juice (see example in Fig. 8). Conversely, in two experiments in which the monkey was fed to satiety with fruit juice, the responses of the neurons decreased to fruit juice but not to glucose (Rolls et al., 1989). This evidence shows that the reduced acceptance of food that occurs when food is eaten to satiety and the reduction in the pleasantness of its taste (Cabanac, 1971; Rolls, 1986; Rolls and Rolls, 1977, 1982; Rolls et al., 1981a,b, 1982, 1983) are not produced by a reduction in the responses of neurons in the nucleus of the solitary tract or frontal opercular or insular gustatory cortices to gustatory stimuli. Indeed, after feeding to satiety, humans reported that the taste of the food on which they had been satiated tasted almost as intense as when they were hungry, though much less pleasant (Rolls et al., 1983). This comparison is consistent with the possibility that activity in the frontal opercular and insular (primary) taste cortices, as well as in the nucleus of the solitary tract, does not reflect

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by satiety, and it is presumably in areas such as these that neuronal activity may be related to whether a food tastes pleasant and whether the food should be eaten. In the amygdala, the impact of feeding on taste cells is intermediate (Yan and Scott, 1996). Some neurons are fully suppressed by feeding the monkey to satiety, whereas others are unmodified. The implication is that reward value is reflected in the activity of amygdaloid neurons, but that these responses are less responsive than those in the OFC to a rapidly changing condition which affects taste reward such as a glucose meal. The results above help clarify the neural mechanisms that underlie sensory-specific satiety. Apparently, this phenomenon cannot be attributed to receptor adaptation, nor to reduced activity in peripheral nerves or in the central gustatory system through the level of primary taste cortex. Rather, it rests with higher-order cortical processes of the OFC and beyond. Gustatory information in primates is kept distinct from its reward value until it reaches OFC. Consistent with this, OFC lesions in primates alter food preferences (Baylis and Gaffan, 1990). Not only do OFC neurons in primates represent the primary reward or reinforcing value of taste stimuli, but they are also involved in learning and correcting associations of visual and olfactory with taste reward and punishment stimuli (Rolls, 1999; Rolls et al., 1996a; Thorpe et al., 1983).

IV. MULTIMODAL REPRESENTATIONS IN THE TASTE SYSTEM

Figure 8 The effect of feeding to satiety with glucose solution on the responses of two neurons in the secondary taste cortex to the taste of glucose and of black currant juice (BJ). The spontaneous firing rate is also indicated (SA). Below the neuronal response data for each experiment, the behavioral measure of the acceptance or rejection of the solution on a scale from 2 to 2 is shown. The solution used to feed to satiety was 20% glucose. The monkey was fed 50 mL of the solution at each stage of the experiment as indicated along the abscissa until he was satiated as shown by whether he accepted or rejected the solution. Pre: The firing rate of the neuron before the satiety experiment started. The values shown are the mean firing rate and its SE. (From Rolls et al., 1989.)

the pleasantness of the taste of a food, but rather its sensory qualities independently of motivational state. On the other hand, the responses of the neurons in the caudolateral orbitofrontal cortex taste area and in the lateral hypothalamus (Burton et al., 1976; Rolls et al., 1986) are modulated

At some stage in taste processing, it is likely that taste representations are brought together with inputs from different modalities, for example, with olfactory inputs to form a representation of flavor. Takagi and colleagues (Tanabe et al., 1975a,b) have found an olfactory area in the medial orbitofrontal cortex. In a mid-mediolateral part of the caudal orbitofrontal cortex is the area investigated by Thorpe et al. (1983), in which are found many neurons with visual and some with gustatory responses. Recordings in the caudolateral orbitofrontal cortex taste area were different from those in the frontal opercular and insular primary taste cortices, in that there were neurons with responses in other modalities within or very close to the caudolateral orbitofrontal taste cortex (Rolls et al., 1990). It was therefore investigated systematically whether there are neurons in the secondary taste cortex and adjoining more medial orbitofrontal cortex that respond to stimuli from other modalities, including the olfactory and visual modalities, and whether single neurons in this cortical region in some cases respond to stimuli from more than one modality. It was noted that anatomically olfactory

Central Taste Anatomy and Neurophysiology

inputs reach this orbitofrontal cortex region in macaques from the pyriform (primary olfactory) cortex, which projects to area 13a of the medial orbitofrontal cortex, which in turn has onward projections to the lateral orbitofrontal cortex area, which has been shown to be the secondary taste cortex, and to more rostral parts of the orbitofrontal cortex (area 11) (see Fig. 2) (Carmichael et al., 1994; Price, 2001; Price et al., 1991). A. Olfactory and Visual Representations in the Orbitofrontal Cortex Influenced by Taste In this investigation of the orbitofrontal cortex taste areas (Rolls and Baylis, 1994), it was found that of 112 single neurons that responded to any of these modalities, many were unimodal (taste 34%, olfactory 13%, visual 21%) but were found in close proximity to each other. Some single neurons showed convergence, responding for example to taste and visual inputs (13%), taste and olfactory inputs (13%), and olfactory and visual inputs (5%). Some of these multimodal single neurons had corresponding sensitivities in the two modalities. Thus, one set responded best to sweet tastes (e.g., 1 M glucose) and responded more in a visual discrimination task to the visual stimulus that signified sweet fruit juice than to the one that signified saline. Another set responded to sweet taste and responded in an olfactory discrimination task to fruit odor. An example of one such bimodal neuron is shown in Figure 9. The neuron

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responded best among the tastants to NaCl (N), best among the odors to onion odor (On), and well also to salmon (S). The olfactory input to these neurons was further defined by measuring their responses while the monkey performed an olfactory discrimination task. In the task, if one odor was delivered through a tube close to the nose, then the monkey could lick to obtain fruit juice (reward trials). If a different odor was delivered, the monkey had to avoid licking, otherwise he obtained saline (saline trials). The responses of neurons in this task showed that they could have relatively short latency (e.g., 150 msec) responses to the odor stimuli and did not respond in a visual discrimination task, so that their responses were specifically related to the olfactory stimuli, and not just to performance of a task (Rolls and Baylis, 1994). The different types of neurons (unimodal in different modalities and multimodal) were frequently found close to one another in tracks made into this region (see Fig. 10), consistent with the hypothesis that the multimodal representations are actually being formed from unimodal inputs to this region. To investigate the rules that underlie the formation of these multimodal representations, analyses were performed on responses of these neurons in an olfactory discrimination task, in which one set of odors delivered from an olfactometer were associated if the correct lick response was made with the delivery of glucose taste reward and another set of odors was associated with the delivery of

Figure 9 The responses of a bimodal neuron recorded in the caudolateral orbitofrontal cortex. G, 1.0 M glucose; N, 0.1 M NaCl; H, 0.01 M HCl; Q, 0.001 M quinine HCl; M, 0.1 M monosodium glutamate; Bj, 20% black currant juice; Tom, tomato juice; B, banana odor; Cl, clove oil odor; On, onion odor; Or, orange odor; S, salmon odor; C, control no-odor presentation. The mean responses  SE are shown. The neuron responded best to the tastes of NaCl and monosodium glutamate and to the odors of onion and salmon. (From Rolls and Baylis, 1994.)

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Figure 10 Examples of tracks made into the orbitofrontal cortex in which taste (T) and olfactory (O) neurons were recorded close to each other in the same tracks. Some of the neurons were bimodal (T/O). (From Rolls and Baylis, 1994.)

saline if the monkey incorrectly licked to that set of stimuli. It was found that 25% of the neurons responded to the taste reward–associated odors more than to the saline punishment associated odors; that 35% responded to the saline punishment–associated odors more than to the reward taste–associated odors; and that 40% of the orbitofrontal cortex olfactory neurons did not categorize the odors based on the taste with which they were associated (Critchley and Rolls, 1996a). In a further test of how the taste with which an odor is associated influences the representation of odor in the primate orbitofrontal cortex, associated was reversed in the taste with which an odor was associated in a Go/NoGo olfactory discrimination task. Rolls et al. (1996a) found that 68% of orbitofrontal cortex odor-responsive neurons modified their responses in some way following changes in the taste reward associations of the odorants during olfactory-taste discrimination reversals.* Full reversal of the neuronal responses was seen in 25% of the neurons analyzed. In other words, in these cases the odor to which

Rolls and Scott

the neuron responded reversed when the taste with which it was associated reversed. Data from one such experiment performed on a single neuron in the macaque orbitofrontal cortex are shown in Figure 11. Extinction of the differential neuronal responses after task reversal was seen in 43% of these neurons; i.e., these neurons simply stopped discriminating between the two odors after the reversal. These findings demonstrate directly a coding principle in primate olfaction whereby the responses of some orbitofrontal cortex olfactory neurons are modified by and depend upon the taste with which the odor is associated. It was of interest that this modification was less complete, and much slower, than the modifications found for orbitofrontal visual neurons during visual-taste reversal (Rolls et al., 1996a). This relative inflexibility of olfactory responses is consistent with the need for some stability in odor-taste associations to facilitate the formation and perception of flavors. In addition, some orbitofrontal cortex olfactory neurons did not code in relation to the taste with which the odor was associated (Critchley and Rolls, 1996a), so that there is also a taste-independent representation of odor in this region. Many of the neurons with visual responses in this region also show dependency of the visual response on the taste with which the visual stimulus is associated. For example, many of the visually responsive neurons in the orbitofrontal cortex responded only to a visual stimulus associated with a rewarding taste (e.g., glucose as compared to saline); if the experimenter altered the taste with which the visual stimulus was linked (i.e., reversal of the visual discrimination task), these visual neurons typically altered the visual stimulus to which they responded, so that their visual responses remained associated with a given taste. This part of the orbitofrontal cortex thus seems to implement a mechanism that can alter flexibly the responses to visual stimuli depending on the reinforcement (e.g., the taste) associated with the visual stimulus (see Thorpe et al., 1983). This enables prediction of the taste associated with ingestion of what is seen, and thus in the visual selection of foods (see Rolls, 1986, 1999). This cor-

*In an olfactory discrimination experiment, if the monkey makes a lick response when one odor, the S, is delivered, he obtains a drop of glucose reward; if the monkey incorrectly makes a lick response to another odor, the S, he obtains a drop of aversive saline. At some time in the experiment, the contingency between the odor and the taste is reversed. The monkey relearns the discrimination, showing reversal. It is of interest to investigate in which parts of the olfactory system the neurons show reversal, for where they do, it can be concluded that the neuronal response to the odor depends on the taste with which it is associated.

Central Taste Anatomy and Neurophysiology

695 Figure 11 The activity of a single orbitofrontal olfactory neuron during the performance of a two odor olfactory discrimination task and its reversal. Before reversal a lick to the odor of amyl acetate produced a taste of salt, and after the reversal a taste of glucose. Before reversal a lick to the odor of cineole produced a taste of glucose, and after the reversal a taste of salt. Each point represents the mean poststimulus activity of the neuron to approximately 10 trials of the different odorants. The standard errors of these responses are shown. The odorants were amyl acetate (initially S) and cineole (initially S). After 80 trials of the task, the taste reward associations of the stimuli were reversed. This neuron reversed its responses to the odorants following the task reversal, showing that the neuron learned to respond with a greater increase of firing rate to whichever odor was associated with salt taste. (From Rolls et al., 1996.)

tical region is implicated more generally in a certain type of learning, namely in extinction and in the reversal of visual discriminations. It is suggested that the taste neurons in this region are important for these functions, for they provide information about whether a reward has been obtained (see Rolls, 1986, 1990, 1992a; Thorpe et al., 1983). The ability of this part of the cortex to perform rapid learning of associations between visual stimuli and primary reinforcers such as taste provides the basis for the importance of this part of the brain in food-related and emotion-related learning (see Rolls, 1990, 1992a, 1994, 1999). Consistent with this function, parts of the orbitofrontal cortex receive direct projections from the inferior temporal visual cortex ( Barbas, 1988; Seltzer and Pandya, 1989), a region important in high-order visual information processing (Rolls, 1992b, 1999, 2000d; Rolls and Treves, 1998). These results show that there are regions in the orbitofrontal cortex of primates where the sensory modalities of taste, vision, and olfaction converge; in many cases the neurons have corresponding sensitivities across modalities. It appears to be in these areas that flavor representations are built, where flavor is taken to mean a representation best evoked by a combination of gustatory and olfactory input. This orbitofrontal region does appear to be an important region for convergence, for there is only a low proportion of bimodal taste and olfactory neurons in the primary taste cortex (Rolls and Baylis, 1994).

The interactions between olfactory, visual, and taste stimuli found in the orbitofrontal cortex may underlie the interactions found in psychophysical experiments [such as the finding that the rated flavor intensity of umami (protein) was enhanced by adding methyl furyl disulfide (garlic odor) to monosodium glutamate (Rolls, 2000c; Rolls et al., 1998)]. B. Olfactory, Visual, and Taste Sensory-Specific Satiety It has also been possible to investigate whether the olfactory representation in the orbitofrontal cortex is affected by hunger. In satiety experiments, Critchley and Rolls (1996b) showed that the responses of some olfactory neurons to a food odor are decreased when the monkey is fed to satiety with a food (e.g., fruit juice) with that odor. In particular, seven of nine olfactory neurons that were responsive to the odors of foods, such as black currant juice, were found to decrease their responses to the odor of the satiating food. The decrease was typically at least partly specific to the odor of the food that had been eaten to satiety, potentially providing part of the basis for sensory-specific satiety. It was also found for eight of nine neurons that had selective responses to the sight of food that they demonstrated a sensory-specific reduction in their visual responses to foods following satiation. These findings show that the olfactory and visual representations of

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food, as well as the taste representation of food, in the primate orbitofrontal cortex are modulated by hunger. Usually a component related to sensory-specific satiety can be demonstrated. These findings link at least part of the processing of olfactory and visual information in this brain region to the control of feeding-related behavior. This is further evidence that part of the olfactory representation in this region is related to the hedonic value of the olfactory stimulus and, in particular, that at this level of the olfactory system in primates, the pleasure elicited by the food odor is at least part of what is represented. As a result of the neurophysiological and behavioral observations showing the specificity of satiety in the monkey, experiments were performed to determine whether satiety was specific to foods eaten in humans. It was found that the pleasantness of the taste of food eaten to satiety decreased more than for foods that had not been eaten in the meal (Rolls et al., 1981). One consequence of this is that if one food is eaten to satiety, appetite reduction for other foods is often incomplete, which will lead to enhanced eating when a variety of foods is offered (Rolls et al., 1981a,b, 1984). Because sensory factors such as similarity of color, shape, flavor, and texture are usually more important than metabolic equivalence in terms of protein, carbohydrate, and fat content in influencing how foods interact in this type of satiety, it has been termed “sensory-specific satiety” (Rolls, 1990a; Rolls and Rolls, 1977, 1982; Rolls et al., 1981a,b, 1982). It should be noted that this effect is distinct from alliesthesia, in that alliesthesia is a change in the pleasantness of sensory inputs produced by internal signals (such as glucose in the gut) (see Cabanac, 1971; Cabanac and Duclaux, 1970; Cabanac and Fantino, 1977), whereas sensory-specific satiety is a change in the pleasantness of sensory inputs, which is accounted for at least partly by the external sensory stimulation received (such as the taste of a particular food), in that, as shown above, it is at least partly specific to the external sensory stimulation received. The parallel between these studies of feeding in humans and of the neurophysiology of hypothalamic and orbitofrontal cortex neurons in the monkey has been extended by the observations that in humans, sensoryspecific satiety occurs for the sight as well as for the taste of food (Rolls et al., 1982). Further, to complement the finding that in the hypothalamus neurons are found that respond differently to food and to water (see Rolls, 1999) and that satiety with water can decrease the responsiveness of hypothalamic neurons that respond to water, it has been shown that in humans motivation-specific satiety can also be detected. For example, satiety with water decreases the pleasantness of the sight and taste of water but not of food (Rolls et al., 1983).

Rolls and Scott

To investigate whether the sensory-specific reduction in the responsiveness of the orbitofrontal olfactory neurons might be related to a sensory-specific reduction in the pleasure produced by the odor of a food when it is eaten to satiety, Rolls and Rolls (1997) measured human responses to the smell of a food eaten to satiety. It was found that the pleasantness of the odor of a food, but much less significantly its intensity, was decreased when the subjects ate it to satiety. It was also found that the pleasantness of the smell of other foods (i.e., foods not eaten in the meal) showed much less decrease. This finding has clear implications for (1) the control of food intake, (2) ways to keep foods presented in a meal appetizing, and (3) effects on odorpleasantness ratings that could occur following meals. In an investigation of the mechanisms of this odor-specific sensory-specific satiety, Rolls and Rolls (1997) allowed humans to chew a food, without swallowing, for approximately as long as the food is normally in the mouth during eating. They demonstrated a sensory-specific satiety with this procedure, showing that the sensory-specific satiety does not depend on food reaching the stomach. Thus, at least part of the mechanism is likely to be produced by a change in processing in the olfactory pathways. The earliest stage of olfactory processing at which this modulation occurs is not yet known. It is unlikely to be in the receptors, because the change in pleasantness found was much more significant than the change in the intensity (Rolls and Rolls, 1997). When a variety of foods is available, the enhanced eating due to the operation of sensory-specific satiety may have been advantageous in evolution by ensuring that different foods with important different nutrients were consumed. However, for humans today, when a wide variety of foods is readily available, this factor may lead to overeating and obesity. In a test in the rat, it has been found that variety itself can lead to obesity (Rolls et al., 1983; see also Rolls and Hetherington, 1989). C. Taste and Texture, Including Astringency and the Texture of Fat 1.

Texture

The orbitofrontal cortex of primates is also important as an area of convergence for somatosensory inputs, related, for example, to the texture of food, including that of fat in the mouth. It has been shown in recent recordings that single neurons influenced by taste in this region can in some cases have their responses modulated by the texture of the food. This was shown in experiments in which the texture of food was manipulated by the addition of methyl cellulose or gelatine or by puréeing a semi-solid food (Rolls, 1997, 1999; Rolls and Critchley, unpublished). The somatosensory inputs may reach this region via the rostral insula does pro-

Central Taste Anatomy and Neurophysiology

ject into this region, and which Mesulam and Mufson (1982a,b; Mufson and Mesulam, 1982) have shown does receive somatosensory inputs. 2.

Fat

Texture in the mouth is an important indicator of whether fat is present in a food, which is important not only as a high-value energy source, but also as a potential source of essential fatty acids. In the orbitofrontal cortex, Rolls et al. (1999) found a population of neurons that responds when fat is in the mouth. An example of such a neuron is shown in Figure 12. The neuron illustrates that information about fat, as well as taste, can converge onto the same neuron in this region. The neuron responded to taste, in that its firing rate was significantly different within the group of tastants sweet, salt, bitter, and sour. However, its response to fat in the mouth was larger. The fat-related responses of these neurons are produced, at least in part, by the texture of the food rather than by receptors sensitive to certain chemicals, in that such neurons typically respond not only to foods such as cream and milk containing fat, but also to paraffin oil (which is a

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pure hydrocarbon) and to silicone oil [Si(CH3)2O)n]. Some of the fat-related neurons do have convergent inputs from the chemical senses, in that in addition to taste inputs, some of these neurons respond to the odor associated with a fat, such as the odor of cream (Rolls et al., 1999). Feeding to satiety with fat (e.g., cream) decreases the responses of these neurons to zero on the food eaten to satiety. This is a sensoryspecific texture-dependent effect in that if the neuron receives a taste input from, for example, glucose taste, then the response of the neuron to glucose is not decreased by feeding to satiety with cream. Thus, there is a representation of the macronutrient fat in this brain area, and the activation produced by fat is reduced by eating fat to satiety. The perception of lipids is a function of both taste and texture. Given that the taste system is crucial for the identification of nutrients, it is curious that it appeared for many years to take no part in the perception of lipids, the most calorically rich of nutrients. Recently, however a role of taste in lipid detection has been identified in rats (Herness and Gilbertson, 1999). Lingual lipase reduces triglycerides to mono- and diglycerides and free fatty acids, which are transported to the receptors by proteins

Figure 12 A neuron in the primate orbitofrontal cortex responding to the texture of fat in the mouth. The cell (Be047) increased its firing rate to cream (double and single cream), and responded to texture rather than the chemical structure of the fat in that it also responded to 0.5 ml of silicone oil (Si(CH3)2O)n) or paraffin oil (hydrocarbon). The cell has a taste input too, in that it had a consistent but small response to umami taste (monosodium glutamate, MSG). Gluc, glucose; NaCl, salt; HCl, sour; Q-HCL, quinine, bitter. The spontaneous firing rate of the cell is also shown. (After Rolls et al., 1999.)

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produced by von Ebner’s glands (Kock et al., 1992). Free fatty acids have been shown to inhibit delayed-rectifying potassium channels in taste cells (Liu et al., 1998), resulting in depolarization, increased intracellular calcium, and a presumed release of neurotransmitter. The channels are sensitive only to essential fatty acids, reinforcing their role in the detection of nutrients. However, this system is unlikely to be involved in fat detection in humans, both because of the low concentration of lingual lipase in humans (Herness and Gilbertson, 1999) and because fat in the mouth can be detected very rapidly (1–2 sec), whereas the free fatty acid–detection system described has a time course of many seconds. 3. Astringency Another somatosensory input to the orbitofrontal cortex that combines with taste inputs is produced by tannic acid, which produces the “taste” of astringency. Tannic acid is a member of the class of compounds known as polyphenols, which are present in a wide spectrum of plant matter, particularly in foliage, the skin and husks of fruit and nuts, and the bark of trees. The tannic acid in leaves is produced as a defense against insects. There is less tannic acid in young leaves than in old leaves. Large monkeys cannot obtain the whole of their protein intake from small animals, insects, etc., and thus obtain some of their protein from leaves. Tannic acid binds protein (hence its use in tanning) and amino acids, and thus prevents their absorption, rendering them indigestible. Thus it is adaptive for monkeys to be able to taste tannic acid, so that they can select food sources without too much tannic acid (Hladik, 1978 and personal communication). In humans, tannic acid elicits a characteristic astringent taste. Oral astringency is perceived as the feeling of longlasting puckering and drying sensations on the tongue and membranes of the oral cavity. High levels of tannic acid in some potential foods makes them unpalatable without preparative techniques to reduce its presence (Johns and Duquette, 1991), yet in small quantities tannic acid is commonly used to enhance the flavor of food. In this context tannic acid is a constituent of a large range of spices and condiments, such as ginger, chilies, and black pepper (Uma-Pradeep et al., 1993).* Tannic acid is a natural antioxidant by virtue of its chemical structure (see Critchley and Rolls, 1996c). In order to investigate whether astringency is represented in the cortical taste areas concerned with taste, *Tannic acid itself is not present in tea, yet a range of related polyphenol compounds are, particularly in green tea (Graham, 1992).

Rolls and Scott

Critchley and Rolls (1996c) recorded from taste-responsive neurons in the orbitofrontal cortex and adjacent insula. Single neurons were found that were tuned to respond to tannic acid (0.001 M) and represented a subpopulation of neurons that was distinct from neurons responsive to the tastes of glucose (sweet), NaCl (salty), HCl (sour), quinine (bitter), and monosodium glutamate (umami). In addition, across the population of taste-responsive neurons, tannic acid was as well represented as the tastes of NaCl, HCl, quinine, or monosodium glutamate. Multidimensional scaling analysis of the neuronal responses to the tastants indicated that tannic acid lies outside the boundaries of the four conventional taste qualities (sweet, sour, bitter, and salty) (Critchley and Rolls, 1996c). Taken together, these data indicate that the astringent taste of tannic acid should be considered as a distinct flavor quality, which receives a separate representation from sweet, salt, bitter, and sour in the primate cortical taste areas. Although the sensors for astringency are probably somatosensory rather that gustatory, single neurons in the primate orbitofrontal cortex activated by tannic acid were also typically activated by some taste inputs, so that convergence between the astringency and taste systems does occur, and astringency can contribute to the flavor of food.

V.

IMAGING STUDIES IN HUMANS

Human neuroimaging studies using function magnetic resonance imaging (fMRI) have shown that taste stimuli activate an area of the anterior insula, which is probably the primary taste cortex, and part of the orbitofrontal cortex, which is probably the secondary taste cortex (Francis et al., 1999; Small et al., 1999). The orbitofrontal cortex taste area is distinct from areas activated by odors and by pleasant touch (Francis et al., 1999). It has been shown that within individual subjects separate areas of the orbitofrontal cortex are activated by sweet (pleasant) and salt (unpleasant) tastes (O’Doherty et al., 2000b). Zald et al. (1998) found activation of the human amygdala by the aversive taste of salt. Francis et al. (1999) also found activation of the human amygdala by the taste of glucose. Extending this study, O’Doherty et al. (2000b) showed that the human amygdala was as much activated by the affectively pleasant taste of glucose as by the affectively negative taste of NaCl, and thus provided evidence that the human amygdala is not especially involved in processing aversive, as compared to rewarding, stimuli. It is of interest that, in humans, there is an area of the far anterior insula that is activated by olfactory stimuli (Francis et al., 1999; O’Doherty et al., 2000). It is not clear whether this area is separate from the region of the insula

Central Taste Anatomy and Neurophysiology

activated by taste. In macaques, the primary taste cortex (in the anterior insula and adjoining frontal operculum) does not appear to be strongly activated by olfactory stimuli (though further studies on this are in progress), and the human anterior insular olfactory area may thus correspond to what, in macaques, is the caudal transitional area of the orbitofrontal cortex where it adjoins the insula, area Ofdg, where part of the secondary taste cortex is located (Baylis et al., 1994). In humans, there is strong and consistent activation of the right orbitofrontal cortex by olfactory stimuli (Francis et al., 1999; Zatorre et al., 1992). In an investigation of where the pleasantness of olfactory stimuli might be represented in humans, O’Doherty et al. (2000) showed that the activation of an area of the orbitofrontal cortex to banana odor was decreased (relative to a control vanilla odor) after bananas were eaten to satiety. Thus activity in a part of the human orbitofrontal cortex olfactory area is related to sensory-specific satiety.

VI. THE NEURAL REPRESENTATION OF TASTE AND FLAVOR Gustatory neurons are broadly tuned, a fundamental characteristic that demands that the afferent code for taste quality be read across more than one neuron type. Such a distributed or ensemble code offers advantages of redundancy, precision, and high information-carrying capacity [discussed in Chapter 35 and by Rolls and Treves (1998)]. In addition, a distributed representation offers an advantage in forming associative memories, where associations must be learned between stimuli in different modalities, using a Hebbian associative synaptic modification rule. Such an ensemble-encoded representation allows many associations to be stored, yet enables the important properties of generalization, and of graceful degradation upon sustaining damage, to be expressed (Rolls and Treves, 1998). Gustatory neurons in the primate remain rather broadly tuned through to the level of the primary taste cortex (see Rolls, 1997; Scott and Plata-Salamán, 1999). The highly distributed nature of the representation here may be appropriate for a representation of the identity of the taste stimulus, that is, exactly how sweet, bitter, salt, sour, or umami-tasting it is, together with information about its intensity. Consistent with this, hunger and sensory-specific satiety do not affect the firing to tastants of neurons in the primate primary taste cortex (see Sec. III. B), so the representation can be about the identity and strength of the tastant. In the secondary taste cortex in the OFC, the neurons become more selective to taste quality, but are also likely to be multimodal, extending their sensitivity

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beyond taste to olfactory, textural, thermal, and visual components of the stimulus, so that in the secondary taste cortex of primates flavor representations are built. The responses of any neuron are usually kept in register across the modalities by associative learning. In particular, olfactory stimuli and visual stimuli become secondary or learned reinforcers because of their learned association in the primate orbitofrontal cortex with the primary reinforcer of taste or texture (see Secs. IV. A and IV. C). Because neurons respond to combinations of inputs received through these different modalities, their tuning can become quite selective. In addition, in the primate secondary taste cortex, the reward or hedonic value of the taste (and flavor) of food is represented, in that the responses of neurons here decrease after the monkey has fed to satiety. This decrease is in part specific to the particular food eaten, and this sensory-specific satiety is implemented in the orbitofrontal cortex. Sensory-specific satiety may be implemented by a process of synaptic habituation in the orbitofrontal cortex (but at no earlier stage of processing), which has a time course of the 5–10 minutes that it may take to eat a food to satiety. The effect of this functional architecture may be to allow in primates the hedonic or reward value of a food to decrease with satiety, while at the same time an assessment of the identity and intensity of the taste, performed in the primary taste cortex, would be largely unmodified. By this architecture, stimulus identification remains distinct from hedonics in primates, a phenomenon suggested by the results of psychophysical studies in humans. The evidence from rodents suggests that early analyses of the taste input at the hindbrain level control reflexes for acceptance or rejection and autonomic reflexes that anticipate digestion (nucleus of the solitary tract). These early analyses also enable the associative processes involved in conditioning (parabrachial nucleus). The thalamic relay may serve gustatory memories that do not arise from taste-gut interactions. The functional architecture of the rodent taste system may be rather different from that of primates, in that rodents do not have obligatory routing of taste information from the nucleus of the solitary tract to the thalamus and this to the cortex, but have additional connections from the nucleus of the solitary tract to the parabrachial nucleus, which in turn has connections with many subcortical structures. Further, and probably related to this difference in the anatomy, the effects of satiety on taste seem to be implemented at least in part in lower order parts of the taste system of rodents such as the nucleus of the solitary tract, and consistent with this, the identity and intensity of taste in rodents do not appear to be kept very distinct from the hedonic value of the taste.

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703 Rolls, E. T. (1999). The Brain and Emotion. Oxford University Press, Oxford. Rolls, E. T. (2000a). The orbitofrontal cortex and reward. Cerebral Cortex 10:284–294. Rolls, E. T. (2000b). Taste, olfactory, visual and somatosensory representations of the sensory properties of foods in the brain, and their relation to the control of food intake. In Neural and Metabolic Control of Macronutrient Intake, H. R. Berthoud and R. J. Seeley (Eds.). CRC Press, Boca-Raton, FL, pp. 247–262. Rolls, E. T. (2000c). The representation of umami taste in the taste cortex. J. Nutrition 130:9605–9655. Rolls, E. T. (2000d). Functions of the primate temporal lobe cortical visual areas in invariant visual object and face recognition. Neuron 27:1–20. Rolls, E. T. (2000e). Neurophysiology and functions of the primate amygdala, and the neural basis of emotion. In The Amygdala: A Functional Analysis, J. P. Aggleton (Ed.). Oxford University Press, Oxford, pp. 447–478. Rolls, E. T., and Baylis, L. L. (1994). Gustatory, olfactory and visual convergence within the primate orbitofrontal cortex. J. Neurosci. 14:5437–5452. Rolls, E. T., and Rolls, B. J. (1977). Activity of neurons in sensory, hypothalamic and motor areas during feeding in the monkey. In Food Intake and Chemical Senses, Y. Katsui, M. Sato, S. Takagi, and Y. Oomura (Eds.). University of Tokyo Press, Tokyo, pp. 525–549. Rolls, E. T., and Rolls, B. J. (1982). Brain mechanisms involved in feeding. In Psychobiology of Human Food Selection, L. M. Barker (Ed.). AVI Publishing Company, Westport, CT, pp. 33–62. Rolls, E. T., and Rolls, J. H. (1997). Olfactory sensory-specific satiety in humans. Physiol. Behav. 61:461–473. Rolls, E. T., and Treves, A. (1998). Neural Networks and Brain Function. Oxford University Press, Oxford. Rolls, E. T., Burton, M. J., and Mora, F. (1980). Neurophysiological analysis of brain-stimulation reward in the monkey. Brain Res. 194:339–357. Rolls, E. T., Rolls, B. J., and Rowe, E. A. (1983). Sensory-specific and motivation-specific satiety for the sight and taste of food and water in man. Physiol. Behav. 30:185–192. Rolls, E. T., Murzi, E., Yaxley, S., Thorpe, S. J., and Simpson, S. J. (1986). Sensory-specific satiety: food-specific reduction in responsiveness of ventral forebrain neurons after feeding in the monkey. Brain Res. 368:79–86. Rolls, E. T., Scott, T. R., Sienkiewicz, Z. J., and Yaxley, S. (1988). The responsiveness of neurones in the frontal opercular gustatory cortex of the macaque monkey is independent of hunger. J. Physiol. 397:1–12. Rolls, E. T., Sienkiewicz, Z. J., and Yaxley, S. (1989). Hunger modulates the responses to gustatory stimuli of single neurons in the orbitofrontal cortex. Eur. J. Neurosci. 1:53–60. Rolls, E. T., Yaxley, S., and Sienkiewicz, Z. J. (1990). Gustatory responses of single neurons in the orbitofrontal cortex of the macaque monkey. J. Neurophysiol. 64: 1055–1066.

704 Rolls, E. T., Critchley, H., Mason, R., and Wakeman, E. A. (1996a). Orbitofrontal cortex neurons: role in olfactory and visual association learning. J. Neurophysiol. 75:1970–1981. Rolls, E. T., Critchley, H., Mason, R., and Wakeman, E. A. (1996b). Responses of neurons in the primate taste cortex to the glutamate ion and to inosine 5-monophosphate. Physiol. Behav. 59:991–1000. Rolls, E. T., Critchley, H. D., Browning, A., and Hernadi, I. (1998). The neurophysiology of taste and olfaction in primates, and umami flavor. Ann. Acad. Sci. 855:426–437. Rolls, E. T., Critchley, H. D., Browning, A. S., Hernadi, A. and Lenard, L. (1999). Responses to the sensory properties of fat of neurons in the primate orbitofrontal cortex. J. Neurosci. 19:1532–1540. Roper, S. N., Lévesque, M. F., Sutherling, W. W., and Engle, J. Jr. (1993). Surgical treatment of partial epilepsy arising from the insular cortex. J. Neurosurg. 79:226–229. Sakai, N., and Yamamoto, T. (1998). Role of the medial and lateral parabrachial nucleus in acquisition and retention of conditioned taste aversion in rats. Behav. Brain Res. 93: 63–70. Sanghera, M. K., Rolls, E. T. and Roper-Hall, A. (1979). Visual responses of neurons in the dorsolateral amygdala of the alert monkey. Exp. Neurol. 63:610–626. Scalera, G., Grigson, P. S., and Norgren, R. (1997). Gustatory functions, sodium appetite, and conditioned taste aversion survive excitotoxic lesions of the thalamic taste area. Behav. Neurosci. 111:633–645. Schneider, R. J., Friedman, D. P., and Mishkin, M. (1993). A modality-specific somatosensory area within the insula of the rhesus monkey. Brain Res. 621:116–120. Scott, T. R., and Erickson, R. P. (1971). Synaptic processing of taste-quality information in the thalamus of the rat. J. Neurophysiol. 34:868–884. Scott, T. R., and Giza, B. K. (1987). A measure of taste intensity discrimination in the rat through conditioned taste aversions. Physiol. Behav. 41:315–320. Scott, T. R., and Perrotto, R. S. (1980). Intensity coding in the pontine taste area: gustatory information is processed similarly throughout rat’s brainstem. J. Neurophysiol. 44: 739–750. Scott, T. R., and Plata-Salamán, C. R. (1999). Taste in the monkey cortex. Physiol. Behav. 67:489–511. Scott, T. R., Yaxley, S., Sienkiewicz, Z. J., and Rolls, E. T. (1986a). Taste responses in the nucleus tractus solitarius of the behaving monkey. J. Neurophysiol. 55:182–200. Scott, T. R., Yaxley, S., Sienkiewicz, Z. J., and Rolls, E. T. (1986b). Gustatory responses in the frontal opercular cortex of the alert cynomolgus monkey. J. Neurophysiol. 56: 876–890. Scott T. R., Karadi Z., Oomura Y., Nishino, H., Plata-Salaman, C. R., Lenard, L., Giza, B. K., and Aou, S. (1993). Gustatory neural coding in the amygdala of the alert macaque monkey. J. Neurophysiol. 69:1810–1820. Seltzer, B., and Pandya, D. N. (1989). Frontal lobe connections of the superior temporal sulcus in the rhesus monkey. J. Comp. Neurol. 281:97–113.

Rolls and Scott Shimura, T., Grigson, P. S., and Norgren, R. (1997a). Brain stem lesion and gustatory function. 1. The role of the nucleus of the solitary tract during a brief intake test in rats. Behav. Neurosci. 111:155–168. Shimura, T., Tanaka, H., and Yamamoto, T. (1997b). Salient responsiveness of parabrachial neurons to the conditioned stimulus after the acquisition of taste aversion learning in rats. Neuroscience 81:239–247. Small, D. M., Zald, D. H., Jones-Gotman, M., Zatorre, R. J., Pardo, J. V., Frey, S. and Petrides, M. (1999). Human cortical gustatory areas: A review of functional neuroimaging data. NeuroReport 10:7–14. Steiner, J. E. (1979). Human facial expressions in response to taste and smell stimulation. In Advances in Child Development, H. W. Reese and L. Lipsett (Eds.). Academic Press, New York, pp. 57–295. Tanabe, T., Yarita, H., Iino, M. Ooshima, Y., and Takagi, S. F. (1975a). An olfactory projection area in orbitofrontal cortex of the monkey. J. Neurophysiol. 38:1269–1283. Tanabe, T., Iino, M., and Takagi, S. F. (1975b). Discrimination of odors in olfactory bulb, pyriform-amygdaloid areas, and orbitofrontal cortex of the monkey. J. Neurophysiol. 38:1284–1296. Thompson, D. R., Moskowitz, H. R., and Campbell, R. G. (1976). Effects of body weight and food intake on pleasantness for a sweet stimulus. J. Appl. Physiol. 41:77–83. Thorpe, S. J., Rolls, E. T. and Maddison, S. (1983). Neuronal activity in the orbitofrontal cortex of the behaving monkey. Exp. Brain Res. 49:93–115. Turner, B. H., Mishkin, M., and Knapp, M. (1980). Organization of the amygdalopetal projections from modality-specific cortical association areas in the monkey. J. Comp. Neurol. 191: 515. Uma-Pradeep, K., Geervani, P., and Eggum, B. O. (1993). Common Indian spices: nutrient composition, consumption and contribution to dietary value. Plant Foods Hum. Nutr. 44:138–148. Verhagen, J. V., Giza, B. K., and Scott, T. R. (1999). Taste in the rat thalamus. Neurosci. Abstr. 25:2184. Wallis, G., and Rolls, E. T. (1997). Invariant face and object recognition in the visual system. Prog. Neurobiol. 51:167–194. Weusten, B. L. A. M., Franssen, H., Wieneke, J. H., and Smout, A. J. P. M. (1994). Multichannel recording of cerebral potentials evoked by esophageal balloon distention in humans. Digest. Dis. Sci. 39:2074–2083. Wilkins, L., and Richter, C. P. (1940). A great craving for salt by a child with corticoadrenal insufficiency. J. Am. Med. Assoc. 114:866–868. Williams, G. V., Rolls, E. T., Leonard, C. M., and Stern, C. (1993). Neuronal responses in the ventral striatum of the behaving monkey. Behav. Brain Res. 55:243–252. Wilson, F. A. W., and Rolls, E. T. (1993). The effects of stimulus novelty and familiarity on neuronal activity in the amygdala of monkeys performing recognition memory tasks. Exp. Brain Res. 93:367–382. Wilson, F. A. W., and Rolls, E. T. (2003). The primate amygdala and reinforcement: a dissociation between rule-based and associatively-mediated memory revealed in amygdala neuronal activity. (In press.)

Central Taste Anatomy and Neurophysiology Wolf, G., and DiCara, L. V. (1974). Impairments in sodium appetite after lesions of gustatory thalamus—replication and extension. Behav. Biol. 10:106–112. Yamaguchi, S. (1967). The synergistic taste effect of monosodium glutamate and disodium 5’-inosinate. J. Food Sci. 32:473–478. Yamaguchi, S., and Kimizuka, A. (1979). Psychometric studies on the taste of monosodium glutamate. In Glutamic Acid: Advances in Biochemistry and Physiology, L. J. Filer, S. Garattini, M. R. Kare, A. R. Reynolds, and R. J. Wurtman (Eds.). Raven Press, New York, pp. 35–54. Yamamoto, T. (1993). Neural mechanisms of taste aversion learning. Neurosci. Res. 16:181–185. Yamamoto, T., Shimura, T., Sako, N., Yasoshima, Y., and Sakai, N. (1994). Neural substrates for conditioned taste aversion in the rat. Behav. Brain Res. 65:123–137. Yan, J., and Scott, T. R. (1996). The effect of satiety on responses of gustatory neurons in the amygdala of alert cynomolgus macaques. Brain Res. 740:193–200.

705 Yaxley, S., Rolls, E. T., Sienkiewicz, Z. J., and Scott, T. R. (1985). Satiety does not affect gustatory activity in the nucleus of the solitary tract of the alert monkey. Brain Res. 347: 85–93. Yaxley, S., Rolls, E. T., and Sienkiewicz, Z. J. (1988). The responsiveness of neurones in the insular gustatory cortex of the macaque monkey is independent of hunger. Physiol. Behav. 42:223–229. Yaxley, S., Rolls, E. T., and Sienkiewicz, Z. J. (1990). Gustatory responses of single neurons in the insula of the macaque monkey. J. Neurophysiol. 63:689–700. Zald, D. H., Lee, J. T., Fluegel, K. W., and Pardo, J. V. (1998). Aversive gustatory stimulation activates limbic circuits in humans. Brain 121:1143–1154. Zatorre, R. J., Jones-Gotman, M., Evans, A. C., and Meyer, E. (1992). Functional localization of human olfactory cortex. Nature 360:339–340.

34 Molecular Physiology of Gustatory Transduction Timothy A. Gilbertson Utah State University, Logan, Utah, U.S.A.

Robert F. Margolskee Howard Hughes Medical Institute and The Mount Sinai School of Medicine, New York, New York, U.S.A.

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code typically utilizing cyclic nucleotides (cNMPs) and inositol trisphosphate (IP3). These signaling cascades usually involve effector enzymes downstream of the G proteins to generate or regulate second messengers leading to TRC depolarization and Ca2 release. In the case of ionic stimuli (e.g., Na, K, H), the tastant itself constitutes all or part of the initial intracellular signal: these ions pass through or block apical ion channels leading to TRC depolarization and/or hyperpolarization. The great chemical and physical diversity of tastants has necessitated multiple transduction mechanisms (see Fig. 1), in contrast to visual and olfactory systems where multiple receptors in iterated but fundamentally similar transduction pathways transduce variants of one general type of stimulus (photons or small volatile molecules) into an intracellular signal. The vertebrate taste bud is a polarized ovoid structure containing between 50 and 150 TRCs. The TRCs are elongated neuroepithelial cells that stretch from the bottom of the taste bud to the top. TRCs make contact with the oral cavity through the bud’s apical taste pore. The apical surface of TRCs is rich in convoluted microvilli; it is here that receptors and channels involved in taste transduction are presumably concentrated. At the base of the taste bud are the basal cells that are thought to be precursors to mature TRCs (see Chapter 32).

INTRODUCTION

The aim of this chapter is to provide an overview of the molecular mechanisms involved in taste transduction. Human taste perception can be divided into five major categories: sweet, sour, salt, bitter, and umami (glutamate). Acids and sodium salts, which elicit sour and salt taste, respectively, are fluxed by and/or regulate the activities of ion channels expressed in taste receptor cells (TRCs). Sugars, artificial sweeteners, bitter plant alkaloids, and other bitter compounds primarily stimulate G protein–coupled receptors (GPCRs) to initiate one or more second messenger signaling cascades. Transduction of the taste of amino acids such as L-glutamate appears to depend on both GPCR cascades and stimulation of amino acid–gated ion channels. During the past 10 years major new insights in taste transduction have been gained from diverse studies utilizing electrophysiology, biochemistry, molecular cloning, and transgenic and gene knockout animal models. The sensation of taste is initiated by the interaction of sapid molecules (“tastants”) with receptors and ion channels in the apical microvilli of TRCs. TRCs use a variety of mechanisms to transduce chemical information into intracellular signals. Those taste transduction pathways utilizing G proteins and their coupled receptors convert chemical information into a cellular second messenger

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BITTER TRANSDUCTION MECHANISMS

A. Diversity of Bitter Compounds and Their Detection Mechanisms The human sense of bitter taste is thought to be an evolutionarily selected mechanism for the avoidance of poisons, particularly toxic plant alkaloids and glycosides,

which are detected by humans as bitter in the nanomolar to micromolar range. Other vertebrates also detect and avoid many of these same compounds in similar ranges, leading to the inference that the human sense of bitterness correlates with other vertebrate animals’ aversive responses to the same toxic compounds that humans characterize as bitter. Support for this idea comes from the identification in humans, rats, and mice of conserved

Figure 1 Proposed transduction mechanisms in vertebrate taste receptor cells. All taste pathways converge on the common elements of a rise in intracellular Ca2 followed by neurotransmitter (NT) release. Sodium salts depolarize taste cells directly through Na influx through amiloride-sensitive ENaC. Acids, in the form of protons, also permeate ENaC, activate proton-activated cation channels (MDEG and, perhaps, ASIC) and inhibit apical K channels. L-Glutamate (L-Glu), which is the stimulus eliciting umami taste, activates the T1r1/T1r3 heterodimer and the taste form of mGluR4, a GPCR linked to decreases in cAMP concentration via phosphodiesterase (PDE) activation. The decrease in cAMP may lead to decreased inhibition of cNMP-gated channels and a rise in intracellular Ca2. Other amino acids, like arginine (L-Arg), activate ionotropic receptors causing TRC depolarization. Artificial sweeteners activate ionotropoic receptors linked to cation channels and GPCR linked via phospholipase C (PLC) to IP3 production and release of Ca2 from intracellular stores. Natural sugars apparently activate GPCRs linked via adenylyl cyclase (AC) to cAMP production, which in turn may inhibit basolateral K channels through phospohorylation by cAMP-activated protein kinase A (PKA). Bitter compounds, such as denatonium and propylthiouracil (PROP), activate the GPCR T2R (TRB). Activated T2R stimulates production of PDE via the -gustducin and the concomitant decrease in cAMP. The G subunits of (3, 13) released upon activation of heterotrimeric gustducin by T2R lead to IP3 production via PLC2 and eventual rise in intracellular Ca2. Other bitter compounds, including quinine and divalent cations, have been demonstrated to inhibit apical K channels in some species. DAG, diacylglycerol; AP, action potentials. (Adapted from Gilbertson et al., 2000.)

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families of genes encoding apparent bitter-responsive receptors (Adler et al., 2000; Matsunami et al., 2000; Chandrashekar et al., 2000). Furthermore, and with some notable exceptions, detection thresholds for many bitter compounds are similar among humans and other vertebrates. On the whole, bitter agents comprise an incredibly diverse group of compounds (e.g., charged-hydrophilic denatonium; neutral-hydrophobic quinine; divalent cations Ca2 and Ba2; amino acids L-tryptophan and Lphenylalanine; peptides Ser-Leu-Ala and Phe-Phe-Phe; modified sugar and sweetener analogs MAD-diCl-Gal). This broad diversity is reflected in the multiple receptors and multiple transduction mechanisms used by taste cells. B. Genetic Studies Suggesting the Existence of Multiple Bitter Detection Mechanisms Inherent interindividual differences in the ability to taste certain bitter compounds have been demonstrated in genetic studies of humans and mice (for reviews see Miller and Bartoshuk, 1991). It was reported in 1932 that phenylthiocarbamide (PTC) tastes bitter to certain individuals, but is tasteless to others (Fox, 1932). Family studies indicated that insensitivity to PTC (PTC “taste blindness”) is inherited as a simple Mendelian recessive trait. PTC nontasters are also insensitive to many other bitter compounds, including 6-n-propyl-2-thiouracil (PROP). PTC/PROP tasters are more sensitive than nontasters to the bitter tastes of caffeine, KCl, saccharin, and benzoate. However, tasters and nontasters do not differ in their responses to other bitter compounds (e.g., quinine) (Hall et al., 1975; Bartoshuk, 1979). Interestingly, PTC/PROP tasters are also more sensitive than nontasters to the sweet tastes of saccharin, sucrose, and neohesperidin (Bartoshuk, 1979; Gent and Bartoshuk, 1983). The variety of compounds that tasters and nontasters differentially respond to suggests that the molecular basis for differences between these two populations is not simply due to a single taste receptor; rather, this difference may relate to coordinate expression of multiple genetically linked receptors or to a common site of action further along in the transduction cascade (e.g., a G-protein subunit, a second messenger–regulating enzyme, or an end target such as an ion channel). Polymorphisms in the ability to taste various bitter compounds have been identified and mapped in inbred strains of mice. Independent autosomal dominant loci were identified for strychnine and sucrose octaacetate (SOA) (the soa locus) (Lush, 1981); raffinose undecaacetate (rua) (Lush, 1986); quinine (qui) (Lush, 1984); copper glycinate (glb) (Lush and Holland, 1988; Lush et al.,

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1995); and cycloheximide (cyx) (Lush and Holland, 1988). Other data suggest that these are not all independent loci, but in some cases pleiotropic manifestations of the soa locus (Harder et al., 1992). The soa locus has been mapped to mouse chromosome 6 (Capeless et al., 1992). The murine genetic data argue for the existence of three or more unlinked loci that affect bitter taste: (1) soa, (2) PTC aversion, and (3) several loci that affect quinine aversion (Harder et al., 1992). SOA nontaster mice display little or no whole nerve response to SOA (although they respond normally to other bitter compounds), indicating that the defect is peripheral in nature (i.e., concerning the ability of the TRC to generate a depolarizing response to the tastant). In recent genomics-based studies (Adler et al., 2000; Matsunami et al., 2000), clusters of 40–80 candidate taste receptors were identified and mapped to mouse chromosomes 6 and 15 (see below). One of these GPCRs was shown to function as a cycloheximide (bitter) receptor and may correspond to the cyx locus; presumably the other candidate receptors among this large gene family may also be bitter-responsive and correspond to the rua, soa, glb, and qui loci, as well as to other as yet unidentified bitter response loci. C.

Receptor-Mediated Bitter Transduction Pathways

The membrane impermeant compound denatonium benzoate is for humans the most intensely bitter compound known (Saroli, 1984). In rat circumvallate (CV) papillae TRCs, denatonium caused the release of Ca 2 from internal stores (Akabas et al., 1988). Denatonium caused a rapid transient increase in IP3 levels within dissected CV papillae (Spielman et al., 1994), and by autoradiographic visualization it was shown that calcium was depleted from the papillae upon the addition of IP3 (Hwang et al., 1990) (as would be expected if IP3 caused the opening of Ca2 channels within taste cells). It had been thought previously that the denatonium-responsive receptor activated Gq or a Gq-like G protein (both of which have been identified in rodent TRCs (McLaughlin et al., 1992a,b; Kusakabe et al., 1998, 2000), to activate phospholipase C (PLC) to generate IP3. Furthermore, the IP3 receptor, PLC2, and other components of the GPCR/G protein-responsive phosphatidylinositol signaling pathway have been shown by immunological and/or histochemical methods to be present in rodent CV papillae TRCs (Hwang et al., 1990; Rossler et al., 1998; Clapp et al., 2001; Miyoshi et al., 2001). It is now known that denatonium-responsive GPCRs mediate this response via activation of PLC2 by gustducin’s  component (see below).

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Gustducin is a transducin-like heterotrimeric G protein selectively expressed in ~30% of TRCs (McLaughlin, et al., 1992a; Boughter et al., 1997). In the presence of any one of several bitter compounds, bovine taste receptor–containing membranes activated transducin or gustducin, but not the related G protein Gi (Ruiz-Avila et al., 1995; Ming et al., 1998). Among the bitter compounds that activated transducin/gustducin were denatonium, quinine, nicotine, atropine, naringen, but not caffeine or urea. In vivo assays also have demonstrated clearly that gustducin’s -subunit (-gustducin) plays a key role in TRC responses to numerous bitter compounds (Wong et al., 1996). In comparison to their littermate controls, -gustducin knockout mice displayed markedly reduced behavioral and nerve responses to the bitter compounds denatonium benzoate and quinine sulfate. Based on its close relatedness to -transducin, particularly within the regions of -transducin that are known to interact with PDE6, it was originally proposed that -gustducin’s role in bitter transduction is mediated by activation of a taste PDE (McLaughlin et al., 1992a). Consistent with this proposal, Price (1973) had shown that the bitterness of several compounds correlated well with their ability to activate PDE. Furthermore, very high levels of PDE activity are present in taste tissue (Kurihara, 1972; Law and Henkin, 1982) and two cAMP PDEs have been cloned from taste tissue and shown to have elevated expression in taste tissue (McLaughlin et al., 1994). More recently, it was shown in vitro that -gustducin and transducin activate taste-expressed Ca2/calmodulinsensitive type I PDEs (Ruiz-Avila et al., 1995; M. M. Bakre, R. Lupi, and R. F. Margolskee, unpublished). A direct demonstration of the importance of the gustducin-PDE interaction in bitter taste transduction comes from the recent work of Yan et al., (2001): several bitter compounds were shown to cause a decrease in cNMP levels in taste tissue, which was blocked selectively by antibodies against -gustducin. The subsequent steps in this transduction pathway are speculative (reviewed in Spielman, 1998; Gilbertson et al., 2000): decreased cNMPs may act on protein kinases, which in turn may regulate TRC ion channel activity, or cNMP levels may regulate directly the activity of cNMPgated and cNMP-inhibited ion channels expressed in TRCs. Many of the bitter compounds shown to activate gustducin in vitro lead to pertussis toxin–sensitive generation of IP3 (Spielman et al., 1994), yet neutralizing antibodies directed against -gustducin did not block the bitter-induced rise in IP3 levels (Yan et al., 2001), suggesting that another G protein -subunit might mediate this response. The resolution of this apparent conflict comes from the demonstration that the IP3 effect depends on the activation of PLC2 by gustducin’s  constituents (Huang

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et al., 1999). A novel G-protein  subunit, G13, was cloned and shown to colocalize in TRCs with -gustducin and G3. G13 was shown in vitro to interact with gustducin, G1, and denatonium-responsive taste receptors. It had been shown previously that rat TRCs expressed PLC2 (Rossler et al., 1998), an isoform particularly sensitive to activation by G protein  subunits (Katz et al., 1992). Using quench flow measurements and neutralizing antibodies to PLC2, it was shown that this PLC isoform mediated the generation of IP3 in taste tissue (Rossler et al., 1998). Similar studies showed that the TRC-expressed G subunits G13 and G3 were also required to mediate the rise in taste tissue IP3 in response to denatonium (Huang et al., 1999; Rossler et al., 2000). Thus, it appears that heterotrimeric gustducin mediates two responses in TRCs: a decrease in cNMPs via its -subunit and a rise in IP3 via its G313 component. The subsequent steps in the /PLC2/IP3 pathway are apparently activation of IP3 receptors and release of Ca2 from internal stores followed by neurotransmitter release (Bernhardt et al., 1996).

D. Identification of Gustducin-Coupled Taste Receptors Following up on genetic mapping of bitter response loci of humans and mice and utilizing the partially completed, at that time, human genomic DNA sequence databases, a family of ⵑ25 human GPCRs (named T2Rs or TRBs) was identified recently as candidate taste receptors (Adler et al., 2000; Matsunami et al., 2000). The T2R/TRB receptors map within multigene clusters on human chromosome regions 5p15, 12p13, and 7q31 and the syntenic regions of mouse chromosomes 6 and 15. Consistent with the inference that T2R/TRB receptors might be responsive to bitter compounds, PROP sensitivity maps to human 5p15 and 7q31 (Reed et al., 1999), while SOA sensitivity (soa) maps to the distal region of mouse chromosome 6 (syntenic with human 12p13) (Capeless et al., 1992; Lush et al., 1995). Members of the T2R/TRB multigene family share 30–70% identity, but are only distantly related to other known GPCRs. T2R/TRB receptors are most highly conserved in their three predicted cytoplasmic loops (presumptive sites of G protein interaction) and the adjacent transmembrane segments; these receptors display the greatest divergence in the extracellular regions (predicted regions of ligand binding). Using in situ hybridization, it was shown that the T2R/TRB receptors are only expressed in gustducinpositive TRCs. Furthermore, the in situ hybridization signal with single vs. multiple T2R/TRB probes identified largely the same TRCs, implying that most or even all T2R/TRB receptors are expressed in the same gustducin-positive

Molecular Physiology of Gustatory Transduction

TRCs. This finding would suggest that activation of gustducin by one or another bitter-responsive T2R/TRB receptor would have the same output and that at equipotent concentrations one bitter compound should not be distinguishable from another—generally the case. Most of the gustducin-positive TRCs in the CV, foliate, or palate were also T2R/TRB-positive, although most gustducin-positive TRCs in the fungiform papillae were T2R/TRB-negative. It may be that other receptors are expressed in the gustducinpositive/T2R/TRB-negative TRCs; given that gustducin is implicated in bitter and sweet responses, this subset of gustducin-positive TRCs might express sweet-responsive taste receptors unrelated to the T2R/TRB receptors. A regionally variable pattern of expression in rat and mouse TRCs was noted with several T2R/TRB clones examined. Most taste buds of CV, foliate, geschmacksstreifen (“taste stripe”), and epiglottis contained T2R/TRB-positive TRCs; typically~15–20% of TRCs were positive. However, fewer than 10% of fungiform taste buds contained any T2R/TRBpositive TRCs, although in the positive buds ~15% of the TRCs were positive. Chimeras of two of the T2R/TRB receptors (containing the N-terminal 39 amino acids of rhodopsin) were successfully expressed in HEK 293 cells and shown to be activated by bitter compounds (Chandrashekar et al., 2000). Cells transfected with the mouse receptor mT2R-5 responded only to cycloheximide from among 55 tastants tested. Consistent with this receptor mediating in vivo responses to this bitter compound, the cycloheximide concentration needed to elicit a half-maximal response in mT2R-5-transfected cells was comparable to the murine threshold for aversion. Multiple amino acid differences were noted in mT2R-5 isolates from cycloheximide taste-sensitive (CBA/Ca, BALB/c, C3H/He) vs. -insensitive (C57BL/6, 129/Sv) strains of mice. Furthermore, transfected cells expressing mT2R-5 from the nontaster strain required a nearly 10-fold higher concentration of cycloheximide to elicit a response, similar to the difference in in vivo responses between the taster and nontaster strains. mT2R-5 was shown to selectively couple to -gustducin vs. i, s, o, or q, providing additional support to it being a cycloheximide-responsive bitter receptor and suggesting that the other T2R/TRB receptors are most likely additional bitter-responsive taste receptors. To finally prove that mT2R-5 encodes a (or the) cycloheximide-responsive taste receptor will require the generation and testing of the appropriate knockout or transgenic mouse model. Cells transfected with either hT2R4/mT2R-8, an orthologous pair of human/mouse of receptors, showed responses to denatonium and PROP. However, the concentration of denatonium required was more than three orders of magnitude higher than the

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human threshold for detection of denatonium, suggesting that another ligand is probably the preferred stimulus for this receptor pair and that another human T2R/TRB receptor may be more sensitive to denatonium. E. Other G Protein–Dependent and Independent Bitter Transduction Pathways G proteins other than gustducin have been inferred to play a role in bitter (and sweet) transduction since -gustducin knockout mice have diminished, but not totally abolished, responses to both bitter and sweet compounds (Wong et al., 1996). Transgenic expression in the gustducin lineage of TRCs of a dominant-negative form of -gustducin that interferes with T2R/TRB-G protein interactions further decreased the residual responses to bitter compounds of -gustducin knockout mice, consistent with another G protein expressed in gustducin-positive TRCs mediating these responses (Ruiz-Avila et al., 2001). Gi2, Gi3, G14, G15, Gq, Gs, and -transducin are possible candidates to mediate these responses since they are expressed in TRCs (McLaughlin et al., 1992a; Ruiz-Avila et al., 1995; Kusakabe et al., 1998, 2000). Based on in vitro taste assays with bovine taste membranes, the bitter compounds caffeine and theophylline are not transduced by the T2R/TRB-gustducin pathway (Ming et al., 1998). Based on quench flow studies with murine taste tissue, it was determined that caffeine and theophylline, known PDE inhibitors, act directly on taste PDEs to raise TRC cGMP levels (Rosenzweig et al., 1999). Soluble guanylyl cyclase (GC) is the presumptive source of the cGMP; both GC and nitric oxide synthase (NOS) are known to be present in TRCs (Asanuma and Nomura, 1995; Kretz et al., 1998). Presumably, these two bitter compounds also raise TRC cAMP levels. In addition to bitter transduction mediated by GPCR/G protein pathways, there is considerable evidence for bitter transduction mediated by ion channels. Several bitter compounds are known K channel blockers and cause TRC depolarization by blocking K channels (e.g., TEA, 4-aminopyridine, and Ba2) (reviewed in Lindemann, 1996; Herness and Gilbertson, 1999). Quinine leads to TRC depolarization along with an increase in membrane resistance to K (Sato and Beidler, 1982). In patchclamped frog TRCs, quinine completely blocked Na currents and partially blocked K currents (Avenet and Lindemann, 1987); the addition of EGTA or CaCl2 to the patch pipette solution did not affect the quinine blockade of currents, implying that Ca2 is not involved. In another study using intracellular recording of frog TRCs, quinine was shown to depolarize the cells by inducing the active secretion of Cl across the basolateral membrane

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(Okada et al., 1988). Although denatonium has been shown to activate heterotrimeric gustducin via coupled T2R/TRB receptors, leading to G13/PLC2-mediated generation of IP3 and Ca2 release from internal stores, it has also been shown in guinea pig TRCs to cause Ca2 influx (Orola et al., 1992), and in the mouse denatonium blocks delayed rectifier K currents (Spielman et al., 1989). In bullfrog TRC membrane patches it was shown that quinine, denatonium benzoate, and other bitter compounds directly gated a TRC cation channel (Tsunenari et al., 1999). In mudpuppy (Necturus maculosus) TRCs denatonium benzoate led to an increase in intracellular Ca2, derived predominantly from internal stores (Ogura et al., 1997). Thapsigargin, GDPS, and a PLC inhibitor inhibited this response, but IBMX, membrane-permeant cNMPs, and pertussis toxin did not affect this reponse, arguing against the involvement of pertussis toxin-sensitive G proteins (e.g., gustducin, transducin, or Gi). Dextromethorphan also led to Ca2 release from mudpuppy TRCs; this response was independent of G proteins and was not blocked by a PLC inhibitor, suggesting a direct action on Ca2 stores (Ogura and Kinnamon, 1999).

3.

For those components of these proposed pathways that have been molecularly cloned or otherwise identified in TRCs, it should be possible to confirm their presence and functionality in bitter-responsive TRCs. Among the proposed transduction components, T2R/TRB receptors, -gustducin, -transducin Gq, G14, G1, G3, G13, PLC2, and cNMP-gated channels have been shown by molecular methods to be present in TRCs. The cNMP-inhibited channel identified electrophysiologically has not yet been cloned. All ~25 of the T2R/TRB receptors are candidates for involvement in bitter transduction, although strong functional data exist only for mT2R5 as a cycloheximide receptor.

III. F. Proposed Models for Bitter Transduction There are at least three possible bitter transduction pathways. 1.

2.

A T2R/TRB-gustducin/transducin-PDE-↓cNMP pathway—bitter compounds such as cycloheximide bind to and activate one or more T2R/TRB receptors, which are coupled to heterotrimeric gustducin (and/or transducin); activated -gustducin activates PDEla isoforms, leading to decreased TRC levels of cNMPs. The subsequent steps are speculative: one possibility is that decreased cNMPs lead to opening of cNMPinhibited cation channels, causing TRC depolarization; another possibility is that decreased cNMPs lead to closure of cNMP-gated cation channels, causing TRC hyperpolarization. A T2R/TRB-gustducin/transducin-PLC2-↑IP3/DAG pathway—bitter compounds such as denatonium bind to and activate one or more T2R/TRB receptors, which are coupled to heterotrimeric gustducin; activation of gustducin releases its  subunits (G3G13), which activates PLC2; PLC2 generates IP3 and DAG, which lead to Ca2 release from internal stores and TRC depolarization and transmitter release from vesicles. It is possible that a receptor-mediated/gustducin-independent pathway may utilize the -subunit of Gq or G14 to activate PLC.

GPCR/G protein–independent mechanisms may transduce responses to bitter compounds via direct effects upon TRC ion channels. For example, TEA and quinine block apical K channels, leading to TRC depolarization. These three models are not mutually exclusive and may occur in the same TRCs.

SWEET TRANSDUCTION MECHANISMS

A. Diversity of Sweet Compounds and Sweet Modifiers Sweet compounds are extremely diverse in their chemical composition: examples include simple carbohydrates (monosaccharides and disaccharides), D- and L-amino acids (most D-amino acids are sweet; only some L-amino acids are sweet), artificial sweeteners (saccharin, cyclamate, aspartame, acesulfame K, etc.), plant proteins (monellin and thaumatin), chloroform, and simple salts of beryllium and lead. Sweet transduction is thought to utilize seven transmembrane-helix receptors coupled to G proteins and/or an amiloride blockable Na channel. Sugars and high-potency artificial sweeteners have been shown to bind to the taste cell surface and to taste cell membrane fractions (Cagan and Morris, 1979), consistent with there being a cell surface receptor for sweet compounds. Biochemical studies with membranes derived from the anterior tongue of the rat (containing fungiform papillae) or with intact taste buds from rat CV papillae showed that sweet compounds activated adenylyl cyclase (AC) to elevate intracellular levels of cAMP (Striem et al., 1989, 1991). The competitive sugar antagonist SOA inhibited sweet induced elevation of cAMP (Striem et al., 1991). In membrane extracts from the rat tongue, AC was activated by sucrose in a GTP-dependent fashion (Striem et al., 1989). These observations argue for the presence of specific receptors that upon activation by sweeteners

Molecular Physiology of Gustatory Transduction

activate Gs, which in turn activates AC to generate cAMP as the intracellular second messenger. Monellin and thaumatin are two naturally occurring sweet proteins that are on a molar basis 104–105 times as sweet as sucrose (van der Wel and Loeve, 1972). Thaumatin consists of 207 amino acids and has a molecular weight of 22,200 (Iyengar et al., 1979). Monellin consists of two chains, both of which are required for sweetness. Both thaumatin and monellin are perceived as sweet only by humans and old world primates: rat, pig, hamster, dog, and rabbit do not exhibit neural responses to these proteins (Brouwer et al., 1973, Hellekant, 1976). Presumably, the sweet-responsive receptor that binds to these proteins is present only in primates or amino acid differences in this receptor in non-primates affect its ability to bind these protein sweeteners. The protein miraculin is tasteless by itself but induces sweetness in the presence of acid (Bartoshuk et al., 1969; Kurihara et al., 1969). Miraculin, like thaumatin and monellin, is limited in its action to humans and certain other primates. Miraculin caused sweet responsive nerve fibers to respond to acid stimuli, but it did not affect sourresponsive fibers (Brouwer et al., 1983). Responses to monellin, thaumatin, and miraculin cross-adapt, indicating that these proteins act at the same site. Perhaps miraculin binding induces a conformational change in the sweet receptor that does not fully activate the receptor except at low pH. Alternatively, two independent events may be required to activate sweet-responsive cells: binding of miraculin to the receptor and lowering of pH. Another protein taste modifier, curculin, has sweet taste by itself, but its sweetness is greatly enhanced in the presence of acid or in the absence of divalent cations (Yamashita et al., 1990); presumably curculin and miraculin act by similar mechanisms at the same receptor target. Several different types of compounds specifically inhibit sweet taste. Gymnemic acid, ziziphin, and holdulcin are triterpine saponins that preferentially inhibit sweet (Stocklin, 1968; Saul, Kennedy and Stevens, 1985; Yamada, Imoto and Yoshiokai, 1985). Other surface-acting agents such as the detergent SDS reduce sweetness, but SDS also reduces the intensity of salt and bitter tastes (DeSimone et al., 1980). In addition, low concentrations (0.1 mM) of CuCl2 and ZnCl2 selectively suppress sweet without affecting bitter or salt tastes (Iwasaki and Sato, 1984). The sweet receptors are also very sensitive to proteolysis: treatment of the apical surface of the rat tongue with proteases results in the loss of responsiveness to sucrose, but not to other tastants (as assessed by chorda tympani nerve recordings) (Hiji, 1975). That detergents and saponins had their most profound effects upon sweet responses is consistent with the sweet receptor being

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a transmembrane protein with a readily accessible extracellular domain. Receptors with large extracellular ligandbinding domains, such as T1r1, T1r2, T1r3, and mGluR, might be expected to be particularly sensitive to these treatments. Genetic mapping and heterologous expression have identified T1r2 and T1r3 as components of a sweet receptor (see below). B.

Physical Characterization of Sweet Receptors

Direct biochemical analysis of sweet receptors has been hindered by the lack of suitable high-affinity ligands. Sugars are unsuited for biochemical purification of their cognate receptor(s) since they have apparent Kd of 0.1–1.0 M (Cagan, 1971). However, more useful ligands may be found among high-potency artificial sweeteners such as superaspartame and the intensely sweet proteins thaumatin and monellin that are ~100,000-fold sweeter than sucrose on a molar basis (van der Wel and Loeve, 1972; Tinti and Nofre, 1991). Attempts to use these high-affinity sweeteners for the biochemical purification of sweet receptors have not yet yielded a sufficiently pure preparation to determine the sequence and thereby molecularly clone the receptor(s). A more fruitful approach to cloning the receptors underlying sweet taste responses has been to use the genetics of sweet taste responses in combination with genomics-based analysis of candidate genes (Max et al., 2001; Montmayer et al., 2001; Sainz et al., 2001). Labeled thaumatin was used to physically characterize the nature of its binding site (Farbman et al., 1987): thaumatin bound to microvilli and vesicles shed from microvilli of rhesus monkey foliate papillae. It was subsequently shown that thaumatin binding elicited shedding of microvilli vesicles into the pores of foliate and vallate papillae (Farbman and Hellekant, 1989). Repeated stimulation with thaumatin led to a decline in the neural response to thaumatin or sucrose, but not to citric acid (sour), suggesting that the thaumatin-and sucrose-binding sites are in the same TRCs and in close proximity. In another set of studies, monellin was shown to bind to membranes from human and bovine CV papillae with a Kd of about 105 M (Cagan and Morris, 1979). A photo-affinity derivative of thaumatin was used to label monkey taste papillae: polyacrylamide gel electrophoresis indicated that a 50,000 MW protein present in taste papillae but absent from nontaste papillae bound to the derivatized thaumatin (Shimazaki et al., 1986). C.

SAR-Inferred Models of Sweet Receptors

Based upon common features of sweeteners, several different models have been developed for the structure of the

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sweet receptors’ binding pocket. In the A-H/B model of Schallenberger and Acree (1967), a hydrogen bond donor within the sweet tastant (A-H: A is an electronegative atom, H is a hydrogen atom) interacts with an electronegative hydrogen bond acceptor within the sweet receptor; and an electronegative atom within the tastant (B: usually a N or O atom) interacts with an electropositive hydrogen within the receptor. According to the original model, an A-H/B pharmacophore must be present in all sweeteners with the distance between the A and B groups between 2.5 and 4 Å. This model also posits that the sweet receptor (or a family of closely related receptors) has complementary regions that recognize and bind to the A-H/B sites of the sweet compounds: the initial event in sweet detection then is the simultaneous interaction of the A-H/B sites with their complements in the sweet receptor. Two modifications of the original A-H/B model propose the presence of a third binding site: Kier (1972) suggested that a dispersion interaction () at the receptor is necessary for high potency; Schallenberger (1978) proposed a third interaction () that is hydrophobic in nature and required for high potency. Recognition by the receptor in these tripartite models is predicted to be chiral, with specific assigned distances between each of the three constituents of the sweet pharmacophore. One problem with all of these models is that almost any organic compound with OH, NH, or CH (A-H groups), an electronegative group (B), and a hydrophobic group () is predicted to be sweet—and this is not the case! Several high-potency sweeteners do not seem to fit in with these models (DuBois et al., 1993). Furthermore, slight structural alterations of these sweeteners that do not affect the A-H/B groups lead to loss of sweetness or the appearance of bitterness. Based upon ultra high-potency guanidine sweeteners such as superaspartame, a model has been proposed (DuBois et al., 1993) that suggests the presence of complementary sites within the receptor that interact with the following structures: a carboxylate region, a major N-H region, a minor N-H region, a hydrophobic region, and a  stacking region. One problem for a sweet receptor model involving classical receptor-ligand type interactions is the fact that enantiomeric hexoses, including D- and L-glucose, elicit identical taste behaviors (Schallenberger et al., 1969); this argues against stereospecific recognition and led to the suggestion of a mechanism based upon colligative properties of carbohydrates (DuBois et al., 1993). Now that theT1r2/T1r3 sweet-responsive receptors have been molecularly cloned and expressed (Kitagawa et al., 2001; Max et al., 2001; Montmayeur et al., 2001; Nelson et al., 2001; Sainz et al., 2001) (see below), it will be possible to biochemically characterize the receptors’

Gilbertson and Margolskee

ligand binding properties and determine if these receptors underlie all sweet responses. Experiments with T1r2/T1r3 knockout mice will be particularly informative regarding the existence of multiple sweet detection mechanisms. D. Evidence for Multiple Sweet Receptors and Multiple Transduction Pathways Studies with mouse taste cells (Tonosaki and Funokosaki, 1984), hamster taste buds and taste fibers (Faurion and Vaysettes-Courchay, 1990; Cummings et al., 1993), gerbil and hamster chorda tympani (Jakinovich and Goldstein, 1976, Rehnberg et al., 1992), chimpanzee taste fibers (Hellekant and Ninomiya, 1991), and human psychophysics (Schiffman et al., 1981) all argue for multiple sweet receptors and/or mechanisms. In human psychophysical experiments, it was found that partial cross adaptation or no cross adaptation occurred between highpotency sweeteners (neohesperidin dihydrochalcone, aspartame, and saccharin) and sucrose when the highpotency sweeteners were the preadapting stimuli (Schiffman et al., 1981). Electrophysiological studies have shown that sucrose causes a subset of mouse TRCs to depolarize and undergo an increase in membrane resistance (Tonosaki and Funokoshi, 1988a,b). Injection of cAMP, cGMP, EGTA, or the channel blocker TEA into this subset of mouse TRCs also elicited depolarization with decreased K conductance (Tonosaki and Funokoshi 1988a). In about one third of whole-cell recordings from isolated rat TRCs, saccharin caused a decrease in outward K current sufficient to depolarize the intact TRC (Behe et al., 1990). Furthermore, in on-cell recordings from rat TRCs, saccharin caused action potentials in about one third of the cells (Behe et al., 1990). In gerbil TRCs, sucrose and saccharin caused a decrease in outward K currents (Uchida and Sato, 1997ab). In isolated TRCs from frog (Rana esculenta) cAMP, forskolin and methyl xanthines caused a decrease in outward K currents (Avenet and Lindemann, 1987). It was subsequently shown by whole-cell and excised patch recordings that a cAMP-dependent kinase closes a basolateral K conductance (Avenet et al., 1988ab). These results from various vertebrate TRCs suggest that cNMPs serve as the intracellular second messenger for the sweet pathway and elicit depolarization of these cells by K channel closure, followed by Ca2 influx through voltage-dependent Ca2 channels. It is uncertain that protein kinase A (PKA) is actually the mediator of cNMP-elicited K channel closure since the PKA inhibitor H-89 did not block sucrose-elicited action responses of hamster fungiform TRCs (Varkevisser and Kinnamon, 2000). It may even be that sweetener-elicited cNMPs

Molecular Physiology of Gustatory Transduction

directly regulate this channel or act indirectly through some other mechanism independent of PKA. Using a “loose patch-in situ” recording method, Cummings et al., (1993) recorded action currents in response to sucrose and several artificial sweeteners: with time (1–3 min ), the action currents abated, suggesting that an adaptive response occurred. Subsequent to this adaptation, the same sweetener could not elicit an action current; however, other sweeteners did elicit currents (i.e., they did not cross-adapt). Membrane-permeant cNMP analogs elicited action currents in sweet-responsive taste buds (28/28 with 8 cpt-cAMP; 7/8 for dibutyryl cGMP) and did not elicit such currents in sweet-nonresponsive taste buds (0/12 with 8 cpt-cAMP; 0/8 with dibutyryl cGMP). In this study it was shown that neither amiloride nor the absence of permeant apical cations had an effect upon taste bud action currents; this argues that cNMPs are involved as a second messenger in taste cell responses to sweet and that this response does not involve an apical cNMP-responsive channel. In additional studies of hamster fungiform TRCs using whole-cell recording, it was demonstrated that all sweet-responsive cells (~12.5% of the total) were also responsive to cNMPs (31/31 cells tested); conversely, all sweet-unresponsive cells were likewise unresponsive to cNMPs (0/206 cells tested) (Cummings et al., 1996). Outward K currents of sweet-responsive TRCs were reduced by sweet compounds or cNMPs and potentiated by the PDE inhibitor IBMX, suggesting that the sweet compounds elicited cNMPs that led to reduction of the K currents. In addition to the TRC K channels, which are thought to be indirectly regulated by cNMPs, cNMP-gated channels, such as are well known in olfactory receptor neurons and photoreceptor cells, may also serve in a sweet/cNMP transduction pathway. Molecular biological (Misaka et al., 1997, 1998, 1999) and electrophysiological studies (Herness, 1993; Sugimoto, 1997) have provided direct evidence for the existence in taste cells of a cNMP-responsive channels. A novel channel that is inhibited by cNMPs has been identified in TRCs from frog (Kolesnikov and Margolskee, 1995) and mouse (S. S. Kolesnikov and R. F. Margolskee, unpublished). These observations leave open the possibility that cNMPs may act indirectly in TRCs via protein kinase A and directly via cNMP-gated and cNMP-inhibited ion channels. Additional molecular biological studies to identify other sweet transduction components are described in the next section. There is evidence for the presence of an altogether different sweet transduction mechanism in both dogs and humans. Psychophysical studies in humans have shown that the diuretic amiloride reduces the perceived intensity of both NaCl and sweet compounds (Schiffman et al.,

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1983); presumably amiloride exerts its effect upon an epithelial type Na channel or a structurally related channel. This may explain the psychophysical observation that very low concentrations of NaCl are perceived by humans as sweet (Beets, 1978; Bartoshuk et al., 1978). Ion transport studies with isolated canine lingual epithelium showed that both mono- and disaccharides increased a transepithelial short circuit current that was partially blocked by amiloride (DeSimone et al., 1984, Mierson et al., 1988). Amiloride was also found to inhibit the canine chorda tympani responses to sucrose and fructose (Mierson et al., 1988); however, other studies failed to demonstrate an amiloride sensitive chorda tympani response to sugars in rat, gerbil, and hamster (Brand et al., 1985; Jakinovich, 1985; Herness, 1987). The canine response to sucrose was unaffected by cAMP, cGMP, or Ca2, arguing against these second messengers playing a role in sucrose response in the dog (Simon et al., 1989). It is clear that different species use different mechanisms for sweet transduction; furthermore, some species (e.g., humans) use multiple mechanisms for sweet transduction (see Jakinovich and Sugarman, 1988). E. Biochemical and Molecular Biological Identification of Sweet Transduction Elements In biochemical studies using membrane vesicles derived from the anterior tip of the rat tongue (containing fungiform papillae, connective tissue, and some underlying muscle), sucrose caused a twofold stimulation of AC activity (Striem et al., 1989). The effect on AC required GTP and was dependent upon the sucrose concentration. Among nonsugar sweeteners, saccharin, but not aspartame or neohespederidin, elevated cAMP production in the tongue tip membranes. Sucrose did not elevate cAMP production in membrane vesicles derived from skeletal muscle, olfactory cilia, or nonsensory tongue epithelium. However, vesicles from tongue muscle did show an elevation of cAMP in response to sucrose. In a subsequent series of experiments (Naim et al., 1991), membrane vesicles derived from the CV papilla of the pig showed a GTP-dependent elevation of cAMP in response to sucrose: control experiments with nonsensory epithelium from pig tongue showed no rise in cAMP in response to the addition of sucrose. However, membranes from cow or pig fungiform papillae (devoid of underlying muscle) showed no sucrose-dependent rise in cAMP; instead, the addition of sucrose led to a decrease in cAMP levels (as was also found with control nonsensory epithelium). These results suggest that sweet receptors activate AC (via Gs) in the CV papillae of cow and pig, but not in the fungiform papillae from these animals. Either the AC response in rat

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fungiform differs from those of the cow and pig, or the muscle present in the rat tongue tip preparation is responsible for much of the sucrose-induced stimulation of AC. In biochemical studies with intact rat CV taste buds, sucrose caused a dose-dependent rise in cAMP (Striem et al., 1991). A competitive antagonist of sucrose, methyldichloro-dideoxy--D-galactopyranoside (MAD-diClGal), inhibited the cAMP elevation by about 65%. MAD-diCl-Gal had previously been shown to competitively inhibit sucrose activation of the chorda tympani response and sucrose stimulation of AC in rat anterior tongue tip (Striem et al., 1990). Control nontaste lingual epithelium did not respond to either sucrose or the competitive inhibitor. The artificial sweetener saccharin had no significant effect upon cAMP levels (in contrast to its above-cited effect on tongue tip membranes). In patchclamp experiments with TRCs from rats, saccharin was shown to cause depolarization by a decrease in K permeability (Behe et al., 1990). However, only 40% of the saccharin-responsive cells also responded to cAMP; supporting the idea that saccharin does not exert its effects via AC. It has been proposed that saccharin (and certain other amphiphilic sweet compounds) directly activates TRC-expressed G proteins (Naim et al., 1994). In other studies it was shown that saccharin acted by taste membrane receptors to inhibit bitter-responsive receptor activation of gustducin/transducin (Ming, et al., 1999). The recent demonstration that heterologously expressed T1r2/T1r3 responds to saccharin and other sweeteners (Nelson et al., 2001) confirms that this is a receptor— mediated phenomenon. Gs -subunit mRNA and protein are elevated in TRCs vs. the surrounding nontaste tissue (Kusakabe et al., 2000; S. K. McLaughlin and R. F. Margolskee, unpublished), consistent with a proposed role for Gs in sweet taste transduction. Gs presumably activates AC in response to sweeteners, since the only known G proteins that stimulate AC are Gs and Golf (Jones and Reed, 1989). Golf is known not to be expressed in taste tissue (P. McKinnon, unpublished), whereas Gs is expressed in taste buds (McLaughlin et al., 1992a, b; Kusakabe et al., 2000). The Gs gene generates a great diversity of transcripts via differential splicing (Kozasa et al., 1988; Swaroop et al., 1991); preliminary data indicate that at least one Gs splice product is expressed in taste tissue but not in nonsensory portions of the tongue (Kusakabe et al., 2000; S. K. McLaughlin and R. F. Margolskee, unpublished). Potential targets for cNMPs generated by Gs/AC are protein kinase A–regulated K channels and cNMP-gated channels, described above. PDEs and ACs are implicated in sweet and/or bitter transduction via cNMP regulation. Although caffeine is

Gilbertson and Margolskee

bitter, it also potentiates the sweetness of certain compounds in humans (Schiffman et al., 1986), perhaps by inhibition of cAMP PDE activity. The PDE inhibitor IBMX causes depolarization of cAMP-responsive TRCs from frog (Avenet and Lindemann, 1987) and sweetresponsive TRCs from hamster (S. C. Kinnamon, personal communication). PDE has been shown to be present at high levels in taste tissue (Law and Henkin, 1982); two cloned cAMP type PDEs (PDE4 isoforms) were shown to have elevated expression in taste tissue (McLaughlin et al., 1994). Most recently, PDE1A, PDE1C, PDE4, and PDE5 were identified in bovine taste tissue by immunoblots of chromatography fractions (M. M. Bakre, R. Lupi, and R. F. Margolskee, unpublished). PDE1A isoforms were shown by immunohistochemistry to be highly expressed in a subset of -gustducin–positive and–negative TRCs. PDE1A is well known to be activated by Ca2/calmodulin (Zhao et al., 1997); it is now known to be activated by transducin and/or gustducin (Bakre et al., 2001). Given the supposition that elevated cNMPs are involved in sweet responses, transducin/gustducin-activated PDE1A would decrease cNMP levels and in all likelihood be antagonistic to sweet and/or involved in sweet signal termination. This provides a possible explanation for how the -gustducin knockout mouse could be deficient in sweet responsiveness even if -gustducin is not directly involved in the sweet transduction pathway. If tonic activation of PDE1A by -gustducin helps to set the basal level of TRC cNMPs, then elevation of TRC cNMP levels in the absence of -gustducin may interfere with normal sweet transduction responses. AC has been shown to be present in bovine taste tissue (CV and fungiform regions) (Kurihara and Koyama, 1972) and in foliate papillae of the rabbit (Nomura, 1978; Asanuma and Nomura, 1982); however, specific AC isoforms have not been cloned from TRCs to date. In addition to the role proposed for cNMPs in sweet responses, there is also evidence that IP3 may act as a second messenger for some sweet compounds. Furthermore, this pathway may also regulate the same K conductance targeted by cNMPs generated in response to sugars. The artificial sweeteners saccharin and SC45647 caused a robust increase in IP3 levels in CV tissue and a rapid rise in CV TRC Ca2 via release from intracellular stores (Bernhardt et al., 1996). Sucrose elicited only a modest rise in CV IP3 levels and elevated Ca2 in the sweetener-responsive TRCs via influx, not store release. In this study a clear segregation was noted between sweet-responsive (sucrose, saccharin and SC45647) vs. bitter-responsive (denatonium) TRCs. An important conclusion was that IP3- and cNMP-dependent sweet pathways exist in the same TRC. Recent results with hamster taste buds determined that inhibition of protein kinase C

Molecular Physiology of Gustatory Transduction

(PKC) blocked artificial sweetener-elicited action currents in fungiform taste buds (Varkevisser and Kinnamon, 2000). Apparently the same TRC K conductance sensitive to cNMPs generated in response to sugars is phosphorylated and closed by PKC activated in response to the artificial sweetener-stimulated taste transduction pathway. It is interesting to consider these results in the context of heterotrimeric gustducin-mediated responses to the bitter compound denatonium: -gustducin via PDE1A decreases cNMP levels, while G313 via PLC2 increases IP3 levels. Perhaps in the case of the sweetresponsive TRCs heterotrimeric Gs activates AC to generate cAMP, while its  component activates PLC2 to generate IP3. Although gustducin is clearly implicated in bitter responses based on results from in vitro and in vivo studies, it also is thought to play a role in sweet. -Gustducin null mice have markedly diminished chorda tympani and glossopharyngeal nerve responses to sucrose and SC45647. In two bottle preference tests the -gustducin null mice showed diminished preference for both sucrose and SC45647. There are also biochemical data to directly implicate gustducin in sweet transduction: although neither sucrose nor artificial sweeteners activate gustducin in the presence of bovine taste receptor-containing membranes (as is the case with many bitter compounds) (Ruiz-Avila et al., 1995; Ming et al., 1998), a number of artificial sweeteners competitively inhibit bitter receptor activation of gustducin, suggesting that these compounds may act as bitter antagonists (Ming et al., 1999). This type of interaction may underlie peripheral aspects of bittersweet mixture suppression and the observation that quinine suppresses chorda tympani nerve responses to sucrose (Formaker and Frank, 1996). F. Genetic Mapping of Sweet Taste Loci and the Search for Sweet Receptors Gs, -gustducin, AC, PLC2, basolateral K channels, and cNMP-gated channels are all potential components of one or more sweet transduction pathways. However, the sweet-responsive receptors themselves were only recently cloned. A number of laboratories used genomics-based approaches such as were used to clone the mammalian T2R/TRB receptors to identify sweet taste receptors (Max et al., 2001; Montmayeur et al., 2001; Sainz et al., 2001). In mouse, sac and dpa are genetically mapped loci that provide major contributions to differences between sweetsensitive and sweet-insensitive strains (Lush et al., 1995). Sac has been finely mapped to the distal end of mouse chromosome 4, while dpa has been less finely mapped to the proximal portion of mouse chromosome 4

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(Bachmanov, et al., 1997; Blizzard et al., 1999). It was initially proposed that the candidate taste receptor T1r1 was sac due to its position on distal 4 (Hoon et al., 1999). However, by an analysis of the recombination frequency between T1r1 and those markers closest to sac in F2 mice, it is clear that T1r1 is rather distant (~5 cM) from sac (D18346 is an STS marker that maps most closely to sac) (Li et al., 2000). Furthermore, by analysis of regions of the sequenced human genome syntemic to that of the mouse in the region of D18346 and other markers linked to sac, it was determined that a novel GPCR, termed T1r3, is within ~15,000 bp of D18346/sac (Max et al., 2001). From this same analysis it was determined that T1R1 is several hundred thousand base pairs away from D18346/sac. Unlike the T2R/TRB receptor family, T1r3 is a single receptor, not part of a multigene cluster. At the amino acid sequence level T1r3 is ~30% related to T1r1 and T1r2 (Hoon et al., 1999). T1r3 is expressed selectively in TRCs in fungiform, foliate and circumvallate papillae and geschmacksstreifen (Max et al., 2001; Montmayeur et al., 2001; Sainz et al., 2001). T1r3 is coexpressed with T1r1 in anterior TRCs and with T1r2 in posterior TRCs (Nelson et al., 2001; Montmayeur et al., 2001). A comparison of the sequence of T1r3 from several strains of mice identified two polymorphisms that differentiated all sweet sensitive “taster” strains of mice from all sweet-insensitive “nontaster” strains (Bachmanov et al., 2001; Max et al., 2001; Montmayeur et al., 2001; Nelson et al., 2001; Sainz et al., 2001). These polymorphisms are within the N-terminal extracellular region of T1r3 that based on homology to the mGluR1 glutamate receptor is predicted to be involved in GPCR dimerization and ligand binding (Kunishima et al., 2000; Max et al., 2001). Confirmation that T1r3 is indeed sac came from the conversion of nontaster mice into tasters by the transgenic expression of the taster form of T1r3 (Nelson et al., 2001; M. Rong, W. He, S. Damak, and R. F. Margolskee, unpublished). Heterologous expresion of T1r3 together with T1r2 demonstrated that this combination responds to several natural and artificial sweeteners, suggesting that T1r2 and T1r3 form a heterodimeric receptor (Nelson et al., 2001). How T1r2/T1r3 can bind to such diverse sweeteners is presently unknown. G. Proposed Models for Sweet Transduction There are at least three possible sweet transduction pathways: 1.

A GPCR-Gs-cNMP pathway—sugars are thought to bind to and activate one or more GPCRs (e.g., T1r2/T1r3) coupled to Gs; activated Gs would

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2.

3.

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activate AC to generate cAMP; cAMP activates protein kinase A, which phosphorylates a basolateral K channel, leading to closure of the channel, depolarization of the taste cell, voltage-dependent Ca2 influx, and neurotransmitter release. A GPCR-Gq/G-IP3 pathway—artificial sweeteners presumably bind to and activate one or more GPCRs (e.g., T1r2/T1r3) coupled to PLC2 by either the  subunit of Gq or by G subunits; activated Gq or released G would activate PLC2 to generate IP3 and DAG; IP3 and DAG elicit Ca2 release from internal stores, leading to depolarization of the taste cell and neurotransmitter release. A ligand-gated cation channel has been identified in dog and human—sweet compounds lead to cation influx via an amiloride-blockable Na channel; this pathway is insensitive to second messengers and is not known to occur in rats, hamsters, or mice.

Direct evidence suggests that at least the first two pathways exist in the same subset of TRCs. For those components of these proposed pathways that have been molecularly cloned or otherwise identified in TRCs, it should be possible to confirm their presence and functionality in sweet-responsive TRCs. Among the proposed transduction components, T1r2, T1r3, Gs, Gq, G (of various types), and PLC2 have been shown by molecular methods to be present in TRCs. The amilorideblockable Na channel inferred to be involved in sweet responses has not yet been cloned. No TRC-expressed ACs have been cloned or otherwise characterized. Eventually it should be possible to reconstitute the entire sweet-responsive pathway in vitro using molecularly cloned and/or purified components.

IV. SODIUM SALT TRANSDUCTION MECHANISMS A. Sodium Salt Transduction Utilizing Ion Channels The earliest studies of taste transduction involved those aimed at elucidating the mechanism for the detection of sodium salts, which are generally thought of as the prototypical ‘salty’ stimuli in humans. These initial reports using transepithelial current recording (DeSimone et al., 1981), afferent nerve recording (Brand et al., 1985), and human psychophysics (Schiffman et al., 1983) were consistent in showing that the response to NaCl was at least partially sensitive to the diuretic amiloride. Amiloride’s action as a diuretic depends on its well-known activity to inhibit voltage-insensitive Na channels in transporting

epithelia (Garty and Palmer, 1997). Thus, it was hypothesized that the apically localized epithelial Na channel found in the kidney and in other Na-conserving tissues was utilized by TRCs in the recognition of Na salts. The mechanism involves Na influx through the apical amiloride-sensitive sodium channels leading to a direct depolarization of the TRCs and subsequent neurotransmitter release, similar to the model in Na-transporting epithelia proposed decades earlier (Koefoed-Johnsen and Ussing, 1958) (Fig. 2A). The involvement of amiloride-sensitive pathways in the transduction of sodium salts subsequently has been documented in numerous studies carried out in a variety of species (for detailed reviews, see Lindemann, 1996; Boughter and Gilbertson, 1999; Lindemann et al., 1999; Gilbertson et al., 2000). Two important points have been demonstrated in these studies. First, the amiloride-sensitive Na channels in TRCs appear structurally and functionally similar to the well-described epithelial Na channels (ENaC) found in other tissues. Second, amiloride-sensitive pathways cannot account for the entire gustatory response to NaCl. B.

Taste Cells with ENaC-Like Channels

The properties of ENaC in Na-conserving epithelia have been described in detail (for review see Palmer, 1992; Garty and Palmer, 1997). Electrophysiological recordings have demonstrated that the amiloride-sensitive currents in TRCs, like the native ENaC in most Na-transporting epithelia, are sensitive to amiloride in the submicromolar range (inhibition constants  1 M). Benzamil, a more specific inhibitor of ENaC than amiloride (Kleyman and Cragoe, 1988), also inhibits amiloride-sensitive Na currents in TRCs (Gilbertson and Fontenot, 1998) consistent with effects on ENaC-like channels and not other amiloride-sensitive targets (Kleyman and Cragoe, 1988). Though there has been no detailed biophysical characterization of the amiloride-sensitive Na channels in mammalian TRCs, a preliminary report (Avenet, 1992) showed that they have the small unitary conductance (~5 pS) predicted of the common ENaC in the kidney. Another similarity between the common ENaC and the amiloride-sensitive Na channels in TRCs is in the saturation of the channel with increasing concentrations of mucosal Na. In transporting epithelia, this saturation has been attributed to feedback inhibition due to increases in intracellular Na that activate secondary, Ca2-dependent mechanisms (Ling and Eaton, 1989), self-inhibition due to a direct inhibitory effect of Na on ENaC (Fuchs et al., 1977), decreases in driving force due to large-scale changes in intracellular Na (Lindemann,

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Figure 2 Models for Na current flow in taste cells. (A) Apical amiloride-sensitive Na channels allow influx of Na ions directly depolarizing the taste receptor cell (transcellular pathway). Net current flow is inward through these ENaC-like channels and outward via the Na/K ATPase. The closed current loop (dotted line) is through the tight junction permeable to small ions. (B) For influx of Na in taste cells lacking apical ENaC-like channels, Na ions must first permeate the tight junctions (paracellular pathway). Na ions may then enter the cell through basolateral amiloride-sensitive channels. Since amiloride cannot permeate the tight junctions, this mechanism would appear insensitive to mucosal amiloride. In this case two current loops are present: one through the tight junctions as in (A) and another through the basolateral membrane. (Adapted from Lindemann et al., 1999.)

1984), and saturation at the Na-binding site within ENaC itself. Amiloride-sensitive Na conductances recorded both in isolated TRCs and from the lingual epithelium show evidence of saturation. On the basis of the time course of the development of saturation and its sensitivity to the sulfhydryl reagent p-hydrozymercuribenzoate, this phenomenon, which is found only in cells containing amiloride-sensitive Na channels, has been attributed to self-inhibition (Gilbertson and Zhang, 1998). The presence of additional mechanisms (e.g., feedback inhibition, or changes in driving force) has not been ruled out. Taken together, the amiloride-sensitive Na channels in TRCs appear functionally similar in many respects to the ENaC found in a variety of other Na-conserving epithelia Recent molecular approaches have also demonstrated the presence of both ENaC-like protein and RNA in taste tissue. ENaC, which is a member of the Degenerin/ENaC superfamily of ion channels (see Benos and Stanton, 1999), consists of 3 homologous subunits(, , and ), each containing only two transmembrane regions, making them the structurally simplest of ion channels found to date. The  subunit is capable of making functional (i.e., Na-conductive) channels when expressed alone, but the amiloride-sensitive Na currents are enhanced by two orders of magnitude when the  and  subunits are coexpressed with  (Canessa et al., 1994). All of the ENaC subunits (, ,

and ) are expressed in the taste buds from the fungiform papillae in the anterior rat tongue (Lin et al., 1999; Kretz et al., 1999) and the fungiform taste buds also exhibit significant amiloride-sensitive Na currents (Gilbertson and Zhang, 1998; Gilbertson and Fontenot, 1998). Conversely, in the rat, taste buds from the CV papillae express primarily the  subunit and relatively little  or . These taste buds have voltage-independent Na conductances qualitatively similar to those expected from ENaC channels, yet these conductances are insensitive to amiloride and benzamil (Gilbertson and Fontenot, 1998; Gilbertson and Zhang, 1998). One interpretation of these data is that the relative expression of ENaC subunits in the gustatory system may be critical for establishing the set point for salt responsiveness in the oral cavity. Moreover, the regulation of the expression of these molecular components by natriferic hormones may contribute to the adaptive nature of the gustatory system, at least in terms of sodium salt recognition. Consistent with this interpretation, a recent report demonstrated that aldosterone caused increased expression of the  and  subunits of ENaC in all papillae and concomitantly led to an increase in the amplitude of the amiloride-sensitive Na currents recorded electrophysiologically (Lin et al., 1999). Thus, the relative expression of ENaC subunits in the oral cavity may affect both the magnitude as well as the pharmacology of the response to Na salts.

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Differential ENaC subunit expression may also explain some of the species differences with respect to the contribution of amiloride-sensitive Na currents to overall salt responsiveness. These differences are significant and range from those, like rat, in which a large degree of the NaCl response is amiloride-sensitive (Brand et al., 1985; Doolin and Gilbertson, 1996; Gilbertson and Fontenot, 1998), to those in some mouse strains that are apparently completely insensitive to this inhibitor (Ninomiya et al., 1989; Miyamoto et al., 1998, 1999). Apparently, those ENaClike channels expressed at the apical membrane may be the ones important for imparting a high degree of salt sensitivity. Miyamoto and colleagues (1999) have shown that C57B1/6 mice, which are more salt responsive than BALB/c mice, have greater amiloride sensitivity at their apical receptive membranes than do the BALB/c mice. Despite this difference, when the whole cell (i.e., apical and basolateral membranes) was exposed to amiloride, no difference between amiloride sensitivity in the two strains was found. Within a species this difference is noted as well. Recordings from the rat chorda tympani nerve that innervates the anterior tongue including the fungiform taste buds are more salt responsive than those of the glossopharyngeal nerve innervating the CV taste buds and posterior tongue (Harada and Smith, 1992; Ninomiya et al., 1994). Both epithelial transport studies (Gilbertson and Zhang, 1998) and patch clamp recordings (Gilbertson and Fontenot, 1998) have shown that the high specificity, apical amiloride-sensitive conductance found in the fungiform TRCs is lacking in those from the CV papillae. Clearly, more systematic analyses of the strain and taste bud differences in salt responsiveness and expression of ENaC subunits is needed to strengthen this intriguing correlation. C. Amiloride-Insensitive Pathways in Salt Taste Transduction A common theme emerging in the study of taste transduction is that there is no single mechanism for the detection of any individual class of taste stimuli. Consistent with this idea, apical amiloride-sensitive Na channels cannot completely account for sodium salt responsiveness. Some of the evidence suggesting the presence of additional salt transduction pathways has come from experiments that showed an insensitivity of salt responses to mucosal applications of amiloride (Formaker and Hill, 1991). One of the mechanisms that has been proposed to account for these responses involves the movement of Na (and other small ions) through the tight junctions between the TRCs (Elliot and Simon, 1990; Ye et al., 1991). This “paracellular pathway” for Na flux would lead to changes in interstitial

Gilbertson and Margolskee

Na concentrations around the basolateral membrane of the TRCs, where it may enter TRCs through amiloridesensitive Na channels situated on this surface (see Fig. 2B). The differential permeability of the tight junctions to various anions has been hypothesized to account for the differences in the intensity and taste of various sodium salts (i.e., sodium chloride vs. sodium gluconate). One caveat of this mechanism is that there may be amiloride-sensitive Na channels on the basolateral membrane of TRCs. Though such high-affinity amiloridesensitive Na channels have been found in frog TRCs (Avenet and Lindemann, 1989), evidence for their presence in rodents, for example, is equivocal. As alluded to above, antibodies against ENaC have labeled TRC profiles on their basolateral margins, indicative of the presence of an ENaC-like protein on this surface. Consistent with this, Mierson et al. (1996) found a low-affinity (Ki ~ 50 M), amiloride-sensitive Na conductance using transepithelial recording. Gilbertson and colleagues failed to identify either a high- or low-affinity basolateral amiloridesensitive conductance in rat or hamster using both transepithelial and patch clamp recording (Doolin and Gilbertson, 1996; Gilbertson and Zhang, 1998; Gilbertson and Fontenot, 1998). The correlation between ENaC expression, particularly specific ENaC subunit expression, and functional amiloride-sensitive Na channels remains an open question that needs to be systematically addressed. Additional mechanisms, independent of apical and/or basolateral amiloride-sensitive Na channels, also likely contribute to salt transduction. Though the molecular components of these mechanisms have not been identified, several independent investigations have concluded that a non-specific apically localized cation channel contributes to sodium salt transduction (Doolin and Gilbertson, 1996; Gilbertson and Zhang, 1998; Miyamoto et al., 1999). This balance between amiloride-sensitive, amiloride-insensitive and paracellular pathways appears variable among different species and may account for some of the strain and species differences in salt responsiveness reported. D.

Proposed Models for Salt Transduction

The transduction of sodium salts, like that for other modalities, involves more than a single transduction scheme. The clearest example of a mechanism for sodium salt transduction in taste receptor cells involves the movement of Na ions through apically localized epithelial Na channels, similar to the ENaC channels found in other Na-absorbing epithelia. Simply, Na ions in the oral cavity diffuse down their electrochemical gradient, entering the taste cell through these open ENaC-like channels leading to a direct depolarization of the cell. As alluded to earlier, this

Molecular Physiology of Gustatory Transduction

depolarization activates voltage-dependent Ca2 influx and eventually release of the taste cell neurotransmitter onto gustatory afferents. The involvement of paracellular pathways in sodium salt transduction may also lead to taste cell activation. In this mechanism, the selective permeability of the intercellular tight junctions between taste cells allows the movement of small ions (Na, Cl, H) into the basolateral margins of the taste buds. Once in the interstitial space below the tight junctions, Na ions may diffuse through putative ENaC-like channels present on the basolateral membrane leading to a direct depolarization of the taste cell. The entire sodium salt response in taste cells cannot, however, be completely accounted for by the apical ENaC-like channels and by the aforementioned paracellular pathway. Thus, though there is little direct evidence for their existence, additional mechanisms have been hypothesized to account for these ENaC and paracellular independent pathways. To date, none of the molecular components of the salt transduction pathway have been cloned from taste tissue. Though the ENaC in other transporting epithelia have been cloned and functionally expressed, this has not been done for the ENaC-like channels of TRCs. Given the increase in molecular approaches being used in the study of the taste system, this shortcoming likely will be rectified in the near future. It will be of particular interest to know whether multiple variants of ENaC-like channels are present in taste cells that may explain the data showing the disjunction between ENaC expression and functional amiloride sensitivity in TRCs, as well as identify variants of the ENaC that may have different affinities for amiloride.

V.

ACID TRANSDUCTION MECHANISMS

A. Effect of Acidic Stimuli on Multiple Ion Channels and Other Targets The proton is primary stimulus for the transduction of acids, which humans refer to as sour (Settle et al., 1986). Acidic stimuli activate TRCs, afferent nerve fibers, and central gustatory neurons in a concentration-dependent fashion (for review, see Herness and Gilbertson, 1999). Protons have widespread actions, affecting a variety of pHsensitive cellular targets and coupled with their high permeability through many types of ion channels and intercellular junctions; it is not surprising that a variety of potential transduction mechanisms for these compounds have been described (see Fig. 1). One of the first descriptions of a mechanism underlying acid transduction involved a direct proton-mediated inhibition of an apical K channel in mudpuppy (Kinnamon et al., 1988;

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Cummings and Kinnamon, 1992). This conductance, partially open at the cells’ resting potential, would lead to a direct depolarization of the TRC. The applicability of this mechanism beyond lower vertebrates remains in doubt, however, since the evidence suggesting that there is an apical K conductance in mammals is scant (Gilbertson et al., 1992) and may be highly species dependent (Gilbertson and Zhang, 1998). Other mechanisms have been proposed in lower vertebrates that similarly have not been demonstrated in higher organisms to date. These include the ability of acids to alter the electrical coupling between TRCs (Bigiani and Roper, 1994) and to activate a cationic conductance (Okada et al., 1987, 1994; Miyamoto et al., 1988). B. Acid-Sensing ENaC-Like Channels and Acid Transduction The recent cloning of several new members of the Degenerin/ENaC superfamily of ion channels (Benos and Stanton, 1999) that have the capability to sense and respond to changes in acidity implicated their involvement in acid sensing in TRCs as well (Lindemann et al., 1999). The ENaC-like channel found in hamster TRCs had previously been implicated in acid transduction, owing to its significant proton permeability (Gilbertson et al., 1992, 1993), which is a hallmark of ENaC-like channels everywhere (Palmer, 1992). Significant proton influx through amiloride-sensitive Na channels apparently only occurs under conditions of low apical (mucosal) Na concentrations, as is typically the case in hamster (Rehnberg et al., 1992). This may partially explain why some species like rat and mouse do not show a strong inhibitory effect of amiloride on acid-induced responses in the taste system (Kinnamon et al., 1993, DeSimone et al., 1995) while others do (Gilbertson et al., 1993; Gilbertson and Gilbertson, 1994). Expression of alternative combinations of ENaC subunits, as discussed above, may also be predicted to alter the properties of acid responses in TRCs. Other amiloride-sensitive elements have also been implicated in the acid response in TRCs. Waldmann et al. (1997), in their report describing the cloning and functional expression of another member of the Degenerin/ENaC family, the acid-sensing channel, or ASIC, suggested that this channel may be responsible for acid transduction in the taste system as well. To date, several ASICs, including ASIC1, ASIC2a, ASIC2b, and ASIC4, have been identified in taste tissue (Liu and Simon, 2001; Ugawa et al., 1998). Using reverse transcription polymerase chain reaction, Liu and Simon (1999) failed to find ASIC (ASIC-) and another acid-sensitive member of the family, DRASIC (dorsal root acid-sensing channel), in

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TRC RNA, yet they did find a sensory-specific splice variant of ASIC (Chen et al., 1998), ASIC-, and another intriguing candidate for a role in taste transduction, the acid-sensitive vanillinoid receptor (VR1), that responds to capsaicin, heat, and low pH (Welch et al., 2000). Another candidate that has been identified in TRCs by in situ hybridization that may play a role in acid sensing is the mammalian degenerin-1 channel (MDEG1). This channel, also known as the brain-type Na channel or BNaCl, when expressed in heterologous systems like Xenopus oocytes, expresses large acid-induced currents (Ugawa et al., 1998). Recently, Lin et al. (2002) demonstrated that acid-induced currents in rat vallate taste receptor cells are functionally similar to ASIC2-mediated currents in both native cells and heterologous expression systems. Interestingly, acetic acid generates larger responses from MDEG-1 than does HCl at an equivalent pH, which correlates with the perceived intensities of these two acids by humans. Though it is not clear yet what roles these identified elements (ASIC, VR1, MDEG-1) actually play in acid taste, the ability of all members of this family to function as proton-gated cation channels that are sensitive to amiloride is consistent with the hypothesized mechanisms for acid taste transduction (Fig. 2). More detailed experiments linking these molecular approaches with the physiological properties of acid responses in TRCs are needed. Other acid-gated conductances have also been implicated as contributing to acid responses in mammalian TRCs. Two recent reports have independently demonstrated that acid responses in mouse (Miyamoto et al., 1998) and rat (Lin et al., 2000) TRCs are sensitive to the compound NPPB (5-nitro-2-[3-phenylpropylamino]benzoic acid). While Miyamoto et al., (1998) concluded the NPPB sensitivity was consistent with the activation of a Cl conductance, Lin et al. (2000) showed evidence consistent with acids activating primarily a NPPB-sensitive cation current, which was Cl independent. Finally, as mentioned above, the high permeability of protons through the paracellular pathways in the lingual epithelium has been hypothesized to play a role in acid taste (DeSimone et al., 1995). The data suggest that protons alter the permeability of the tight junctions between TRCs from cation-selective to anion-selective, changing the local ionic environment around the taste bud proper. Clearly, the properties of the paracellular pathway have a profound influence on the performance of the TRCs themselves (Stewart et al., 1997). TRCs also have the ability to track changes in extracellular pH intracellularly (Stewart et al., 1998), and these changes in intracellular pH have the potential to affect a variety of pH-sensitive intracellular targets (ion channels, enzymes). However, specific effects of intracellular changes in pH in TRCs

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have not been detailed. Finally, a recent study identified the presence of hyperpolarization-activated and cyclic nucleotide-gated channels (HCN1, HCN4) in taste tissue where they were shown to be responsive to low pHs associated with sour taste (Stevens et al., 2001). As with the case for sodium salt transduction, it has become clear that there are multiple mechanisms responsible for the transduction of acids by the peripheral gustatory system. C.

Proposed Models for Acid Transduction

Several different transduction pathways have been reported to be involved in acid sensing in TRCs. In amphibia, apically localized K channels have been shown to be inhibited directly by acids (i.e., protons) in a concentration-dependent fashion, leading to taste cell depolarization. Activation of cation channels by acids has also been reported and would be expected to depolarize taste cells via cation influx at normal TRC resting potentials. Direct proton influx through the ENaC-like channels has been reported in mammalian TRCs. Proton influx would directly depolarize the TRC, though this process apparently requires a low concentration of mucosal Na ions in order for sufficient proton entry to activate the TRC. The molecular identification of several acid-sensing channels in taste cells may prove promising to our understanding of acid transduction, but there is little physiological evidence to support a functional role for these candidate transduction elements. The recent finding that NPPB-sensitive conductances in TRCs may contribute to acid taste implicates the involvement of other ion channels in acid transduction. Finally, similar to the contribution of the paracellular pathway to sodium salt taste transduction, the proton permeability of the tight junctions in taste buds and the ability of TRCs to track these pH changes may contribute to acid sensing. It is clear that the ubiquity of proton effects on ion channels and other acid-sensitive intracellular targets will result in the identification of numerous candidates for acid transduction components.

VI. AMINO ACID TRANSDUCTION MECHANISMS Monosodium glutamate (MSG) has often been considered to have a unique taste, separate from the other four generally agreed-upon basic taste qualities of sweet, bitter, salty, and sour (Yamaguchi, 1991). Yet until very recently, little was known about how the amino acid glutamate is detected in TRCs. Most of the early research on amino acid transduction mechanisms centered on the channel catfish, Ictalurus punctatus, which has an exquisitely sensi-

Molecular Physiology of Gustatory Transduction

tive system for the detection of amino acids. The catfish has at least three broad classes of amino acid receptors. Individual receptors for L-arginine and L-proline activate cation channels, while those for the third major class, L-alanine, activate metabotropic receptor(s) that ultimately lead to production of the second messengers, cAMP and IP3 (for reviews on amino acid transduction in catfish, see Brand et al., 1991; Caprio et al., 1993). More recently, however, the emphasis on amino acid transduction mechanisms has shifted to mammalian systems and the mechanism underlying umami taste. Though L-glutamate activates both ionotropic and metabotropic receptors in mammalian TRCs, the latter is believed to represent the mechanism for the taste of umami (Chaudhari and Roper, 1998). Consistent with the ability of L-glutamate to activate two types of receptors, two types of responses are recorded from rat TRCs during glutamate stimulation. In patch clamp recordings of rat TRCs, Lin and Kinnamon (1999) showed that depolarizing responses to Lglutamate were mimicked by N-methyl-D-aspartate (NMDA), inhibited by NMDA antagonists like AP-5 (D-2amino-5-phosphonobutyric acid) and potentiated by glycine. This response was thought to be representative of synaptic responses to L-glutamate. Hyperpolarizing responses to L-glutamate, on the other hand, were thought to represent the transduction mechanism for umami taste. Consistent with activity at metabotropic glutamate receptors (mGluR), these hyperpolarizing responses were inhibited by the mGluR antagonist CPPG ([RS]--cyclopropyl4-phosphophenyl-glycine) and mimicked by the mGluR agonist L-2-amino-phosphonobutyric acid (L-AP4). Evidence for a role of cAMP in the hyperpolarizing response was demonstrated by the ability of a membrane permeant analog of cAMP (8-bromo-cAMP) to inhibit this response. Thus, one might propose a mechanism for umami taste involving mGluR activation, leading to activation of a PDE and a subsequent decrease in intracellular cAMP concentration (Fig. 1). The ultimate target that is modulated by this decrease in cAMP is not known, but has been hypothesized to be the disinhibition of a cNMP-suppressible channel (Chaudhari et al., 1996). The hypothesis that the umami receptors may be related to glutamate receptors in the mammalian brain was initially posed during experiments that demonstrated that ligands of brain glutamate receptors (ibotenate, aspartate, tricholomic acid, homocysteinic acid) could elicit umami taste in humans (Maga, 1983) and induce responses in the chorda tympani nerve (Faurion, 1991). Because of their ability to bind peptides that can elicit umami taste, type 4 metabotropic glutamate receptors (mGluR4) have been suggested to play a role in the taste response to MSG (Monastryskaia et al., 1999). Identification of a taste-spe-

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cific variant of mGluR4 has provided support for this theory. Chaudhari and colleagues (2000) successfully cloned and expressed this mGluR4 variant, called taste-mGluR4, that has all the hallmarks expected of the umami receptor. Consistent with Lin and Kinnamon’s electrophysiological study on native rat TRCs (1999), the heterologously expressed taste-mGluR4 bound glutamate in the millimolar range was inhibited by L-AP4 and led to decreased production of cAMP. In addition, taste-mGluR4 activation by L-glutamate was potentiated by 5’-ribonucleotides, another property of umami responses in vivo (Yamamoto et al., 1991). Though not definitively proven, it appears that the taste-mGluR4 is an excellent candidate for the receptor mediating umami taste. Two recent studies have shed more light on the mechanism(s) underlying the response to L-amino acids like Lglutamate. The heterodimer T1r1/T1r3 is expressed in a subset of taste receptors cells and responds to a variety of L-type amino acids (Li et al., 2002; Nelson et al., 2002). Moreover, as predicted for an umami receptor, responses of heterologously expressed T1r1/T1r3 to L-type amino acids were potentiated by 5’-ribonucleotides and were elicited by concentrations of amino acids that were physiologically relevant. Thus, the T1r1/T1r3 heterodimer appears to be a broadly tuned amino acid receptor that may contribute to umami taste. The relative contributions of mGluR4 and T1r1/T1r3 to umami taste remains to be determined.

VII. UNASSIGNED CANDIDATE TRANSDUCTION ELEMENTS In a number of cases, ion channels, G proteins, and GPCRs have been molecularly cloned from TRCs or otherwise shown to be expressed in TRCs but, lacking functional data, could not be assigned even tentatively to any transduction pathway(s). In the absence of such corroborating evidence, these potential taste transduction elements must be considered candidates only. Prior to the identification of the T2R/TRB GPCRs taste receptors, two groups had cloned seven transmembrane-helix receptors from taste tissue (Abe et al., 1993a,b; Matsuoka et al., 1994): these cloned receptors were quite similar to the family of odorant receptors cloned by Buck and Axel (1991). However, it seems unlikely that these particular receptors play any prominent role in taste transduction: in one case it was shown that the receptor is not expressed in the taste buds, but rather in the surrounding nontaste epithelium (and in this case only under conditions of reduced stringency) (Abe et al., 1993b). In the other case (Matsuoka et al., 1994), Northern blot analysis showed that the predominant site of expression was in the olfactory epithelium (orders

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of magnitude higher than in tongue epithelium). Furthermore, mRNA expression levels in tongue containing CV papillae, foliate papillae, or no taste papillae were approximately equal, suggesting that this receptor is expressed at only very low levels and in the nontaste epithelium of the tongue. To prove that a receptor protein plays a role in taste transduction requires the demonstration of its presence in the TRCs themselves, followed by a demonstration of the biological function of the protein in taste transduction. VIII.

FUTURE PROSPECTS

During the past 10 years our knowledge of the molecular components underlying taste transduction has increased dramatically. The powerful combination of biochemistry, molecular biology, electrophysiology, genetics, and transgenic mouse models has provided several important new insights into the cellular and molecular mechanisms of taste transduction. The identification and molecular cloning of gustducin’s  subunits, followed by the generation and in vivo analysis of -gustducin knockout mice provided an understanding of the physiology of taste transduction at the molecular level. The use of molecular cloning and genomics to identify and clone taste receptors responsive to bitter (T2R/TRB), sweet (T1r2, T1r3), and glutamate (T1r1, T1r3, mGluR4) has opened up new avenues to approach these pathways. Behavioral and electrophysiological analysis of transgenic and knockout mice altered in these (and other) taste transduction components will provide new insights into the functions of these particular proteins and taste pathways in general and provide a means to test specific hypotheses regarding the involvement of particular components in specific taste transduction pathways. During the next 5 years we expect it to be possible to reconstitute entire taste transduction pathways in vitro using cloned and/or biochemically purified reagents. ACKNOWLEDGMENTS This research was supported by the following grants: NIH DC03055 and NIH DC03155 to RFM; NIH DC02507, NIH DC00353, and a Novartis Research Award to TAG. RFM is an Associate Investigator of the Howard Hughes Medical Institute. REFERENCES Abe, K., Kusakabe, Y., Tanemura, K., Emori, Y., and Arai, S. (1993a). Multiple genes for G protein-coupled receptors and their expression in lingual epithelia. FEBS Lett. 316:253–256.

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35 Gustatory Neural Coding David V. Smith University of Tennessee Health Science Center, Memphis, Tennessee, U.S.A.

Thomas R. Scott San Diego State University, San Diego, California, U.S.A.

I.

INTRODUCTION

II. TASTE-GUIDED BEHAVIOR AND INFORMATION EXTRACTION

By providing sensory information important for decisions about ingestion and rejection, the sense of taste serves as the gateway to the body’s internal milieu. Gustatory neurobiologists generally agree on the existence of at least four basic taste qualities—sweet, salty, sour, and bitter—although they disagree about the possible existence of others (e.g., the taste of amino acids). These qualities and the behaviors they elicit help to ensure the animal’s energy supply (carbohydrates, amino acids), maintain the proper electrolyte and pH balance (salts, acids), and avoid the ingestion of toxins (acids, alkaloids). Of considerable debate is the way in which taste quality is represented in the activity of gustatory neurons. Many investigators accept the idea that peripheral gustatory nerve fibers and central neurons can be classified into groups on the basis of similarities and differences in their response profiles and other biological properties. Taste neurons have typically been characterized by analyses that employ a neuron classification scheme in an attempt to impose some order on these data. However, the breadth of tuning and multimodal sensitivity of central gustatory neurons make it difficult to accept a strict labeled-line code for taste quality, in which each neuron type signals a particular quality. Rather, it is likely that the various neuron types play a critical role in defining unique across-neuron patterns, which can unambiguously represent gustatory quality.

A. The Role of Taste in Behavior: Ingestive Decisions Before examining how information about taste stimuli is coded in the nervous system, it is instructive to consider the functions that taste serves in the life of an organism. The gustatory system functions as the gatekeeper of the internal milieu, acting to guide ingestive and avoidance behaviors. Because the anatomical organization of the central taste pathway parallels that of other visceral sensory input, and because taste stimulation triggers autonomic as well as perceptual responses, it is useful to consider taste as a component of the visceral afferent system (Norgren, 1985). Taste serves both a discriminative and an affective function, allowing animals to make distinctions among potential foods and providing information that guides decisions about the ingestion or rejection of chemical stimuli (Scott, 1987b). 1.

Perception Versus Visceral Function

As humans, it is natural for us to think about the gustatory system in terms of our own experience. Consequently, research on the taste system has been directed primarily toward understanding the neural basis of the human sensations of sweet, salty, sour, and bitter. These qualities are associated with stimuli that are important for animals to 731

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recognize as nutrients or toxins (Carpenter, 1956; Pfaffmann, 1964). Certainly these perceptual categories are important to our investigation of the taste system, but input from gustatory receptors also directly controls an array of reflexive motor responses involved in food ingestion and a range of autonomic responses important for digestive and other homeostatic processes, such as electrolyte balance. Salivary, gastric, and pancreatic secretions and cardiovascular and thermoregulatory responses resulting from gustatory stimulation are referred to as cephalic phase reflexes, which prepare the body for the arrival and utilization of nutrients (Friedman and Mattes, 1991). Because gustatory stimulation produces a variety of responses beyond taste perception, understanding the functional neural organization of the gustatory system requires consideration of the many roles that taste plays in the physiology of the organism, from perception to autonomic function. 2. Ingestion and Rejection: Taste Reactivity Grill and Norgren (1978b) first described the reflexive behaviors in the rat resulting from gustatory stimulation. They termed these ingestive and aversive response sequences, which largely reflect stimulus palatability, “taste reactivity.” Sucrose and other preferred stimuli evoke a pattern of ingestive responses that begins with rhythmic mouth movements followed by midline tongue protrusions, lateral tongue flicks, and swallowing. Aversive stimuli, such as quinine, produce a response sequence that includes oral gapes and a number of somatic motor responses associated with rejection, such as chin rubbing, paw flailing, head shaking, and face washing. These response sequences occur in decerebrate rats (Grill and Norgren, 1978c), suggesting that the neural substrate for these reflexive behaviors is contained within the brainstem. In intact rats, but not in those that are decerebrate, these responses are modifiable by learning, so that a normally ingestive sequence becomes an aversive one after a conditioned taste aversion (Grill and Norgren, 1978a). In general, the results from taste reactivity tests correspond with other short-term palatability measures such as lickrate tests (Schwartz and Grill, 1984). The patterns of taste reactivity responses are quite distinct for stimuli of different taste qualities (Brining et al., 1991). Closely related stimuli produce strikingly similar patterns of taste reactivity, as shown in Figure 1. Patterns of taste reactivity to the four basic stimuli are shown in Figure 1A, and the similarities among patterns for 12 taste stimuli are depicted in the factor space of Figure 1B. Based on correlations among the patterns of taste reactivity, these stimuli fell into four groups: sugars, bitter-tasting substances,

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acids, and sodium salts. These various experiments demonstrate that much of the neural distinction among stimuli of different qualities, as evidenced by specific patterns of motor output, can occur within the hindbrain and does not require the contribution of more rostral levels of the gustatory system. 3.

Generalization and Discrimination

In order to make decisions about ingestion or rejection, the ability to discriminate among different taste stimuli is essential. Behavioral studies have employed operant conditioning techniques or conditioned taste aversion generalization to determine the discriminative capacity of the gustatory system of experimental animals. A taste aversion can be conditioned to a particular tastant by pairing that stimulus with gastrointestinal malaise produced by drugs or radiation (Garcia et al., 1970). Following such learning, the degree to which the aversion generalizes to other stimuli serves as an indication of the gustatory similarity between the conditioned stimulus and any others (Nachman, 1963; Smith et al., 1979; Nowlis et al., 1980; Nowlis and Frank, 1981). The results of these kinds of studies show that hamsters and rats generalize a learned sucrose aversion to several other stimuli described as sweet by humans, including fructose, glucose, and sodium-saccharin (Smith et al., 1979; Nowlis et al., 1980). These techniques also demonstrate that a number of sodium and lithium salts are considered similar by rodents and distinct from nonsodium salts such as KCl or NH4Cl, which are behaviorally similar to acids. Finally, bitter-tasting substances, such as quinine hydrochloride (QHCl) and MgSO4, show cross generalization in these experiments. To a considerable extent, such generalization studies produce groupings of gustatory stimuli that are similar to those produced by analyzing the responses of peripheral or central gustatory neurons (see Fig. 7 below) or the patterns of taste reactivity (Fig. 1). Discriminative operant conditioning has been used to examine both generalization and discrimination among gustatory stimuli and the role of different cranial nerve inputs in taste-guided behavior. Using shock avoidance, Erickson (1963) demonstrated that rats conditioned to avoid KCl would also avoid NH4Cl but not NaCl. A similar pattern of generalization occurred with an appetitive operant task, in which rats were trained to respond on one of two levers, depending upon the taste of a previously conditioned stimulus (Morrison, 1967). Using similar operant techniques, Spector and colleagues have examined the role of the chorda tympani (CT) and glossopharyngeal (IXth) nerves in taste discrimination performance

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Figure 1 Taste reactivity in the hamster. (A) Profiles of taste reactivity responses to the four basic stimuli, depicting the percentage of animals showing each of eight different responses. Abbreviations: LTP, lateral tongue protrusions; TP, tongue protrusions; LO, locomotion; CR, chin rubbing; FF, forelimb flailing; AP, aversive posturing; FR, fluid rejection, and G, gapes. (B) Two-dimensional representation of the correlations of hamster taste reactivity profiles to 12 different stimuli with two common factors (I and II). Each point in the space represents the correlation of one stimulus profile with two factors. Each stimulus profile consisted of eight different measures of taste reactivity, ranging from lateral tongue protrusions to oral gapes (see A). Zero is at the origin, where the axes cross, and  0.50 is marked on each axis by crossing line segments. A value of  1.0 is indicated by the intersections of the circle with the axes. A point would fall on the circumference of the circle if 100% of the variance of its profile were accounted for by these two factors. Stimuli with similar taste quality (e.g., the sugars or the bitter-tasting stimuli) produce similar patterns of taste reactivity, as indicated by their proximity within this factor space. The first factor (I) separated the highly preferred sugars from the highly aversive bitter-tasting substances, which showed opposite patterns of taste reactivity (see A). (Data from Brining et al., 1991.)

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in the rat. Transection of the CT nerve but not the IXth nerve severely impairs the ability of rats to discriminate between NaCl and KCl (Spector and Grill, 1992; St. John et al., 1997). These and other data suggest that information arriving via the CT nerve is important for discriminating NaCl from nonsodium salts. Treatment of the tongue with amiloride, which blocks the response in NaCl-best fibers of the CT nerve to NaCl, completely eliminates the ability of rats to make a NaCl/KCl discrimination (Spector et al., 1996). In addition, when rats are conditioned to avoid NaCl in the presence of amiloride, they generalize that aversion to KCl (Hill et al., 1990). Since rats do not have an amiloride-sensitive response to NaCl in the IXth nerve (Formaker and Hill, 1991) nor even any N-best IXth nerve fibers (Frank, 1991), it is undoubtedly the comparison between N-best and other nerve fibers in the CT and perhaps the greater superficial petrosal (GSP) nerve, serving the palate, that allows this discrimination. Similarly, VIIth nerve transection virtually eliminates the ability of rats to discriminate quinine from KCl, whereas IXth nerve damage has no effect (St. John and Spector, 1998). This latter result was surprising because the IXth nerve is highly responsive to quinine and its individual fibers respond differentially to quinine and KCl (Frank, 1991). These data imply that the VIIth nerve is more important for the discrimination of taste stimuli, whereas the IXth nerve serves a protective function (St. John and Spector, 1998). For example, the IXth nerve clearly contributes to the avoidance of quinine and other stimuli (Spector and St John, 1998; Markison et al., 1999) and is important in the elicitation of aversive taste reactivity (Travers et al., 1987). B. What Information Is Coded by Gustatory Neurons? Most neurophysiological studies of the gustatory system have focused on the neural representation of gustatory quality. Single peripheral gustatory fibers and central neurons are broadly sensitive across taste qualities and stimulus concentrations and often respond to tactile and thermal stimuli as well. Understanding the functional role of a taste-responsive neuron requires an appreciation of the kinds of information that must be coded by these cells. 1. Quality, Intensity, and Hedonic Tone: A Nutritional Dimension The gustatory system codes at least three types of information about chemical stimuli: quality, intensity, and hedonic value. The unique perception of taste quality is the

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defining feature of this sense. Most researchers agree that human gustatory experience includes the sweet, salty, sour, and bitter qualities (McBurney and Gent, 1979). However, there has been considerable debate over whether some other qualities (e.g., umami—the taste of monosodium glutamate) should be included or whether any qualities warrant being designated as “basic” (McBurney, 1974; Erickson, 1977; Yamaguchi, 1979; Schiffman and Erickson, 1980). Intensity is a dimension common to all sensory systems, reflecting the magnitude of the evoked sensation. In the gustatory system, increasing stimulus concentration generally leads to greater action potential frequency in the response of single neurons, as shown in the responses of four neurons in the nucleus of the solitary tract (NST) of the hamster in Figure 2. The responses of individual neurons are not always monotonic across stimulus concentration, which results in some variation in each cell’s response profile. Nevertheless, increases in impulse frequency and the recruitment of additional cells at higher concentrations are the mechanisms by which gustatory neurons likely code information about stimulus intensity. Hedonic value, the perceived pleasantness or unpleasantness of a taste sensation, is a response based on genetic, physiological, and experiential factors as well as the characteristics of the stimulus. Taste is an inherently hedonic sense, with close ties to motivated behavior (Pfaffmann, 1964). Although these three dimensions of taste information (quality, intensity, and hedonic value) can be assessed separately, they are not independent. For example, the perceived qualities of some taste stimuli (e.g., Na-saccharin) change with concentration. Similarly, the hedonic value of a stimulus is largely determined by its quality and intensity. Many omnivorous mammals share a concentration-dependent preference for substances that humans describe as tasting sweet or salty and an avoidance of substances humans term sour or bitter (Richter and Campbell, 1940; Carpenter, 1956; Pfaffmann, 1964). Presumably, these predispositions reflect evolutionary pressures related to the ingestional consequences (i.e., nutritional or toxic) of potential foods. Indeed, omnivores inherit a taste system that permits accurate evaluation of stimulus nutritive value or toxicity across a wide range of chemicals with diverse physical characteristics (Scott and Mark, 1987; Scott and Giza, 2000). The neural code that underlies this behavior consists of afferent activity that parcels chemicals out according to the degree to which they offer sources of nutrition (carbohydrates, proteins) or disrupt physiological activity (extremes of pH, alkaloids, etc.). In retrospect, this is a reasonable organization for the taste system, which was shaped by evolutionary pressure to select nutrients from among the wide array of useless and toxic chemicals in

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Figure 2 Concentration-response functions for four hamster PbN neurons. (A) Responses (impulses/s) to each stimulus at five concentration levels. Zero is the level of spontaneous activity and the dashed line indicates 1 SD above spontaneous rate. (B) Response profiles of each cell at three concentration levels (lowest, middle, and highest of each series). The excitatory breadth of tuning (H) at each concentration level is indicated above each profile. Both the breadth of tuning and the most effective stimulus varied with concentration. (From Van Buskirk and Smith, 1981.)

the environment. Selection among foragers presumably favored those with a taste system that activated the appropriate hedonic tone to match the consequences of ingestion: attraction to nutrients and revulsion by toxins (Scott and Mark, 1987). This organization of the gustatory system is largely determined by genetic and developmental factors that

configure the system to match the available chemicals with the animal’s nutritional needs. However, there are also mechanisms allowing experience and physiological factors to influence gustatory neural processing and perception. Species-specific predispositions that impart hedonic value to a stimulus can be overcome via learned preferences or aversions (London et al., 1979; Grill, 1985; Bertino et al.,

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1986; Rameriz, 1991) or in response to metabolic or pharmacological manipulations (Richter, 1956; Parker et al., 1992). Responses to gustatory stimulation recorded from brainstem cells in rodents are subject to several modulatory influences, including the effects of gastric distension (Glenn and Erickson, 1976), blood glucose and insulin levels (Giza and Scott, 1983, 1987), intraduodenal lipids (Hajnal et al., 1999), degree of sodium deprivation (Jacobs et al., 1988; McCaughey and Scott, 2000) and conditioned taste aversion learning (Chang and Scott, 1984). That is, the responses of rodent brainstem cells are modulated not only by stimulus quality, intensity, and modality, but also by the animal’s physiological state and prior experience. In primates, the responses of taste neurons in the orbitofrontal cortex (Rolls et al., 1988), but not in the NST (Yaxley et al., 1985) or primary taste cortex (Rolls et al., 1988), are modulated by the animal’s state of satiety. Although the circuitry underlying such modulatory effects on taste processing is not well understood, in rodents there are descending connections to brainstem taste nuclei from all of the forebrain areas that receive gustatory input, including the cortex, amygdala, and lateral hypothalamus. Descending influences on brainstem taste responses can be either excitatory or inhibitory (Hayama et al., 1985; DiLorenzo, 1988; Mark et al., 1988; DiLorenzo, 1990; DiLorenzo and Monroe, 1995). Neurophysiological studies employing both in vitro (King et al., 1993; Liu et al., 1993; Wang and Bradley, 1993, 1995; Bradley et al., 1996) and in vivo (Smith et al., 1994, 1998; Davis and Smith, 1997; Li and Smith, 1997; Smith and Li, 1998, 2000) preparations have implicated a number of neurotransmitters and peptides in the modulation of NST neuronal activity. Thus gustatory afferent input provides at least three types of information, interrelated in complex ways. This input guides the organism to make appropriate ingestive decisions, directed by physiological and nutritional needs, which provide modulatory feedback over brainstem gustatory neurons. How taste intensity, quality, and hedonic value are represented in the nervous system is the problem of gustatory neural coding. 2. Taste and Visceromotor Integration The processing of gustatory information is often discussed solely in terms of sensory coding and taste discrimination. However, it is becoming more difficult to discuss the gustatory system outside the context of visceromotor integration. Norgren and colleagues (Norgren, 1985, 1995; Travers, 1993) have provided compelling arguments that a more contemporary view of gustation and the autonomic nervous system should

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include considerations of the numerous anatomical interconnectons between visceral afferent and efferent systems and the brainstem gustatory relay nuclei. Gustation is represented in the rostral pole of a functional column that also receives input related to respiration, blood pressure, blood pH, gastrointestional activity, and pain. Anatomically, this functional column corresponds to the NST, with the gustatory zone located in the rostral half of the nucleus. This visceral afferent column is closely associated with both somatic motor nuclei (i.e., the retrofacial area, motor nucleus of V, nucleus ambiguus, and hypoglossal nucleus) and visceromotor (salivatory and vagal preganglionic parasympathetic neurons) nuclei. Collectively, these cell columns contain motor neurons that initiate chewing, tongue movement, salivation, swallowing, gastrointestional motility, acid secretion, and other reflexes associated with eating, drinking, and painful stimulation. The gustatory and other general visceral afferent systems provide the requisite sensory information to control these assorted visceromotor reflexes. Some reflexes are clearly involuntary, such as salivation or baro- and chemoreceptor regulation of cardiac and respiratory rates. Other actions have a voluntary or somatic motor component that is generally considered visceromotor in function, such as the stereotypic tongue and swallowing movements that occur during ingestion. Activation of the gustatory system is necessary for an organism to assess the palatability of food or drink. Taste input generates activity in the ascending gustatory pathway and presumably evokes a perceptual component that involves an awareness of taste quality. Simultaneously, taste stimulation activates the afferent limb of a variety of somatic and visceromotor acceptance or rejection reflexes associated with eating and drinking (Grill and Norgren, 1978b). Electrophysiological studies have shown that responses to sucrose and quinine generally (but not always) occur in separate subsets of cells, suggesting parallel systems that control the ingestion of nutrients and the rejection of toxic substances (Travers and Smith, 1979; Smith et al., 1994). The relatively high sensitivity of the CT and GSP nerves to sugars could selectively activate a set of second-order neurons in the gustatory NST that ultimately drive brainstem circuits that initiate tongue protrusions and other ingestive responses. In contrast, the IXth nerve shows a relatively greater response to bitter-tasting stimuli like quinine and could activate a separate population of gustatory NST neurons that project to brainstem regions that initiate a sequence of protective responses. These are extreme examples and involve specific sensory inputs that generate opposite stereotypic behaviors (as indicated in Fig. 1). In the case of most NST neurons, which have multiple sensitivities to a wide range

Gustatory Neural Coding

of sapid stimuli (as in Fig. 2), the scenario is more complex because the output of such neurons would have to activate a mixture of appropriate motor repertoires reflecting the hedonic quality of the stimulus. Although the descending projections of the gustatory NST have been studied, the differential contributions of sucrose-or quinine-responsive NST neurons, for example, to this caudally directed pathway are not known. A multisynaptic pathway(s) supports these ingestive and rejection reflexes and probably involves intrinsic projections of the gustatory NST to other parts of the NST and to premotor brainstem areas involved in swallowing, salivation, and chewing (Travers and Norgren, 1983; Travers, 1988; Beckman and Whitehead, 1991; Dinardo and Travers, 1997). The taste-responsiveness of NST neurons must provide a gating mechanism for differentiating palatable from unpalatable substances. III. BASIC RESPONSE PROPERTIES OF GUSTATORY NEURONS A universal characteristic of gustatory neurons is their responsiveness to stimuli representing more than one of the classic four taste qualities. The responses of these neurons are also modulated by changes in stimulus concentration and often by tactile and thermal stimulation. Therefore, any theory of the role of these neurons in coding sensory information must take this breadth of responsiveness into account. Even when gustatory neurons can be shown to be relatively narrowly tuned across taste qualities, as occurs in cells of the primate orbitofrontal cortex (Rolls, 1995), these neurons still can be modulated by stimulus concentration and are likely to be multimodal in their responsiveness. This multiple sensitivity raises a serious issue with regard to taste quality coding by labeled lines, as discussed below. A.

Gustatory Sensitivities of Taste Receptor Cells

Taste transduction involves a variety of mechanisms, including direct permeation or block of ion channels and activation of metabotropic and ionotropic receptors (for recent reviews, see Lindemann, 1996; Herness and Gilbertson, 1999) (see also chapter 34). Whereas most of the data that demonstrate these mechanisms have been obtained either through patch recording of isolated taste receptor cells or from molecular or biochemical methods, there is little information about how these mechanisms are distributed within and across receptor cells. Recent wholecell recording experiments from taste cells in intact tongue epithelium, however, confirm that taste receptor cells are broadly tuned to gustatory stimuli (Gilbertson et al., 2001).

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1.

Multiple Sensitivity of Rat Taste Receptor Cells

Intracellular recording experiments have suggested for several years that taste cells are broadly responsive to stimuli representing different taste qualities (Ozeki and Sato, 1972; Sato, 1972; Tonosaki and Funakoshi, 1984; Sato and Beidler, 1997). However, because of their relatively small membrane potentials and the possibility of leak currents associated with penetrating such small cells with sharp electrodes, many investigators have viewed these intracellular experiments with skepticism (Kinnamon, 1988; Avenet and Lindemann, 1989; Lindemann, 1996; Herness and Gilbertson, 1999). More recent experiments have employed patch-clamp recording methods on isolated taste receptor cells (Akabas et al., 1988; Kinnamon et al., 1988; Gilbertson et al., 1993; Cummings et al., 1996), but the range of stimuli that can be applied to an isolated cell preparation is limited and recording is hindered by having the apical and basolateral membranes in the same bathing medium. Recent experiments, however, have combined patch-clamp recording with apically restricted stimulus application (Gilbertson et al., 2001). Whole-cell recordings were made from 103 cells in rat fungiform taste buds maintained in an intact lingual epithelium. Up to six taste stimuli were applied to the apical membrane of each cell by perfusion through a closed mucosal chamber, which effectively separated the apical from the basolateral taste cell membranes. The data showed that individual taste receptor cells often exhibited a range of chemical sensitivities, confirming the earlier data obtained with intracellular electrodes. Over two thirds of the cells responded with a reversible change in membrane current (5 pA) to more than one of the four basic stimuli (sucrose, NaCl, HCl, or QHCl). However, the receptor cells showed greater stimulus specificity than typically seen in first-or second-order afferent neurons, which become more broadly tuned through convergence (see below). Thus, one source of the multiple sensitivity of peripheral and central gustatory neurons arises at the initial step of stimulus recognition by the taste receptor cells themselves. 2.

Random Distribution of Sensitivities: Maximum Information Transfer

Analysis of the distribution of responses to the four basic stimuli across rat fungiform taste cells demonstrates that the sensitivities to sucrose, NaCl, HCl, and QHCl are randomly distributed and independent of each other (Gilbertson et al., 2001). If the proportion of cells in a sample responding to each stimulus is known, it is possible, using the laws of probability, to predict the frequency of occurrence of various combinations of responses to the stimuli if the sensitivities to each are independent and

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randomly distributed. For example, in this experiment, 0.6 of the cells responded to sucrose and 0.5 responded to QHCl. A random distribution predicts that 0.3 (i.e., 0.6  0.5) should respond to both sucrose and QHCl, which was seen in the data. Similarly, all other possible combinations of sensitivities (NaCl with HCl or sucrose with NaCl or sucrose with HCl and QHCl, etc.) were also predictable by random combination. Similar analyses on the responses of rat CT and IXth nerve fibers (Frank and Pfaffmann, 1969) and on data obtained with intracellular electrodes on rat taste receptor cells (Ozeki and Sato, 1972) have also demonstrated a random distribution of sensitivities. The broad responsiveness of taste receptor cells could result from an overlap in the transduction mechanisms for different classes of taste stimuli within single receptor cells, as reported in hamster taste cells for sodium salts and acids, which both use the amiloride-sensitive Na channel (Gilbertson et al., 1992, 1993). Alternatively, multiple receptors and transduction cascades could be present within a single cell (see Glendinning and Hills, 1979) or there could be cell-to-cell communication within the taste bud (see Roper, 1993) that allows the sharing of information. Recent data showing the co-expression of a large number of a family of putative bitter taste receptors in single taste receptor cells (Adler et al., 2000; Chandrashekar et al., 2000) are not incompatible with the broad tuning of receptor cells, which leaves open the possibility that these same cells could express other receptors as well. Further molecular studies should be able to provide definitive evidence for the origin of the multiple sensitivities of individual taste receptor cells. At first glance, a random distribution of sensitivities seems to be counterintuitive. What possible advantage could there be to such an arrangement? One possibility may lie in the greater capacity of such a system for transmitting information (see Pfaff, 1975). A basic tenet of information theory is that multicomponent messages convey maximum information only when the individual components are independent of each other (Shannon and Weaver, 1959). This means that sensory systems that encode information by the pattern of activity across broadly tuned neural elements are inherently capable of transmitting greater amounts of information than systems using specifically tuned cells (Pfaff, 1975). The amount of information that can be transmitted by a system can be calculated from the probabilities of events, such as the proportions of various response combinations in taste receptor cells. An informational analysis of the response distributions across rat taste receptor cells indicates that the broad and independent distribution of sensitivities across these cells permits almost as much information as possible in the responses to four stimuli (3.93 bits) (Smith et al., 2000).

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The maximum information content, which would occur if all possible response combinations among these four stimuli (n
16) occurred with equal frequency, would be 4 bits (i.e., log2 16
4). On the other hand, if responses to the basic stimuli were restricted to only four kinds of cells, each specific to one of the basic tastes and equally distributed, the maximum information that could be transferred from the receptors to the afferent nerve would only be 2 bits (i.e., log2 4
2). In general, the greater the amount of information transfer, the finer the discriminations that can be made on the basis of the sensory input (see Pfaff, 1975). Even if one were to assume only four taste qualities, the many hundreds of potential gustatory stimuli would be composed of subtle combinations of these four. The ability of rats to make a behavioral discrimination between, for example, the taste of sucrose and maltose (Spector et al., 1997) or QHCl and KCl (St. John and Spector, 1998), depends on a system with more subtle discrimination capabilities than could be expected of one with specifically tuned cells. An information content of 4 bits provides four times the discriminability of one of only 2 bits. Thus, the extensive, independent distribution of taste sensitivities across receptor cells and the resulting broadly tuned afferent fibers and central neurons (see below) provides the substrate for an across-neuron pattern code, which permits relatively subtle behavioral discrimination performance (see also Pfaff, 1975). B. Multiple Sensitivity of Peripheral Taste Fibers The broad responsiveness of CT fibers in the rat, cat, and rabbit referred to above first led Pfaffmann (1941, 1955) to propose that taste quality is represented by the pattern of activity across these afferent nerve fibers. Since gustatory nerve fibers are not specifically tuned to a single taste quality, many investigators have attempted to classify these fibers into functional groups in an effort to impose order on the organization of gustatory afferent information. 1.

Responses to Basic Taste Stimuli: Response Profiles and Fiber Types

Although there have been several schemes for classifying taste fibers into meaningful groups, the most common are those based on similarities and differences in the fibers’ response profiles. Of these, the most widely adopted has been the “best-stimulus” classification, first used by Frank (1973, 1974) for grouping hamster and squirrel monkey CT nerve fibers. When the anterior tongue was stimulated by mid-range concentrations of 0.1 M sucrose, 0.03 M NaCl, 0.003 M HCl, and 0.001 M QHCl, fibers of the

Gustatory Neural Coding

hamster’s CT nerve could be categorized into one of three groups: sucrose (S)-best, NaCl (N)-best, or HCl (H)-best (Frank, 1973). In Frank’s study, only one CT fiber out of 79 responded best to QHCl (Q-best). Interestingly, when the stimuli were arranged in order of taste preference (i.e., S, N, H, and Q), the response profile of each best-stimulus group peaked at a single point (the best stimulus), with the responses to the “side-band” stimuli diminishing on either side. The responses of S-, N-, and H-best CT fibers of the hamster to 13 taste stimuli are shown in Figure 3A. These cells were relatively narrowly tuned to stimuli of a given class, but even at these moderate concentrations, they responded to stimuli of more than one quality. Similar groups were seen at central gustatory nuclei of the hamster (Fig. 3B, C) when the same stimuli were applied to the anterior portion of the tongue (Travers and Smith, 1979; Van Buskirk and Smith, 1981). The homogeneity of the response profiles within a beststimulus group made it relatively easy to predict the

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responsiveness of a taste neuron to other stimuli within the same quality class. That is, if a neuron responded vigorously to sucrose it almost always gave strong responses to other stimuli that are sweet to humans and that are classified as similar to sucrose by hamsters in behavioral experiments. Similar observations followed for the responses of N-best neurons to other sodium and lithium salts and of H-best neurons to other acids and nonsodium salts. Consequently, cluster analysis was applied to responses of taste fibers in the CT nerve (Frank et al., 1988) and to neurons in the NST and PbN (Smith et al., 1983b) of the hamster. The results gave strong support for the classification of these neurons into types on the basis of similarities and differences in their response profiles. The response profiles of S-, N-, and H-best neurons were strikingly different from one another, even when the responses to as many as 18 stimuli were used to define the broad profile of each type. There was enough similarity within each neuron type to warrant considering each to be

Figure 3 Mean neural response profiles for S-, N-, and H-best neurons in the CT nerve (A), the NST (B), and the PbN (C) of the hamster to an array of 13 stimuli, all at midrange concentrations (see Smith et al., 1983b; Frank et al., 1988). The response of each class of neurons to the four basic stimuli (sucrose, NaCl, HCl, and QHCl) are shaded. The mean breadth of tuning (H) is given above each profile. Breadth of tuning increased systematically from the periphery to the pons, especially the tuning of S-best cells. (Data are from Frank et al. (1988) for the CT and Smith et al. (1983b) for the NST and PbN.)

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a single class of neurons. Hierarchical cluster analysis has been applied in many other species and at several levels of the gustatory system (Van Buskirk and Smith, 1981; Frank et al., 1988; Hanamori et al., 1988; Scott and Giza, 1990; Giza and Scott, 1991; Travers, 1993; Rolls, 1995), and most investigators agree on the usefulness of classifying neurons into functional types. At the very least, describing taste neurons by their best stimulus provides a convenient method for imposing some order on the system. Beyond that, there are a number of studies suggesting that these neuron types are biologically relevant, e.g., studies showing that only N-and S-best neurons receive input from the amiloride-sensitive Na transduction mechanism (Hettinger and Frank, 1990; Scott and Giza, 1990; Giza and Scott, 1991; Boughter and Smith, 1998; Boughter et al., 1999; St.John and Smith, 2000). The role that these neuron types play in the coding of taste quality is addressed below. 2. Breadth of Responsiveness Because taste neurons typically respond to stimuli representing more than one taste quality, many investigators have attempted to describe their breadth of responsiveness across these stimuli. Early attempts generally employed idiosyncratic criteria for what constituted a response, making it difficult to compare cells in different species or at different levels of the gustatory system. Smith and Travers (1979) introduced a measure of the breadth of tuning based on the entropy equation used in information theory to measure information transmission. In this procedure, the response of a neuron to each stimulus is expressed as a proportion (pi) of the total response to all four. The breadth of tuning (H) is given by the equation: n

H
 冱 pilog pi i
1

where H is the breadth of responsiveness and K is a scaling constant (1.661 for four stimuli). The value of H varies from 0.0, indicating a neuron that responds exclusively to one of the four stimuli, to 1.0, indicating a neuron that responds equally to all four. Therefore, larger values of H reflect a greater breadth of tuning across the four basic stimuli. This measure has been used extensively to compare the breadth of tuning of gustatory neurons across different levels of the nervous system (e.g., see Fig. 3) (Van Buskirk and Smith, 1981; Rolls, 1995) and across species (Travers, 1993). Because gustatory neurons respond to different stimulus classes and are also modulated by stimulus concentration, the response profile of a cell, and its apparent breadth of tuning, can vary with stimulus intensity. Taste neurons in the

hindbrain typically increase their firing rates as stimulus concentration is increased, but the slopes of these concentration-response functions often vary across stimuli, even in the same neuron (see Fig. 2). Consequently, the shapes of the response profiles may be considerably different across the range of effective stimulus concentrations. As seen in auditory tuning curves (Kiang, 1965) and in other sensory modalities, increasing stimulus intensity generally produces greater breadth of responsiveness (Smith and Travers, 1979). When stimulus concentrations are reduced, cells become more narrowly tuned until they only respond to a single (best) stimulus. Therefore, measures of the breadth of tuning of gustatory neurons, regardless of the method used to quantify this concept, can be greatly confounded by the relative concentrations of the test stimuli. Making comparisons across neural levels, across experiments from different laboratories, and especially across species is a difficult task. There are instances, however, where stimulus concentrations and other factors have been held constant across experiments, allowing a direct comparison of the breadth of tuning of taste neurons under different conditions, as in Figure 3 (Van Buskirk and Smith, 1981; Rolls, 1995). 3.

Ambiguity in the Response of One Cell

Because the responses of taste neurons are broadly tuned across both quality and intensity, the response of any one neuron is entirely ambiguous with respect to either parameter (Pfaffmann, 1955, 1959). In addition, gustatory neurons are often responsive to thermal and tactile stimuli (Ogawa et al., 1968; Travers and Smith, 1984; Hanamori et al., 1987; Travers, 1993), as discussed below. This multimodal sensitivity is not entirely unexpected because taste is only one aspect of oral sensation and both temperature and touch have been shown to influence taste perception (Moskowitz and Arabie, 1970; Bartoshuk et al., 1982; Green and Frankmann, 1987; Cruz and Green, 2000). Nevertheless, impulse traffic in a single neuron may be related to several stimulus modalities, making the unambiguous interpretation of that signal impossible without comparing it to activity in other cells (Crick, 1979; Erickson, 1982a, 1984). 4.

Neuron Types in Different Nerves and Different Species

The response profiles of gustatory nerve fibers have been characterized in several species, but those in the hamster are the easiest to compare because the data were collected under similar conditions in each experiment. Fibers of the facial (VIIth) nerve innervate taste buds on the anterior tongue via the CT nerve and on the soft palate and within the nasoincisor ducts via the GSP nerve. Within the hamster

Gustatory Neural Coding

CT nerve, there are three distinct fiber types: S-, N-, and H-best (Frank, 1973). Mean response profiles for 20 S-, 41 N-, and 17 H-best CT fibers are shown in Figure 3A. In the CT nerve, the S-best fibers are clearly the most specifically tuned (see Fig. 3A), with a mean breadth of tuning (H) of 0.39, compared to 0.59 and 0.67 for the N- and H-best fibers, respectively (Van Buskirk and Smith, 1981). The S-best fibers are quite narrowly tuned to sucrose and other sweet-tasting stimuli, with some sensitivity to NaCl and very little response to HCl or QHCl. In comparison to the rat, the CT nerve of the hamster is considerably more responsive to sweet stimuli. The greater breadth of tuning of the N- and H-best fibers in the hamster CT arises predominantly from the fact that these fiber types respond to both NaCl and HCl, albeit to different degrees. There is considerably more sensitivity to sweet stimuli in the GSP nerve of both the rat (Nejad, 1986) and hamster (Harada and Smith, 1992) than in the CT nerve of either species. This difference is more striking in the rat than the hamster. In addition, single-neuron recordings have been made from cells in the rat NST that respond to stimulation of the nasoincisor ducts (Travers and Norgren, 1991), showing a considerable number of S-best neurons, many of which receive converging input from both GSP and CT nerves. Behavioral experiments have also shown that taste receptors innervated by the GSP contribute substantially to the rat’s ability to respond to sweet-tasting stimuli (Krimm et al., 1987; Spector et al., 1993, 1997). Bitter stimuli, such as QHCl, are relatively ineffective in driving fibers of either the CT or the GSP, at least at mid-range concentrations. Fibers of the glossopharyngeal (IXth) nerve, however, are much more responsive to QHCl in both the rat and hamster. In comparison to the hamster’s CT nerve, where there are few Q-best and many S- and N-best fibers, the distribution of these fiber types is reversed in the IXth nerve. The magnitudes of the responses to these chemicals are also reversed between the CT, where sucrose and NaCl produce much larger responses, and the IXth nerve, where the response to QHCl is considerably greater than that to either sucrose or NaCl. In the rat, many Q-best fibers are found in the IXth nerve and no N-best fibers at all (Frank, 1991). Consequently, the information projected to the NST in both of these species by the VIIth nerve is dominated by preferred stimuli (sucrose and NaCl), whereas that from the IXth nerve is more concerned with aversive stimuli (QHCl). As noted above, however, behavioral discrimination among all types of stimuli appears to depend predominantly on input from the VIIth nerve, rather than the IXth. Taste buds within the laryngeal mucosa are innervated by fibers of the internal branch of the superior laryngeal nerve (SLN), a branch of the vagus (Xth) nerve. Unlike

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gustatory fibers of the VIIth and IXth nerves of the hamster, those of the SLN do not discriminate among taste qualities (Smith and Hanamori, 1991). These fibers respond well to water (W), NaCl, and HCl, but poorly to sucrose and QHCl. On the basis of response magnitude, it was possible to identify 26 W-best, 17 N-best, 20 H-best, and 2 Q-best fibers in the hamster SLN, but a hierarchical cluster analysis showed that these fiber types were not at all distinct, as are those in the CT and IXth nerves of this species. Instead, the sensitivities of SLN fibers were graded along a continuum, with no distinct differences among fibers responding best to different stimuli. Laryngeal nerve fibers do not discriminate well among the four basic taste stimuli, but appear to serve a role in protection of the airway from foreign substances. Taste fibers recorded from primates show sensitivities similar to those of the rodent species that have been investigated, except that they appear to be somewhat more narrowly tuned across the four basic taste qualities. Responses of 45 CT fibers of the squirrel monkey were divisible into distinct S-, N-, or H-best categories, very similar to those observed in the hamster CT nerve (Frank, 1974; Pfaffmann et al., 1976). Although the breadth of tuning was not quantified for these fibers, the squirrel monkey CT fibers were somewhat more narrowly tuned to their best stimulus than those of the hamster. Fibers of the macaque CT nerve are divisible into four relatively distinct groups, responding best to sucrose, NaCl, HCl, or QHCl (Sato et al., 1975). Like the squirrel monkey CT fibers, those in the macaque appear to be more specifically tuned to their best stimulus than those in either rat or hamster. Recent data from the chimpanzee also suggest a high degree of specificity of CT afferent fibers (Hellekant and Ninomiya, 1991). Thus, in comparison to rodents, the peripheral gustatory afferent fibers of primates are more narrowly tuned to the basic taste stimuli. C. Convergence and Breadth of Tuning of Central Neurons Gustatory neurons in the CNS are typically more broadly tuned than peripheral nerve fibers in the same species. This increased breadth of tuning is likely due to convergence of peripheral fibers onto brainstem neurons. Neurons in the NST, for example, can often be driven by stimulation of taste receptors in anatomically separate regions of the oral cavity (Sweazey and Smith, 1987; Travers and Norgren, 1991). In addition to greater breadth of tuning to gustatory stimuli, central taste neurons often respond to thermal and tactile stimuli (Travers and Smith, 1984; Travers, 1993) and at cortical levels to visual and olfactory stimuli (Rolls and Baylis, 1995).

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1. Increase in Breadth of Tuning in NST and PbN Experiments in which CT fibers (Frank, 1973) and neurons of the NST (Travers and Smith, 1979) and parabrachial nuclei (PbN; Van Buskirk and Smith, 1981) of the hamster were stimulated by applying the same stimuli (e.g., 0.1 M sucrose, 0.03 M NaCl, 0.003 M HCl, and 0.001 M QHCl) to the anterior tongue have revealed a systematic increase in the breadth of responsiveness of these cells from the periphery to the pons. A comparison of the mean response profiles of S-, N-, and H-best neurons of the hamster CT, NST, and PbN was shown in Fig. 3. The mean breadth of tuning (H) increased from 0.56 for CT fibers to 0.66 in the NST and 0.65 in the PbN. In particular, the breadth of tuning of the Sbest cells, which are the most specifically tuned fibers in the CT nerve, increased from 0.39 in the CT to 0.59 in the NST to 0.74 in the PbN (see Fig. 3), making these neurons more broadly tuned in the pons than the very broadly tuned H-best cells (Van Buskirk and Smith, 1981). This reflects a change from a typical S-best CT fiber that responds predominantly to sucrose with only slight sideband sensitivity to one other stimulus (usually NaCl) to an S-best pontine neuron that responds well to at least three of the four basic stimuli. The breadth of tuning of hamster PbN neurons is quite evident in Figure 4A, which shows the mean responses of S-, N-, and H-best PbN neurons to an array of 18 stimuli. An increase in breadth of responsiveness from periphery to brainstem has been noted in other species as well (Travers, 1993), although the comparisons have been made across data collected in different laboratories and not necessarily employing stimuli at the same concentrations. Across five CT fiber experiments on rat, hamster, and monkey, the mean breadth of responsiveness was 0.56 (range 0.52–0.61), whereas the mean breadth across 10 studies of NST cells in rat, hamster and monkey was 0.74 (range 0.62–0.87). The responses of 100 rat NST neurons to sucrose, NaCl, HCl, and QHCl are shown in Figure 5. The cells are arranged in order of their best stimuli, with S-best cells on the left (shaded bars), N-best cells in the center (open bars), and H-best cells on the right (shaded bars); there were no Q-best cells in this sample. Neurons are arranged within each group in order of the response to their best stimulus; the response of any one cell is read from top to bottom. The mean breadth of tuning (entropy) of these cells was 0.81, reflecting their extremely broad tuning to the four stimuli. Travers (1993) also compared data at peripheral and brainstem levels to those from cells in insular cortex of rats and insular-opercular cortex of monkeys, where the mean breadth of tuning across six experiments was an intermediate level of 0.63 (range 0.54–0.76). Given the caveat that these studies are not completely comparable across

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laboratories or species, these data suggest that the trend of increasing breadth of responsiveness between periphery and brainstem appears to hold across several species. They further suggest no additional increase in the breadth of tuning of taste cells between brainstem and cortex and perhaps even a slight return toward the narrower tuning characteristic of peripheral fibers. Data from several experiments on the macaque (Rolls, 1995) show that central gustatory neurons in this primate decrease their breadth of tuning between the NST and the cortex, with NST cells having a breadth of tuning value of 0.87 (similar to that shown in Fig. 5 for the rat NST), frontal operculum cells a value of 0.67, neurons of the insula a value of 0.56, and the cells in the secondary cortical taste area of the orbitofrontal cortex a breadth of tuning of 0.39. In comparison, CT fibers in the macaque (Sato, 1975) have a mean breadth of tuning of 0.57, showing that the effect of synaptic processing from the CT to the insula is to increase the breadth of tuning and then return it to peripheral levels. Even cells with entropies between 0.5 and 0.6 are relatively broadly tuned (see Fig. 3). Although cells in the orbitofrontal cortex are quite narrowly tuned to tastants, they are often multimodal, responding also to olfactory and visual stimuli (Rolls and Baylis, 1995). Therefore, neurons may become more specific in one modality while extending their sensitivities across others to serve an emergent property, in this case perhaps an appreciation of flavor. 2.

Convergence onto Single NST Cells from Different Fields

There is some convergence of input onto single CT fibers, because each one branches to innervate several fungiform papillae (Miller, 1971; Oakley, 1975). The sensitivities of each branch of a CT axon appear to be the same, suggesting little consequence of this peripheral convergence on taste information processing other than might accrue through spatial summation (Oakley, 1975). Indeed, studies of the effects of the area of stimulation on human taste responses show that spatial summation does occur on the anterior tongue (Smith, 1971). On the other hand, there is considerable convergence of peripheral fiber inputs onto second-order gustatory cells of the NST, often occurring between anatomically separate subpopulations of taste buds. The receptive fields of taste-responsive cells in the NST of the sheep are considerably larger than those of the CT nerve, suggesting that CT fibers converge onto secondary neurons in the NST (Vogt and Mistretta, 1990). About half of the taste-responsive cells of the rat NST receive converging input from separate taste bud regions (Travers, 1993). Travers and colleagues have demonstrated

Gustatory Neural Coding

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Figure 4 (A) Mean responses (impulses/s) of each of three neuron classes in the hamster PbN to 18 stimuli. The sweet-tasting stimuli are shaded in the profile for the S-best cells, the sodium salts are shaded in the profile for the N-best neurons, and the nonsodium salts and acids are shaded in the response profile for the H-best neurons. (Modified from Smith et al., 1983a.) (B) Patterns of activity generated across 31 hamster PbN neurons by two sodium salts (filled symbols) and by sucrose (open symbols). The across-neuron patterns to NaCl and NaNO3 correlated  0.94, whereas that to sucrose was not correlated with the pattern evoked by either sodium salt (e.g., r
0.09 with NaCl). (Data from Smith et al., 1983a.)

that the patterns of convergence of peripheral fibers onto NST neurons in the rat are predominantly among taste buds of the anterior tongue, nasoincisor ducts, or sublingual organ on the one hand, or among those of the foliate papillae, soft palate, or retromolar mucosa on the other

(Travers et al., 1986). That is, taste buds from the anterior or from the posterior oral cavity converge onto NST cells, but there is little evidence that anterior and posterior receptive fields combine in the responsiveness of an NST neuron. These patterns of convergence reflect the orderly

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anterior-to-posterior progression of food through the oral cavity during ingestion, making the simultaneous stimulation of the converging inputs likely (Travers, 1993). Although convergence has been examined mostly in the NST, it has also been noted at other levels of the central gustatory pathway. The available evidence suggests no greater convergence of separate taste bud subpopulations at pontine or thalamic levels (Travers, 1993), although the increased breadth of responding of S-best neurons in the

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hamster PbN suggests that within a receptor population (anterior tongue) there may be further convergence at higher levels (Van Buskirk and Smith, 1981). 3.

Multimodal Sensitivity: Touch and Temperature

In addition to their extensive breadth of responsiveness across stimuli of different taste quality, gustatory neurons at all levels often respond to other sensory modalities, most

Figure 5 Net responses (impulses/s above or below spontaneous rate) of 100 rat NST neurons to the four basic taste stimuli. Neurons are arranged along the abscissa according to their best stimulus, with cells 1–15 being S-best (shaded bars), 16–64 N-best (open bars), and 65–100 H-best (shaded bars); no cells in this sample were Q-best. The mean spontaneous activity of each cell is shown at the bottom of the figure. These cells had a mean breadth of tuning of  0.81. (Data selected from Giza and Scott, 1991; Giza et al., 1991, 1997.)

Gustatory Neural Coding

often to intraoral somatosensory stimuli. Fibers of the CT nerve of rats and hamsters respond to both gustatory and thermal stimulation (Ogawa et al., 1968). This sensitivity to warming and cooling is maintained in central gustatory neurons of the NST (Ogawa et al., 1988), PbN (Travers and Smith, 1984), thalamus (Verhagen et al., 1999), and cortex (Yamamoto et al., 1981). There is a strong correlation at both peripheral and central levels between the responses to warming the tongue and to sucrose and between the responses to cooling and to NaCl or HCl. The responses of a hamster PbN neuron to gustatory and thermal stimulation are shown in Figure 6. This S-best neuron responded also to NaCl and to 37 °C distilled water (Travers and Smith, 1984). Recent human psychophysical experiments show that warming and cooling small areas on the anterior tongue can evoke sensations of sweet and salty or sour, respectively (Cruz and Green, 2000). In the rat NST, one half of the taste-responsive cells with receptive fields in the anterior oral cavity and all of those with receptive fields in the posterior oral cavity are responsive also to mechanical stimulation of their receptive fields (Travers and Norgren, 1995). In fact, across a number of studies it has been shown that about 64% of taste-responsive NST

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neurons receive converging input from gustatory and tactile receptors (Travers, 1993). The few studies that have examined this question at the level of the PbN and thalamus show an average amount of gustatory/mechanical convergence of 77% and 88%, respectively (Travers, 1993). In awake, freely ingesting rats, there is some convergence of both thermal and olfactory stimuli onto cortical gustatory neurons (Yamamoto et al., 1989). Multimodal convergence has been examined in primates most extensively in the secondary cortical taste area in the orbitofrontal cortex, where there are cells responding to combinations of taste and olfactory, visual, or somatosensory stimuli (Rolls, 1995; Rolls and Baylis, 1995). Thus it is clear that taste-responsive cells in the central nervous system are driven often by stimuli differing in taste quality, intensity, and sensory modality. The contribution of the responses of these multimodal cells to the coding of any particular attribute, such as taste quality, must be assessed with this multiple sensitivity in mind. D. Temporal and Spatial Aspects of Taste Processing The responses of taste neurons typically are analyzed by examining the impulse frequency during some defined period of time, such as the 1- or 5-second interval after the arrival of the stimulus at the tongue. The responses to tastants, however, vary over time, and to some extent the temporal parameters of the responses to different stimuli are relatively distinct. As a consequence, some investigators have suggested that temporal characteristics may be important in coding information about taste quality. In addition, there is some evidence that cells responding to different stimuli are differentially distributed in central gustatory nuclei, suggesting the possibility of a chemotopic code for taste quality. Overall, however, the data supporting either a strictly temporal or topographic spatial code for gustatory quality are not compelling. 1.

Figure 6 Oscillographic records for the responses of a hamster PbN neuron to the four basic gustatory stimuli: sucrose (S), NaCl (N), HCl (H), and QHCl (Q) and to warming (37 °C) and cooling (17 °C) of the tongue. The arrows indicate the approximate onsets of the responses. The histograms in the lower right show the mean responses of this neuron to the six stimuli for three tests of the taste and two tests of the thermal stimuli. This neuron responded as well to warm distilled water as to sucrose. (From Travers and Smith, 1984.)

Time Course of Taste Responses: Differences Among Stimuli

The temporal characteristics of gustatory neural responses are often ignored in studies of taste physiology. Where they have been examined, investigators have noted that different classes of stimuli produce responses with different time courses. For example, responses to NaCl are characterized by a brief phasic portion followed by a slowly declining tonic component (Smith et al., 1975). The responses to sucrose, on the other hand, have a much less pronounced, if not absent, phasic component. There are differences among NaCl, HCl, QHCl, and sucrose in

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the time it takes the rat CT nerve to reach its maximum firing rate, the effects of stimulus concentration on that rise time, and the ratio of the phasic to the tonic component of the response (Harada et al., 1983). Responses of rat CT nerve fibers to different tastants are correlated with different temporal patterns, but this correlation is not precise enough to serve as an unambiguous code for taste quality (Nagai and Ueda, 1981). The time course of the response is largely a function of the stimulus, but also depends on which neurons are activated. It is likely, however, that differences in time course can add to the distinctions that occur in the activity across cells to enhance the discrimination among stimuli, resulting in a kind of spatiotemporal pattern that provides a unique signature for each stimulus (Scott, 1987a). Although differences in the temporal patterns of activity elicited by different stimuli may enhance the resolution among stimuli, an unambiguous neural representation of taste quality is provided by the pattern of activity across neurons, as discussed below. 2. Response to Constant Stimulation: Rate of Onset Versus Adaptation The temporal characteristics of the response to a taste stimulus reflect a number of underlying factors, including the rate of stimulus application and the process of adaptation. The response to NaCl in the rat CT nerve has two distinct components: an initial phasic discharge followed by a tonic response component (Pfaffmann and Powers, 1964). This phasic/tonic response pattern is common to many stimuli and occurs in the gustatory responses of all species that have been investigated. Analysis of the time course of the NaCl response in the rat CT nerve shows it to decrease from the initial phasic peak in two exponential stages. The initial phasic response declines with a time constant of about 0.5 second, and this is followed by a tonic response component that decays much more slowly, with a time constant of about 30 seconds (Smith et al., 1975). The ratio of the phasic and tonic components of the CT response to NaCl varies directly with the rate of stimulus onset. Impulse frequency during the phasic response is a power function of the rate of stimulus rise, and if the stimulus is applied slowly enough the phasic response is completely eliminated (Smith and Bealer, 1975). These data show that the gustatory system is exquisitely sensitive to the rate of stimulus change. Taste neurons respond appropriately to increasing stimulus concentration, but the rate at which that concentration changes is a particularly salient feature of the stimulus. Therefore, the time course of the neural response to a tastant reflects not only the chemical stimulus, but also the

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rate at which it flows over the tongue. Beyond this initial rapid decline of the phasic response, there is a secondary decline during the tonic component of the response, which probably reflects adaptation at the level of the taste receptor cell, where a similar slow decline is observed (Ozeki, 1971). Recovery of the rat CT response to NaCl after adaptation follows a time course similar to this slow adaptation process (Smith et al., 1978). 3.

Chemotopic Correlates of Taste in the CNS: Orotopic Representation

The idea that taste quality might be represented in the nervous system by a topographic pattern of activity has been around for many years, although it does not have strong experimental support. There is some evidence that cells responding to different stimuli are differentially distributed in central gustatory nuclei. Topographic distributions of responsiveness have been noted in the NST of the rat (Halpern and Nelson, 1965), hamster (Dickman and Smith, 1989; McPheeters et al., 1990), and monkey (Scott et al., 1986b). Generally, NaCl and sucrose responses are more prevalent in the most rostral portion of the NST, and responses to HCl and QHCl are prominent at more caudal levels. To a large extent, these differences reflect the differential sensitivity of the CT and IXth nerves, since these nerves terminate in a rostral to caudal order, projecting an orotopic map onto the NST. However, even with only anterior tongue stimulation, there is some indication of a differential distribution of best-stimulus categories of cells in the hamster PbN, with 75% of N- and S-best cells located in the caudal half and 65% of H-best neurons in the rostral half of the pontine taste area (Van Buskirk and Smith, 1981). A similar crude topographic organization of chemosensitivity was reported in the macaque NST, but not in the insular-opercular cortex of that species (SmithSwintosky et al., 1991). In the rat cortex, however, distinct spatial patterns of responsiveness are generated by taste stimuli, with sucrose responses most dominant in the anterodorsal region, quinine responses in the posterior region, NaCl responses in the central and ventral regions, and HCl responses evenly distributed throughout the cortical taste area (Yamamoto et al., 1985). These “acrossregion patterns” have been proposed as a code for the representation of taste quality (Yamamoto, 1989). Even if such patterns can be reliably related to taste quality, their generation is dependent to a large extent on the topographic distribution of afferent inputs from different parts of the oral cavity, which serves to create the regional differences in responsiveness seen at various levels of the CNS. Since taste qualities can be reliably identified by humans following stimulation of a single fungiform

Gustatory Neural Coding

papilla (Bealer and Smith, 1975), it is hard to conceive of how such crude topographic representations of the input of the entire oral cavity could serve as a neural code for gustatory quality.

IV. TASTE QUALITY PROCESSING: GUSTATORY NEURAL CODING The principal debate over taste quality coding has focused on two competing theories: the across-fiber pattern theory (Pfaffmann, 1959; Erickson et al., 1965; Erickson, 1968, 1982a) and the labeled-line hypothesis (Pfaffmann, 1974; Pfaffmann et al., 1976). There has been considerable disagreement over the past two decades about the merits of these two ways of representing taste quality. Adherents to the labeled-line hypothesis propose that gustatory neuron types are specific coding channels for taste quality. Proponents of the across-fiber pattern theory, on the other hand, propose that quality is coded by the pattern of activity across neurons. In the following discussion, we examine the role of gustatory neuron types in the definition of the across-neuron patterns. From the result of such an examination, we see that the separate neuron types establish and define the distinctions among the patterns that are necessary for taste discrimination. Therefore, the same neurons are critical for coding taste quality, regardless of whether they are considered a “labeled line” or a necessary part of the “across-fiber pattern.” Taste discrimination is impossible without the contribution of each neuron type. A.

Theories of Quality Coding

Prior to the development of neurophysiological recording methods, a long tradition of human psychophysical research had provided considerable support for the notion that taste experience could be reduced to a few basic qualities, although not necessarily the traditional four (Bartoshuk, 1971). This idea, combined with Müller’s doctrine of specific nerve energies, led to the expectation that the perception of taste quality would arise from the activation of a few specific neuron types, each coding a single taste quality. This strict “labeled-line” theory was discounted by early neurophysiological recordings showing that peripheral taste fibers in several species were responsive to stimuli representing more than one taste quality (Pfaffmann, 1941, 1955). In response, an “acrossfiber pattern” theory was proposed, which held that taste quality is coded by the relative activity across the population of responsive neurons (Pfaffmann, 1959; Erickson,

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1963). This theory accommodated the multiple sensitivity of taste fibers and required neither specific fiber types nor taste primaries. However, the prevailing view of the importance of primary tastes led to a modification of the labeled-line theory, in which it was proposed that taste quality is coded by the activity in a few “best-stimulus” channels, that is, by neurons that respond best, although not specifically, to one of the basic taste qualities (Pfaffmann, 1974; Pfaffmann et al., 1976). Although each coding theory has its strengths, both strain to encompass the full range of data. For example, accumulating evidence suggests that there are indeed functional classes of neurons that correspond in some way to primary taste qualities (Boudreau and Alev, 1973; Frank, 1973; Contreras and Frank, 1979; Smith et al., 1983b; Chang and Scott, 1984; Frank et al., 1988; Hanamori et al., 1988; Jacobs et al., 1988; Ninomiya and Funakoshi, 1988; Hettinger and Frank, 1990; Scott and Giza, 1990; Frank, 1991; Giza and Scott, 1991; Smith and Frank, 1993). On the other hand, analysis of the relative activity of these neuron groups shows that no single class of cells in isolation can reliably discriminate among different taste qualities (Smith et al., 1983b; Smith, 1985; Smith and Frank, 1993). The neural coding problem essentially rests on whether the activity in a given taste neuron, or class of neurons, is an unambiguous representation of the quality of the stimulus applied to its receptors (i.e., a labeled line) or whether this activity is meaningful only in the context of activity in other afferent neurons (i.e., in their relative patterns of response). Here we review the relevant neurophysiological data that bear on this issue and suggest that taste quality is coded in the relative rates of activity across several neuron types (Smith, 1985; Smith and Frank, 1993). 1.

Breadth of Tuning: Across-Fiber Patterns

It was the multiple sensitivity of fibers in the CT nerve that first led Pfaffmann (1941, 1955, 1959) to propose that taste quality is coded by the pattern of activity across taste fibers. According to this hypothesis, taste quality of the basic stimuli remains invariant through the mid-range of intensity, even though any single neuron may increase its breadth of responsiveness. The pattern of activity generated across the entire array of taste neurons rises with increasing concentration, but retains its shape (Erickson et al., 1965; Ganchrow and Erickson, 1970; Erickson, 1982a, 1984). Stimuli with similar tastes, such as sodium salts, generate highly similar patterns of activity across afferent taste neurons, as depicted by the across-neuron patterns for NaCl and NaNO3 across hamster PbN neurons

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shown in Figure 4B. The pattern of response to sucrose, on the other hand (Fig. 4B), is strikingly different from those evoked by the sodium salts. The similarities among these patterns are typically measured by calculating the across-fiber correlation between pairs of stimuli (Erickson et al., 1965), although other indices have been proposed (Gill and Erickson, 1985; DiLorenzo, 1989). Several behavioral investigations have shown that stimuli that evoke highly correlated neural patterns are judged by experimental animals to have similar tastes (Erickson, 1963; Morrison, 1967; Smith et al., 1979; Nowlis et al., 1980; Nowlis and Frank, 1981). This acrossfiber pattern view of quality coding incorporates the multiple sensitivity of gustatory neurons as an essential part of the neural code for taste quality. This theoretical view stresses that the code for quality is given in the response of the entire population of cells (Pfaffmann, 1959; Erickson, 1968, 1982a, 1984), placing little emphasis on the role of an individual neuron. Erickson (1968) has argued that pattern coding is used by many sensory systems. When the across-neuron correlations among the responses to an array of gustatory stimuli are calculated across either peripheral or central neurons, stimuli with similar tastes correlate highly and those with different tastes correlate poorly. Almost every neurophysiological study that has taken this approach to analyzing the responses of gustatory neurons has shown that the acrossneuron patterns reflect the qualitative similarities among taste stimuli (Erickson et al., 1965; Erickson, 1968, 1982a, 1984; Pfaffmann et al., 1976; Smith et al., 1979, 1983a; Travers and Smith, 1979; Van Buskirk and Smith, 1981; Smith, 1985; Scott et al., 1986a, 1986b, 1991; Frank et al., 1988; Scott and Giza, 1990; Giza and Scott, 1991). Often these across-neuron correlations serve as input to a multivariate statistical procedure in order to generate a “taste space,” which represents the neural similarities and differences among the stimuli. A taste space for 18 stimuli is shown in Figure 7; this three-dimensional space was derived from correlations among the responses of 31 hamster PbN neurons and was generated using multidimensional scaling (Smith et al., 1983a). Within this space, there is a clear separation between the sweet-tasting stimuli, the sodium salts, the nonsodium salts and acids, and the two bitter-tasting stimuli. This arrangement of stimuli, based on similarities among their across-neuron patterns, suggests that there is sufficient information within these patterns to discriminate among these four groups of stimuli, even though any one cell in the hamster PbN is likely to respond to stimuli of more than one group (see Fig. 4A) (Van Buskirk and Smith, 1981; Smith et al., 1983a, 1983b).

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2.

Response Profiles: Labeled Lines

Although mammalian taste neurons are broadly tuned, many investigators have attempted to group them into functionally meaningful categories (Pfaffmann, 1941, 1955; Boudreau and Alev, 1973; Frank, 1973; Travers and Smith, 1979; Van Buskirk and Smith, 1981; Smith et al., 1983b; Frank et al., 1988; Hanamori et al., 1988). It is obvious from knowledge about the organization of taste sensitivities that it is possible to group taste neurons into classes based on their best responses to the four prototypical stimuli (see Fig. 3). Indeed, hierarchical cluster analyses of the similarities and differences in the shapes of their response profiles results in distinct groups of neurons that correspond to these “best-stimulus” classes (Smith et al., 1983b; Chang and Scott, 1984; Scott et al., 1986a, 1986b; Frank et al., 1988; Hanamori et al., 1988; Rolls et al., 1988; Frank, 1991). The implication of distinct fiber types in the coding of taste quality began with the categorization of hamster CT fibers into best-stimulus groups (Frank, 1973). This type of categorization became the focus of an ensuing controversy over the neural representation of taste quality when it was proposed that these fiber types code taste quality in a labeled-line fashion (Pfaffmann, 1974; Pfaffmann et al., 1976). This hypothesis suggests that “sweetness” is coded by activity in S-best neurons, “saltiness” by activity in N-best neurons, etc. Pfaffmann (1974) first proposed a labeled-line code for sweetness because activity in S-best fibers of the squirrel monkey CT nerve correlated better with the animal’s preference behavior toward a number of sugars than did activity in the whole nerve. Thus, in contrast to a “population” approach to taste coding (across-neuron patterns), this labeled-line position advocates a “feature extraction” approach, in which particular neurons (or groups of neurons) play specific roles in the representation of taste quality. A labeled-line code, by definition, implies that activity in that “line” carries a specific message, without any reliance on the activity in other “lines” (Perkel and Bullock, 1968). B.

The Role of Neuron Types in Quality Coding

The labeled-line hypothesis requires the existence of neuron types for the coding of taste quality, whereas the across-neuron pattern theory does not. The number of labeled lines would equal the number of discrete taste qualities, which would each be signaled by activity in separate afferent channels. Consequently, the existence of gustatory neuron types has been sharply contested (Woolston and Erickson, 1979; Erickson, 1985), based on

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Figure 7 Three-dimensional “taste space” showing the locations of 18 stimuli, obtained from multidimensional scaling of the correlations in the responses evoked across 31 hamster PbN neurons. Four groups of stimuli are indicated by different symbols: sweet-tasting (open circles), sodium salts (), nonsodium salts and acids (solid circles), and bitter-tasting (open triangles). Although these cells were activated by applying stimuli only to the anterior tongue, there is enough information in the across-neuron patterns to clearly differentiate these four classes of gustatory stimuli, which are clearly behaviorally distinct to the hamster. (Modified from Smith et al., 1983a.)

the assumption that their existence somehow implicates them as labeled lines. However, the mere existence of fiber types (defined by their best stimulus, similarities in their response profiles, or by other criteria such as their amiloride sensitivity) does not imply that these classes of cells compose labeled lines. A classic example where receptor types are evident but where there is general agreement about the existence of a pattern code is in vertebrate color vision (Crick, 1979; Erickson, 1984; Smith, 1985; Smith and Frank, 1993). 1.

Neural Discrimination of Taste Inputs

Because peripheral taste fibers and central neurons are broadly tuned, it is difficult for the activity in a single neuron or even in a single neuron type to discriminate among stimuli that differ in taste quality. Several years ago, Smith and colleagues (Smith et al., 1983a, 1983b; Smith, 1985) examined the roles played by neuron types in the hamster brainstem in the definitions of the acrossneuron patterns and the ability of these neuron types alone or in combination to discriminate among stimuli.

Responses in each neuron type dominate and essentially define the similarities in the across-neuron patterns evoked by their “best” stimulus class, as seen in the responses to several acids in hamster PbN neurons (Fig. 8). Without the contribution of the H-best neurons (shaded), these acids would not produce similar across-neuron patterns. Examination of the contribution of each neuron type to the across-neuron patterns showed that each neuron type was necessary for determining the similarities among stimuli for which it was “best,” but that the reliable discrimination among stimuli of different classes depended upon the activity in more than one neuron type. For example, within a multivariate taste space based on the across-neuron patterns generated from the responses of neurons in the hamster PbN, sodium salts, and nonsodium salts were readily distinguishable (see Fig. 7). However, if either the N-best or the H-best class of cells, which are preferentially sensitive to sodium salts and nonsodium salts, respectively, were missing from the data matrix, the patterns across the remaining cells did not permit distinctions between these two classes of salts. Thus, the discrimination among stimuli with different tastes depended upon the relative activity

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in different neuron types; one neuron type alone was insufficient to discriminate among stimuli with different taste qualities (Smith et al., 1983a). This “across-neuron type” code is simply a restatement of the across-fiber pattern theory first proposed by Pfaffmann (1959), except that it puts an emphasis on the activity in recognizable neuron types. This coding mechanism is similar to the coding of stimulus wavelength by the vertebrate visual system, where three types of broadly sensitive photoreceptor pigments are involved (Marks et al., 1964). The wavelength of light falling on the retina

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can be encoded accurately by considering the relative activity in these three photoreceptors, that is, by a pattern (Erickson, 1968; Boynton, 1971). Deficiencies in one or more of the photoreceptor pigments result in various forms of visual chromatic deficiency, or “color blindness” (Boynton, 1971). For example, the lack of the long-wavelength photopigment results in a confusion in color discrimination (protanopia), where individuals perceive long-wavelength stimuli as one color (yellow) and short wavelength stimuli another (blue), with a neutral (gray) point at 494 nm (Graham et al., 1961; Boynton, 1971).

Figure 8 The responses of 31 hamster PbN neurons to four acids, arranged into across-neuron patterns according to the magnitude of their response to 0.003 M tartaric acid. The H-neuron group is shaded in each pattern, and the correlation of each pattern with that for tartaric acid is shown. Without the contribution of the H-best neurons, the responses to these behaviorally similar stimuli would not be well correlated. (From Smith et al., 1983a.)

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Data on color-blind individuals show that the absence of any one of the three photoreceptor types results in the inability to discriminate among particular sets of wavelengths (Smith, 1985; Smith and Frank, 1993). Similarly, the neural discrimination among different classes of gustatory stimuli depends upon the relative activity in different neuron types.

neuron types that defines the unique patterns that represent taste quality. In that sense, whether these neuron types are viewed as a “labeled line” or the critical part of an “acrossfiber pattern,” they are crucial to the neural code for taste quality. Taste discrimination depends on the differential responses in these separate neuron classes, the activity of which defines the across-neuron patterns.

2.

3.

Comparison of Activity Across Neuron Types

Taste offers no known analogy to the pathology of color blindness, but experiments on the rat NST provide an experimental demonstration of an analogous phenomenon (Scott and Giza, 1990; Giza and Scott, 1991; St. John and Smith, 2000). Blocking the NaCl response of the N-best neurons with amiloride resulted in the inability of the remaining cells to discriminate between sodium and nonsodium salts, that is, their across-neuron patterns were not distinct without the input from the N-best cells. Without considering the differential response of two neuron types (N-best and H-best) to these two classes of stimuli, they were coexistent within the taste space of the amiloridetreated rat (Fig. 9). These results are compatible with the interpretation that taste quality discrimination depends on a comparison of activity across broadly tuned neuron types, comparable to the coding of color vision by broadly tuned photoreceptors (Smith et al., 1983a; Smith, 1985; Smith and Frank, 1993). In both taste and color vision, elimination of any one cell type results in a lack of separation among stimuli of different qualities within the multidimensional space (Smith, 1985; Scott and Giza, 1990; St. John and Smith, 2000) and a loss of behavioral discrimination among the same stimuli (Graham et al., 1961; Boynton, 1971; Hill et al., 1990; Spector et al., 1996). The data cited above show that no one gustatory neuron type is capable of providing information that can reliably distinguish the across-neuron patterns evoked by dissimilartasting compounds. Multiple neuron types must contribute to the pattern for dissimilar stimuli to be coded as being distinct. Thus, in that sense, the various neuron types (S-, N-, H-, or Q-best) are critically important for the discrimination of taste quality. The N-best cells can define the similarities among sodium salts, but activity in both neuron types is required to distinguish sodium salts from nonsodium salts and acids. Behavioral and neural data in rats support this requirement, but there is no evidence to date that N-best cells are labeled lines signaling “saltiness.” On the contrary, N-best cells provide the critical part of a pattern across neuron types that codes saltiness, yet these cells by themselves cannot distinguish sodium salts from other stimuli. It is the activity in these particular

Synthetic Versus Analytic Processing

Drawing parallels between color vision and taste provides an interesting way to understand the broad tuning of gustatory neurons, but is not without its limitations. In his earliest formulations of the across-fiber pattern theory, Erickson (1968, 1973) based many of his ideas on vertebrate photoreceptors, which are broadly responsive across stimulus wavelength and which code color information by their relative patterns of response. Inherent in these early arguments was the idea that if broadly tuned neurons serve to code stimulus information by patterns, then the system must be synthetic rather than analytic (Erickson, 1977). That is, in color vision, a mixture of two colored lights (e.g., red and yellow) results in the synthesis of a new sensation (e.g., orange), whereas the components of a mixture in an analytic modality maintain their individuality (as in a musical chord). Erickson and colleagues have published several papers suggesting that taste is not entirely analytic, as is pitch perception, and may be more akin to the synthetic senses (Erickson, 1977, 1982b; Erickson and Covey, 1980). Bartoshuk and colleagues have countered that human subjects clearly recognize the individual components in taste mixtures, arguing that the gustatory system is analytic in nature (Bartoshuk, 1978; Bartoshuk and Gent, 1985). Still others suggest that taste is neither purely synthetic nor purely analytic but that mixtures exhibit fusion, creating new sensations, as occurs in food flavors, but in which the individual components can often be recognized, especially by trained observers (McBurney, 1986; Halpern, 1997). That separate taste sensations fuse into a new, but ultimately analyzable, sensation may be most compatible with a neural system that codes information by patterns, which themselves are dominated by activity in clearly defined neuronal cell types. That is, both the neurophysiology of the gustatory system and a good deal of psychophysical data support the idea that taste is not entirely synthetic or analytic in nature (see Halpern, 1997, for a thorough discussion of this issue). A challenge to the further understanding of neural coding in this system is to determine precisely the relationship between the activity of these broadly tuned afferent neurons and the psychophysical responses to taste mixtures.

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Figure 9 Three-dimensional taste spaces showing the relative similarity of the across-neuron patterns among 15 stimuli before (A) and after (B) treatment of the tongue and palate with amiloride. Stimulus locations before amiloride are typical of those of the normally functional rat taste system, with sweet-tasting stimuli separated from sodium and lithium salts, which are in turn separated from acids and nonsodium salts and quinine. After amiloride application, all the Na-bearing stimuli, except MSG, migrate to the positions of the acids, nonsodium salts, and quinine, indicating similar across-neuron patterns to these stimuli after amiloride. Abbreviations: N1–N4, 0.01–0.3 M NaCl; Li, LiCl; S, sucrose; G, glucose; Sa, sodium saccharin; H, HCl; Ci, citric acid; Q, qunine hydrochloride; K, KCl; Ca, CaCl2; M, MSG; P, polycose. (Adapted from Giza and Scott, 1991.)

4. Advantages of a Pattern Code: Detecting Signals in Noise The broad tuning of gustatory cells at every level of the nervous system, from the receptor cell to the cortex, makes

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it difficult to accept a pure labeled-line code for gustatory quality. Whereas a labeled-line code might be more compatible with an analytic sensory system than a pattern code would be (Bartoshuk and Gent, 1985), no other aspect of gustatory neurobiology favors a simple labeled-line explanation. The most troubling problem facing the labeled-line approach is the multiple sensitivity of gustatory afferent neurons, especially those in the central nervous system, which are also multimodal in many instances (see above). It was this multiple sensitivity in peripheral afferent fibers that led to Pfaffmann’s (1955, 1959) original formulation of the across-fiber pattern idea and this finding has only been reinforced by subsequent research. Even taste receptor cells are broadly tuned across stimuli representing different taste qualities (e.g., Gilbertson et al., 2001; Ozeki and Sato, 1972). In broadly tuned taste cells, the activity elicited by stimuli with different taste qualities can be interpreted by the labeled-line theory in one of two ways, which are depicted in Fig. 10. The “sideband” activity in a neuron type (e.g., the response to HCl by N-best cells) represents either noise (Fig. 10A) or signal (Fig. 10B). If it represents noise, then there is a very poor signal-to-noise ratio in gustatory neurons (see also Travers and Smith, 1979; Scott and Giza, 2000), making it difficult to conceive of how a signal could ever be reliably detected (Fig. 10A). This problem is especially apparent as the concentration of nonbest stimuli is raised because, as noted by Pfaffmann (1955, 1959), a concentration can usually be found where qualitatively distinct stimuli produce equivalent responses in gustatory neurons (see Fig. 2, above). If, on the other hand, such sideband responses represent signal, then the responses of broadly tuned neurons in the rat NST, for example, would suggest that the “basic” taste stimuli, such as NaCl or HCl, have multiple taste qualities to the rat (Fig. 10B). Behavioral experiments, however, flatly refute this implication. Rats trained to avoid one of the basic taste stimuli do not generalize that aversion to any of the others (Nowlis and Frank, 1981), even though rats will avoid mixtures of the conditioned stimulus (CS) with other stimuli in proportion to the concentration of the CS in the mixture (Smith and Theodore, 1984). Thus it is difficult to conceive of how activity in different neuron types can represent a specific taste quality. Another advantage of pattern coding arises out of the efficiency of stimulus representation by multiple cells. Detection or recognition of a gustatory stimulus requires the nervous system to select a neural signal out of background noise. Each potential signal has some probability distribution associated with it, as does background noise. The task of the system is to detect the signal in the noise and the ability of a system to do this depends upon the size of the signal and the variability of both the noise and sig-

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Figure 10 Schematic diagrams of the implications of a “labeled-line” coding mechanism for the interpretation of the activity in broadly tuned neurons of the rat NST. (A) The mean response profiles of N-best and H-best neurons in the rat NST. If the activity in a given cell type (N-best or H-best) that is elicited by stimuli other than the “best” stimulus is “noise” (shaded area), then there is very little “signal” (solid area) being provided by these broadly tuned neurons. Indeed, there are more impulses generated in these cells by the “sideband” stimuli together than by the best stimulus. (B) The mean responses of each of three neuron types (S, N, and H-best) in the rat NST to 0.1 M NaCl and 0.01 M HCl. If the response to NaCl in H-best neurons or the response to HCl in N-best neurons is “signal” (gray bars), then NaCl should have a large “acid” taste to rats and HCl should have a large “sodium” taste. Behavioral experiments (Nowlis et al., 1980) clearly do not support such a conclusion, even though it has been shown that rats are capable of responding to the presence of a conditioned stimulus in a taste mixture (Smith and Theodore, 1984). (Data from St. John and Smith, 2000.)

nal distributions (Pfaff, 1975). Neural networks composed of multiply sensitive cells are more efficient at detecting signals than systems composed of specific cells (see also Winograd, 1963). As more cells (n) are used in combination to represent a signal, the signal becomes easier to detect because the mean firing rate increases as a function of n, whereas the variability associated with the probability distribution of the signal across the combination of cells increases as a function of the square root of n (see Pfaff, 1975, for a detailed demonstration of this idea). Therefore, the reliability of detection increases with the number of cells involved in the representation of the stimulus. For a given system, the more broadly tuned the cells, the greater the number of cells available to represent any given stimulus and the finer the discriminations possible among

stimuli. These and other considerations, as delineated above, suggest strongly that the gustatory system represents taste quality by the patterns of activity across broadly tuned neurons. REFERENCES Adler, E., Hoon, M. A., Mueller, K. L., Chandrasheskar, J., Ryba, N. J. P., and Zuker, C. S. (2000). A novel family of mammalian taste receptors. Cell 100:693–702. Akabas, M. H., Dodd, J., and Al-Awqati, Q. (1988). A bitter substance induces a rise in intracellular calcium in a subpopulation of rat taste cells. Science 242:1047–1050. Avenet, P., and Lindemann, B. (1989). Perspectives of taste reception. J. Membrane Biol. 112:1–8.

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36 Development of the Taste System: Basic Neurobiology Charlotte M. Mistretta University of Michigan, Ann Arbor, Michigan, U.S.A.

David L. Hill University of Virginia, Charlottesville, Virginia, U.S.A.

I.

INTRODUCTION

fungiform papilla and surrounding lingual epithelium, but not taste buds; (3) the petrosal, innervating taste buds in posterior folds of the foliate papillae, the circumvallate papillae and taste buds, and surrounding posterior lingual epithelium; and (4) the nodose, innervating epiglottal taste buds and epithelium (Mistretta and Hill, 1995). Within the brainstem, central projections of the taste and taste-related nerves from the sensory ganglia reflect the orderly arrangement of taste buds in oral-pharyngeal regions. The emphasis on specialized distributions, or patterns, of receptors and neural projections suggests a high degree of order in the taste system, and we use this concept of ordered arrangements as a broad focus in this revision of our chapter from the first edition of this book (Doty, 1995). Reflecting the models and approaches used in recent studies, discussion is limited primarily to data from the rodent taste system, rat and mouse, and to taste buds and papilla organs on the tongue. Other comprehensive reviews of the developmental biology of the sense of taste, with discussion across species, can be found in Mistretta (1991), Hill (2001), and Mistretta and Hill (1995). There is also an extensive body of experimental results demonstrating that peripheral and central gustatory function and morphology is especially susceptible to environmental manipulations during early development. The reader is referred to recent reviews of these topics by Stewart et al. (1997) and Hill (2001). For referring to embryonic and postnatal ages, throughout the chapter we use the day that the rodent dam is sperm

Taste receptor cells reside in epithelial specializations, the taste buds, which are located primarily in the tongue, soft palate, and epiglottis in mammals (Mistretta, 1991; Mistretta and Hill, 1995) (see Chapter 32). On the tongue taste buds reside solely in specialized sensory structures, the gustatory papillae, and on the soft palate and epiglottis taste buds occur in high density in specialized regions. Thus, mammalian taste buds are not randomly scattered across the mucosae of the mouth and pharynx, but rather are concentrated in specific locations. This provides a basis for functional specialization that contributes to optimal processing in this life-essential sensory system, the gustatory sense. Seated between the peripheral receptors and central nervous system are the cranial, sensory ganglia composed of neurons that extend fibers to synapse with taste bud cells within peripheral oral/facial tissues (Fig. 1). To establish taste pathways during development, neurites extend from neurons within the ganglia to gustatory organs in the tongue in the peripheral nervous system and to the brainstem in the central nervous system. Reflecting the central role of the ganglia in the establishment of taste sensation, this chapter is organized to discuss the ganglia first, then the peripheral taste organs, and lastly the central neural taste regions. Relevant ganglia are (1) the geniculate, innervating taste buds on the soft palate and in fungiform papillae on the tongue; (2) the trigeminal, innervating 759

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Figure 1 Diagram illustrating cells of the trigeminal, geniculate, and petrosal ganglia that extend neurites during development to innervate papilla, taste bud, and lingual epithelial target organs in the tongue and to synapse centrally with neurons in taste nuclei in the brainstem. The sensory ganglia thus have a principal role in establishing taste circuits from peripheral target organs to central neural structures.

positive as gestational day 0 (E0). The day of birth is referenced as postnatal day 0 (P0).

II. THE SENSORY GANGLIA INNERVATING LINGUAL GUSTATORY PAPILLAE AND TASTE BUDS AND CENTRAL GUSTATORY STRUCTURES A.

When and How Do the Ganglia Develop, and What Are the Known Regulatory Elements?

The geniculate, petrosal, and trigeminal ganglia are collections of neurons that extend fibers to innervate specific lingual targets peripherally and localized groups of neurons in the solitary nucleus centrally. These ganglia arise from placodes, local thickenings of cells that will delaminate from the original placode location, migrate, and aggregate to form the presumptive ganglia (Baker and Bronner-Fraser, 2001). The geniculate and petrosal ganglia arise from epibranchial placodes at pharyngeal pouches 1 and 2, whereas the trigeminal ganglion derives from two more rostrally located placodes, the opthalmic and the maxillo-mandibular, near the midbrain-hindbrain junction (Graham and Begbie, 2000). Studies with chick embryo indicate that the epibranchial placodes are induced by

pharyngeal endoderm through signaling actions of BMP-7 (Begbie, 1998). In contrast, the trigeminal placodes are induced by local tissue in the midbrain to rostral hindbrain region. Basic helix-loop-helix neurogenic factors (bHLH) are expressed in precursors of placode-derived sensory neurons. Specifically, neurogenin 2 is essential for development of sensory ganglia derived from epibranchial placodes, including the geniculate and petrosal (Fode et al., 1998). Developing trigeminal neurons depend on neurogenin 1 function (Ma et al., 1998). In each ganglion, other bHLH factors are subsequently expressed including Math3, NeuroD, and NSCL1 (Fode et al., 1998; Ma et al., 1998). Thus, the ganglia that provide sensory innervation to the tongue have different placodal derivations and different early molecular regulators. These differences serve to highlight a distinction in sensory functions—the chemosensory nature of geniculate and petrosal ganglia and the somatosensory nature of the trigeminal. The geniculate, petrosal, and trigeminal ganglia have begun to develop on E8.5 in mouse (Fode et al., 1998; Ma et al., 1998) and E9.5–10 in rat (Altman and Bayer, 1982; White et al., 1995), and the earliest axons emerge from the mouse trigeminal ganglion at E9.5 (Davies, 1997). Ganglion development thus is well underway for a few days in advance of emergence of the tissue swellings that subsequently will coalesce to form the tongue (lingual swellings are apparent on gestational day 11.5 in mouse;

Taste System Development

day 13 in rat) (Kaufman, 1992; Mistretta, 1972). Therefore, the initial formation of ganglion cells is not directed by factors in the tongue (Fig. 2). Only by gestational day 14–15 in the rat have neurites extended into early taste papillae to potentially benefit from targetderived molecules that could support, maintain, and regulate differentiation of ganglion cells (Mistretta, 1998). Once neurites from the ganglia have reached the periphery, what molecules in lingual targets might interact with the growing nerves to influence development of ganglion cells? Among those families receiving much attention in current literature are the neurotrophins. These soluble proteins include nerve growth factor (NGF), brainderived growth factor (BDNF), neurotrophin-3 (NT3), and neurotrophin-4/5 (NT4/5) (Davies, 1997; Farinas and Reichardt, 1996). The neurotrophins have demonstrated roles in ganglion cell survival, maintenance, and morphological and functional differentiation, and effects of the

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various neurotrophins are ganglion-specific and temporally restricted (Snider, 1994). An important influence of neurotrophins in sustaining geniculate and nodose/petrosal ganglion cells was demonstrated in transgenic mice that had a deletion of the gene encoding BDNF, with an approximate 60% loss of neurons in these ganglia (Ernfors et al., 1994; Jones et al., 1994). In contrast, only about 20% of trigeminal ganglion cells were lost in the null mutants. A synthesis of published literature on mutant mice with deletion of genes encoding neurotrophin molecules makes clear the distinctive dependence of the geniculate ganglion, a gustatory ganglion, versus the trigeminal ganglion, which is nongustatory, subserving somatosensation and pain (Table 1). Unfortunately, data for study of the petrosal ganglion alone are not available; rather the petrosal/nodose ganglia have been evaluated as a complex. However, the primary dependence of the petrosal/nodose on BDNF is clear from the literature (Table 1).

Figure 2 Photomicrographs of sagittal sections through embryonic rat heads at gestational days 12 (E12, top two images) and 13 (E13, bottom two images). Trigeminal (tg), geniculate (gg), and petrosal (pg) ganglia already have formed by E12 (top, left), and neurites extend into the maxillary (mx) and mandibular (mn) target zones (top, right). The embryonic tongue has not yet formed at E12. By E13, ganglia have more extensive neurite branches (bottom, left), and some neurites have reached the base of the embryonic tongue (bottom, right; hatched line demarcates base of tongue). (Data from Mbiene and Mistretta, 1997.)

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Table 1 Ganglion Reduction in Neurotrophin Null Mutant Mice Presented as Estimate of Remaining Ganglion Cell Numbers or Volume (% In Mutant Relative to Wild-Type Mice) % Ganglion remaining in null mutantsa

Geniculate Petrosal/Nodoseb Trigeminal

BDNF

NT4

NT3

NGF

55 45 70

50 40 98

65 60 35

na na 30

BDNF/NT4 5 15 70

BDNF/NT3 35 35 25

BDNF/NT4/NT3 0 5 10

na
not available. a Percentages have been averaged across published studies and then rounded to the nearest 5% for ease in comparing across ganglia and neurotrophins. b Studies of petrosal ganglion as a separate entity in neurotrophin mutant mice are not published; data are for combined petrosal/nodose ganglia. Sources: Conover et al., 1995; Ernfors et al. 1994; Farinas and Reichardt, 1996; Jones et al., 1994; Liebl et al., 1997; Liu and Jaenisch, 2000; Liu et al., 1995; Snider, 1994.

There is substantial understanding about the role of neurotrophins in development of the rodent trigeminal ganglion. Based on observations in mouse trigeminal, it is proposed that ganglion neurons survive and extend axons independently of neurotrophins during initial development (Davies, 1997). Later, ganglion neurons are dependent on trophic support from tissues through which their axons extend and then from target organs. For neuron survival, trigeminal dependence on neurotrophins shifts from a requirement for BDNF or NT3 at E10.5–11.5 in mouse, to NGF after E12.5 (Buchman and Davies, 1993; Davies, 1997, Paul and Davies, 1995). Whereas all three of the high-affinity Trk receptors for neurotrophins, Trks A, B, and C, are expressed within the embryonic trigeminal ganglion, neurons with specific Trks develop in two waves (Huang et al., 1999). Trk B–and Trk C–expressing neurons are generated first, with peak numbers at E11.5, and Trk A–expressing neurons later, with peak numbers at E13.5. These investigators have also demonstrated that expression of the Trk receptors is confined to trigeminal neurons and is not in neuronal precursors. Also, by E13.5 there is no coexpression of the Trk receptors in individual trigeminal ganglion neurons. Data on the geniculate ganglion and neurotrophin dependence are not extensive. In the rat, most or all geniculate cells express Trk B mRNA at E13 through E18 (Ernfors et al., 1992). Most cells also express TrkC at E13, but fewer cells express this trk at E16–E18. Neurite outgrowth is supported to some extent by multiple neurotrophins at E13–E16; however, outgrowth is more dense in response to BDNF and NT4, compared to NT3 or NGF (Rao et al., 1997; Rochlin et al., 2000). Neurotrophins have a clear and specific role in functional differentiation of embryonic rat geniculate (Al-Hadlaq et al., 2001, 2002) and trigeminal ganglia (Grigaliunas et al., 2002), as

discussued in later sections of the chapter. The expression of the neurotrophins themselves in sensory ganglia, for example, BDNF in rat geniculate at E14.5 (Schecterson and Bothwell, 1992) and NT3 in mouse at E18 (Ernfors et al., 1992), raises questions about autocrine support within the ganglia and anterograde transport to target tissues. B.

Neurites Growing to the Periphery from Ganglia: How Does Early Innervation of the Tongue Proceed?

A direct demonstration for neurotrophins in guiding nerve outgrowth into the embryonic tongue has not been made with in vivo or in vitro systems. However, from detailed studies of early lingual innervation in embryonic rat tongue, there is substantial understanding of basic regulatory principles in development of tongue pathways. To test the hypothesis that axons growing into the rat tongue distribute homogeneously and then redirect to densely innervate gustatory papillae, the lingual/chorda tympani, glossopharyngeal, and the hypoglossal motor nerve fibers were systematically traced in serial sections at different embryonic ages (Mbiene and Mistretta, 1997). With this study, a new set of working principles for development of tongue innervation was defined: 1.

2.

Tongue nerves do not first grow in widely distributed territories and then retract or redirect to particular tongue areas and to taste papillae. Points of entry and initial tongue pathways are restricted and precise from time of tongue formation through morphogenesis, so there are distinctive lingual territories for each nerve (Fig. 3). Although sensory fibers of the chorda/lingual nerve are within the tongue earlier than the hypoglossal

Taste System Development

3.

4.

motor fibers, sensory and motor nerves distribute independently of each other, neither following a “pioneering” path prepared by the other. Anterior and posterior tongue zones are discrete for nerve entry into and distribution within the lingual tissues. Once fungiform and circumvallate papillae have formed, through nerve-independent processes (Mbiene et al., 1997), they become densely innervated; in contrast, surrounding nonpapilla epithelium does not have periodic clusters of dense innervation. This latter principle provides indirect evidence for the potential role of papilla-derived molecules in attracting sensory nerves once they are within the tongue. Indeed it has been shown that neurotrophin expression is most intense in developing rat gustatory

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papillae during the periods just preceding, and during, nerve ingrowth (Nosrat and Olson, 1995). Recent experiments in mice that express a green fluorescent protein in growing nerves have demonstrated that neurotrophins in vivo guide sensory nerves from dorsal root ganglia into the limb bud (Tucker et al., 2001). Applying such models to studies of embryonic tongue will eventually define precise roles of neurotrophins in directing lingual innervation. Numerous other molecules must play a role in the stereotypic growth of nerves into the embryonic tongue. Chemorepellants, including those in the semaphorin family, have an apparent role in directing the early growing axons away from midline facial structures, from midline tongue, and reportedly away from early tongue epithelium (Rochlin and Farbman, 1998; Rochlin et al., 2000).

Figure 3 Photomicrographs of sagittal sections through the embryonic rat tongue at gestational day 15 (E15). Figures A through D correspond to sections taken from more lateral (A) to progressively more medial (D) tongue regions. Anterior tongue faces left. Points of entry and lingual pathways of the chorda/lingual (ch/l), hypoglossal (hy), and glossopharyngeal (gl) nerves are illustrated. Within the body of the tongue, separate distributions of all nerves are maintained. Near the midline (D) the tongue is essentially devoid of nerves with the exception of a dense plexus of fibers under the developing circumvallate papilla in posterior tongue. (Data from Mbiene and Mistretta, 1997.)

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The presence of C-Kit in taste bud cells of embryonic circumvallate and foliate papillae and in nerve fibers within the core of these papillae suggests interactions that might include tropic or tropic roles (McLaughlin, 2000). C-Kit is one of the protein tyrosine kinase receptors, molecules with known involvement in organogenesis in various systems.

C.

Neurites Growing Centrally from Ganglia: Initial Innervation and Establishment of the Nucleus of the Solitary Tract

In the rat, the peak production of geniculate ganglion neurons that will send central projections into the solitary tract is on day E12 (Altman and Bayer, 1982), somewhat in advance of the petrosal and nodose ganglia, with a peak production at E13 (Fig. 4). Fibers within the solitary tract are identified first at E17, whereas postsynaptic, secondorder taste neurons within the solitary nucleus are produced

between E12 and E15 with a peak at E13 (Altman and Bayer, 1982). This suggests that neurons are produced within the nucleus well in advance of initial entry of taste fibers into the tract (Mistretta and Hill, 1995). Trigeminal axons grow into the brainstem as early as E12 in rat (Mbiene and Mistretta, 1997). Using a slice preparation with ganglion and central and peripheral targets, it was shown that NGF promotes axon elongation without branching in the brainstem, whereas NT3 promotes formation of short axon collaterals (Ulupinar et al., 2000). However, levels of NGF and NT3 mRNA are apparently very low in developing rodent hindbrain (Davies, 1997), so in vivo neurotrophin effects on ganglion neurite growth centrally is far from clear. Interestingly, in DRG neurons studied in NGF/BAX double null mice, a requirement for NGF signaling in peripheral sensory neuron growth and differentiation was established, whereas NGF was apparently not required for extension of central axon projections (Patel et al., 2000). If this result generalizes to other sensory populations, it indicates that peripheral and central ganglion extensions have different molecular dependencies for extension and growth. III. PERIPHERAL TARGETS OF GANGLIA: TONGUE, PAPILLAE, AND TASTE BUDS

Figure 4 Diagram, based on rat embryo, of times of peak production of neurons in sensory ganglia that send processes into brainstem nuclei, including the nucleus of the solitary tract (SL). Peak production in the geniculate ganglion is at 12 days of gestation (E12), somewhat in advance of that in the petrosal ganglion at E13. VG, trigeminal ganglion; VIII Gs, spiral ganglion; VIII Gv, vestibular ganglion; IX-X Gs, superior or proximal, nongustatory ganglia of nerves IX and X (From Mistretta and Hill, 1995.)

Just as sensory ganglion neurons develop and begin to differentiate in advance of target development, and therefore, independently of target-derived support, so too the tongue and lingual papillae develop without initial neural dependence (Farbman and Mbiene, 1991; Mbiene et al., 1997). With continued papilla morphogenesis, and eventual differentiation of taste buds within papillae, nerve-target interactions ensue, and the nature and extent of these interactions is an area of active current research. Because much of the work on papillae and taste bud development derives from studies of rodents, we will summarize these processes for these species. However, there have been major advances in understanding how the peripheral gustatory structures develop in other species, especially human (e.g., see Bradley, 1972; Bradley and Stern, 1967; Ganchrow and Ganchrow, 1985, 1987; Witt and Reutter, 1997, 1998). There are few detailed reports of the morphological development of foliate papillae (Harada et al., 2000; Oakley, 1988); thus, morphogenesis of fungiform and circumvallate papillae only will be discussed. A.

Formation of the Tongue and Timing of Lingual Papilla Development

The rodent tongue emerges from a series of three swellings of mesenchyme tissue in the floor of the mandible (Mbiene

Taste System Development

et al., 1997; Mistretta, 1972). By E14 in rat and about E12.5 in mouse, the three swellings have merged to form the spatulate tongue, followed by extensive growth prenatally (Kaufman, 1992; Mistretta, 1972). Fungiform papilla formation in rat begins at E14 (gestation
21 days), with appearance of small protuberances on the dorsal tongue surface (Mbiene et al., 1997; Mistretta, 1972). By E15, the emerging papillae are clearly evident in that they are elevated from surrounding epithelia, and by E16, fungiform papillae are distinct in rostral to caudal rows. The patterned orientation of rows is maintained at adulthood, although the pattern is much less apparent amid the background of dense filiform papillae that appear on the tongue at about the time of birth. Because of the patterned arrangement, fungiform papillae are ideal targets to study developmentally related cellular and molecular processes in organogenesis. Histologically, fungiform papillae are a cluster of epithelial cells formed from stratification and elongation of cells in more ventral layers of the early embryonic tongue epithelium (Bradley, 1972; Farbman, 1965; Farbman and Mbiene, 1991). The characteristic fungiform papilla morphology is formed by the development of an epithelial thickening, with subsequent epithelial-mesenchymal interactions to form the connective tissue core surrounded by an annular downgrowth of epithelum (Bradley, 1972). As the surrounding epithelium continues its ventral growth, the papilla becomes further raised on the tongue surface. At its mature morphological stage, layers of epithelial cells cover the papilla. Circumvallate papillae located on the posterior tongue have distinct and unique patterning that, like fungiform papillae, originate early in development (Mistretta, 1991). In the adult rat, the single circumvallate located on the midline is relatively large, is located most posteriorly of all lingual papillae, and is surrounded by a trench that invaginates into the tongue. Initially, the circumvallate papilla lacks a surrounding trench, which emerges later as the epithelium grows down along the connective tissue core (Bradley, 1972; Mistretta, 1991). As noted for fungiform papillae, the rat circumvallate papilla forms between E14 and E15. Therefore, morphogenesis of fungiform and circumvallate papillae occurs at about the same time, although adult papilla morphology is very different between the two papilla types. B.

Fungiform Papilla Induction and Differentiation

Organ culture studies have verified that innervation is not necessary for papilla formation and patterned distribution (Mbiene et al., 1997). Fungiform papillae grow and differentiate when entire rat tongues are cultured beginning at

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E13 or 14, and temporal information is retained because papillae form at an equivalent of E15 in vivo, after 2 days in culture from E13, or one day in culture from E14. The patterned rows, characteristic of tongues obtained from older fetuses, are also clearly seen on cultured tongues. In addition to histological integrity, papillae that form on tongues in organ culture retain expression of neurotrophins similar to in vivo stages (Nosrat et al., 2001). Since the cultured tongue is dissected without sensory ganglia or intact trigeminal or gustatory nerves, it is clear that papillae (Farbman and Mbiene, 1991; Mbiene et al., 1997) and their signature neurotrophins (Nosrat et al., 2001) form initially without these nerves. Whereas it can be concluded that nerves do not direct papilla induction and patterning, there are only beginning indications of the gene products that might be central players (Mistretta, 1998). Several morphogens have been identified in the early mouse tongue and forming papillae (Bitgood and McMahon, 1995; Dassule and McMahon, 1998; Hall et al., 1999a; Jung et al., 1999) (Table 2). Preliminary data provide similar results for the sonic hedgehog protein (Shh) in papilla development in rat tongue (Hall et al., 1999b). Just as the Shh, BMP, and FGF families are key morphogens in tooth, hair, and feather formation, these molecules also will undoubtedly be important in papilla formation (Mistretta, 1998). Direct demonstration for a role of the Shh signal transduction pathway in papilla formation and patterning has recently emerged (Mistretta et al., 2000, 2002). Using a specific blocker of the Shh pathway, cyclopamine, in organ cultures of embryonic rat tongue, a doubling of fungiform papillae on the tongue was observed, compared to control cultures (Fig. 5). Because fungiform papillae were distributed even on posterior tongue surrounding the circumvallate papilla, the results also demonstrate a broad competence for lingual epithelium to generate fungiform papilla. This novel finding presents new modes of thinking about the tongue epithelium and its developmental competencies and restrictions. Other molecules that play a role in nerve/target interactions are expressed in specific papilla regions at midTable 2 Expression of “Patterning” Molecules in Embryonic Mouse Tongue Based on In Situ Hybridization Data

E11–12 E13 E14 E15–16

Shh

Ptc

Gli

Bmp2

Bmp4

Fgf8

   



  

   



 

   

Source: Bitgood and McMahon, 1996; Dassule and McMahon, 1998; Hall et al., 1999; Jung et al., 1999.

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Figure 5 Scanning electron micrographs of embryonic whole tongue cultures, begun at gestational day 14 and maintained in organ culture for 2 days. Fungiform papillae develop in the usual pattern in tongues cultured with standard medium (A). However, in tongues cultured in medium with addition of the steroidal alkaloid cyclopamine, which disrupts the sonic hedgehog signaling pathway, the number of fungiform papillae doubles (B). Also, fungiform papillae develop on posterior tongue in front of the circumvallate papilla. In A and B the circumvallate papilla is demarcated with an arrow. (Data from Mistretta et al., 2000, 2002.)

gestational stages of development. In rat fungiform and circumvallate papilla from E14-E16, BDNF is localized to papilla regions that will later form taste buds (Nosrat and Olson, 1995). In contrast, NT-3 is found primarily in the mesenchymal core and in nongustatory epithelium of the papillae (Nosrat et al., 1996). C.

Papilla Maintenance

Although the gustatory papillae develop in organ culture without intact sensory innervation (Farbman and Mbiene, 1991; Mbiene et al., 1997), after 6 days in whole tongue cultures initiated on E13 or E14, large numbers of fungiform papillae are no longer sustained (Mbiene et al., 1997). It appears, therefore, that whereas the early process of papilla induction is not nerve dependent, long-term papilla maintenance is. Further evidence for the role of innervation in papillae maintenance comes from nerve cut studies in early postnatal rats. Sectioning the combined chorda tympani nerve and lingual branch of the trigeminal nerve on postnatal day 1 is followed by degeneration of papillae over the following 21 days (Nagato et al., 1995). Indeed, sectioning only the chorda tympani in postnatal day 10 rats causes 65% of fungiform papillae to degenerate within 30 days, even though the lingual nerve is left intact (Sollars and Bernstein, 2000; Sollars et al., 1996, 2002). Of the fungiform papillae that remain, nearly 80% have unusual morphologies (Sollars and Bernstein, 2000). The morphological changes after early chorda tympani nerve section appear

to be permanent in that papillae do not regenerate at adulthood (Sollars and Bernstein, 2000); however, papillae do regenerate when chorda tympani sectioning occurs at adulthood (St. John et al., 1995). The early developmental effect is most likely attributed to an induced loss of geniculate ganglion cells in young rats due to nerve section (S. Sollars, personal communication); subsequently, the limited number of surviving neurons may not support the postnatal papillae. In contrast, only sectioning the lingual branch of the trigeminal nerve results in a transient degeneration of 44% of fungiform papillae in postnatal day 10 animals, followed by a reappearance of papillae within 50 days after nerve cut (Guagliardo et al., 1999). These findings collectively show that maintenance of the postnatal fungiform papilla is dependent on innervation by both the chorda tympani and trigeminal nerves and that there is likely an interplay between these nerves to maintain papilla structure. D.

Taste Bud Induction

While papilla induction clearly does not require innervation by gustatory and trigeminal nerves, there is considerable controversy concerning the role of these nerves in early taste bud formation. A long-standing view held that lingual taste buds required innervation for formation because nerves reached the dorsal papilla epithelium just before the appearance of rudimentary taste buds (e.g., Farbman, 1965). Thus, due principally to descriptions of timing, nerves have been considered essential to induce taste buds.

Taste System Development

Recent studies on both nonmammalian and mammalian taste bud development, however, have reassessed mechanisms underlying taste bud induction. In classical embryological experiments in salamander, Stone (1933, 1940) reported that taste buds differentiate independently from nerve fibers. In extensions of these experiments, grafts of presumptive oropharyngeal tissue in axolotl that were placed ectopically on the trunk of host embryos prior to innervation and prior to appearance of taste buds formed well-differentiated taste buds, even in the absence of neurites (Barlow et al., 1996). Tissue culture studies in which the presumptive axolotl oropharyngeal region was cultured prior to innervation further supported the idea that taste bud development occurs independently of innervation (Barlow et al., 1996). Experiments with trkB / knockout mice, which lack the tyrosine kinase receptor for brainderived neurotrophic factor, provided support for the idea that taste bud induction does not require innervation. In the absence of trkB, knockout mice lose the geniculate ganglion neurons that usually innervate taste buds in fungiform papillae. Yet taste buds apparently develop, although it is important to note that cytology is atypical, and size and numbers of these taste buds have not yet been specified (Fritzsch et al., 1997). Other studies support the more long-standing neural dependence view. For example, if the glossopharyngeal nerve is avulsed in newborn rat long before all taste buds have begun to form in the circumvallate papilla, the full number of taste buds is never acquired in the denervated papilla (Hosley et al., 1987). In addition, fungiform and circumvallate taste bud numbers in knockout mice missing genes for BDNF are severely reduced, and this is highly correlated with reduced numbers of innervating neurons (Jones et al., 1994; Liu, et al., 1995; Mistretta et al., 1999). This result is consistent with a neural dependence view of taste bud induction in which decreased numbers of innervating neurons should correspond directly with decreased numbers of taste buds. However, since mutant mice were examined only postnatally, after taste buds were induced, it is not clear whether the reduced number of taste buds reflects an initial failure of formation or whether taste buds develop in a rudimentary form and then quickly degenerate. Using intra-amniotic injection in pregnant mice of the neurotoxin -bungarotoxin, another demonstration of neural dependence in the taste system was observed (Morris-Wiman et al., 1999). Taste buds were not found in fungiform and circumvallate papillae in neurotoxininjected animals, compared to controls, and indeed numbers of fungiform papillae were also much reduced. Although some of the current studies challenge the dogma that taste bud induction is dependent upon innervation, induction of taste bud cells is far from understood. The

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axolotl studies would be expected to yield results that speak to different mechanisms from those in mammals (Brockes, 1997). Furthermore, taste bud development in amphibia is complex and highly specialized. The early barrel-shaped, axolotl taste buds from the larval stage are transformed and/or replaced during metamorphosis into taste buds with a disc-like, wider cell mass (Takeuchi et al., 1997). In fact, a separate, more rostral tongue structure, with wide discshaped taste buds on specialized papillae, grows to predominate over and replace the larval tongue with its population of smaller, oval taste buds that eventually disappear. Even the innervation of the two tongues and taste bud populations is not clearly known between nerves VII and IX. The transformation and rearrangement of taste organs on the salamander tongue regions suggest that a totally different set of taste organ structures could be addressed in the studies of Stone (1933, 1940) and Barlow et al. (1996) compared to the postmetamorphosis organism. Whereas chemosensory scientists generally think that mammalian taste buds in all oropharyngeal locations will have identical basic mechanisms for induction, there may indeed be variations between, for example, the epiglottal taste buds and those on the tongue. Mistretta (1991, 1998) has emphasized that on the mammalian tongue, the gustatory papillae are limiting elements for taste bud formation. This makes interpretation of results from knockout mice, where papillae are morphologically disrupted or not present at all, difficult to interpret. There also has been a tendency to use terminology somewhat loosely in some recent papers, so that the process of taste bud “induction” has sometimes been generalized to all of taste bud “development.” Key experiments and cautious data interpretation will be necessary to determine mechanisms of taste bud induction in mammals. This will be a tall order for a sensory receptor organ as complex as the taste bud, in which there is as yet no identified stem cell population or markers. E.

Taste Bud Formation, Numbers, and Size

Taste buds are added to the oropharynx both pre- and postnatally in some mammalian species, such as sheep and human (Mistretta, 1991). In contrast, the full complement of taste bud number in rat and hamster is acquired gradually during postnatal development (Belecky and Smith, 1990; Harada et al., 2000; Hosley and Oakley, 1987; Mistretta, 1972), in each lingual papilla type (Fig. 6). For each species there are differences in the timing of when the “mature” numbers of taste buds are formed. Often researchers use the formation of a taste “pore” to denote when the taste bud is morphologically mature. For example, in the rat, about 50% of taste buds in the soft palate

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Figure 6 Number of taste buds (A) and percentage of taste buds with pores (B) on the soft palate and in lingual fungiform, circumvallate, and foliate papillae, during postnatal development in rat. Development of mature taste bud numbers and pore acquisition have different temporal sequences in different oral regions. (Data redrawn from Harada et al., 2000.)

have taste pores at birth (Harada et al., 2000) (Fig. 6), whereas the majority of fungiform taste buds do not have taste pores until after P12 (Mistretta, 1972), and about 50% of circumvallate papilla taste buds have taste pores by approximately P30 (Harada et al., 2000; Hosley and Oakley, 1987). While using the appearance of a taste pore is useful in denoting the presence of taste buds, it should not be the sole marker of taste bud maturity. Functionally, Mbiene and Farbman (1993) suggest that the absence of a pore during early development may not prevent access of stimuli to taste buds. Instead, the epithelium covering the pore is permeable to stimuli. Therefore, the presence of a pore may not be necessary for the peripheral gustatory system to function at some, albeit immature, level. Various factors have been proposed to regulate final numbers of mammalian taste buds (Mistretta and Hill, 1995), including papilla size constraints for lingual taste buds. The fungiform papillae are not uniform in size

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across the tongue. In rat there is a systematic increase in papilla size, from the anterior quarter of the tongue where diameter averages 135 m, to the posterior quarter with average diameter of 180 m (Mistretta and Baum, 1984). Although the difference in papilla size is substantial, only one taste bud is observed per fungiform papilla in rat, suggesting particular controls on taste bud number. In contrast to rodent, in the human, sheep, and primate tongue there are multiple taste buds per fungiform papilla, and a direct relation between size of papilla and taste bud numbers has been demonstrated from fetal through adult stages in sheep (Mistretta et al., 1988). Furthermore, the numbers of taste buds per papilla increase prenatally and then decrease after birth in sheep, in direct association with an increase and subsequent decrease in number of fibers in the chorda tympani nerve (Mistretta et al., 1988). The important question of factors regulating taste bud number is not understood and may well relate to different cell and molecular factors in lingual versus extralingual taste bud populations; there is experimental evidence for epithelial, neural, and dermal matrix controls (Mistretta and Hill, 1995). Whereas numbers of taste buds vary widely within lingual papillae and in soft palate and epiglottis, taste bud size throughout the oropharynx is more homogeneous than taste bud number within a species. Even without papilla size constraints, extremely large taste buds are not found on the adult epiglottis or soft palate (discussion in Mistretta, 1991). In fact, during development large taste buds apparently “split” to yield more numerous smaller taste buds (Bradley, 1972; Bradley et al., 1980). It has been proposed that very large taste buds may form in fungiform papillae during development under the influence of excess innervation and then divide to yield more numerous, smaller taste buds as neural competition yields appropriate receptive fields (Mistretta, 1998). Furthermore, it will be clear from discussion in a later section that taste bud size in rat fungiform papilla varies directly in relation to innervating elements, with an intriguing developmental progression (Krimm and Hill, 1998). The circumvallate papilla contains a few hundred taste buds in rat, and as a taste organ it contrasts in many ways with the fungiform papilla. Recent work incorporates use of bax null mutant mice to disrupt programmed cell death during development and thereby increase gustatory innervation (Zeng et al., 2000). The excessively innervated circumvallate papilla in bax / mice was larger than in wild-type animals and had larger taste buds, demonstrating a direct link among innervation, papilla size, and taste bud size.

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F.

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Taste Bud Cells: Cycle and Life Span

Aside from the specific factors that regulate taste bud numbers and size, the final site of action is the control of taste receptor cell cycle and life span. Since taste receptors turn over in adult mammals approximately every 10 days, there must be a continual replacement of dying cells by way of new cell divisions (Beidler and Smallman, 1965). Therefore, by altering the rates of cell division, cell life span, and cell death, the size and numbers of taste cells within each taste bud will be affected. Surprisingly, very little attention has been devoted to study of such processes. There now are emerging studies, however, that address these issues. Indeed, Zeng et al. (2000) have recently identified some cell death pathways in circumvallate taste cells by using bax null mutant and wild-type mice. They provide evidence for p53, Bax, and Caspase-2 in taste cell death. This is unusual in that these multiple factors are generally not evident in keratinocytes, cells that also are sloughed from lingual epithelia, and they indicate tight regulation of taste cell death. Perhaps the most interesting, but most difficult to study, aspect of taste cell life span is in the proliferative phase. Specifically, it is not clear what cells give rise to the taste receptor cells, which themselves are almost entirely postmitotic (Beidler and Smallman, 1965). Stone and colleagues (1995), using transgenic X chromosome–inactivated mosaic mice, reported that taste cells and epithelial cells come from a common progenitor and that taste receptor cells may originate from both ectoderm and endoderm. In part because of this complexity, the location and cell types that are taste bud progenitors have yet to be identified. While there have been some advances in understanding taste cell life span in adult taste buds, virtually nothing is known about the earliest formation of taste buds and cell cycle. Recently, however, Hendricks and Hill (1999) provided preliminary data suggesting that the proliferation rates, the rates of dividing cells that are entering the taste bud, and the life span of taste cells are much longer in rats aged 10 days postnatal compared to adults (Fig. 7). Therefore, at least some of the cell cycle parameters of the developing taste system in rat fungiform taste buds seem to be much different than in adults. This is consistent with previous results suggesting that fungiform papillae have very low proliferation rates in late fetal rat that later increase (Farbman and Mbiene, 1991). Similarly, Ganchrow and colleagues (1995) provide evidence that cell proliferation is absent during early periods of chick taste primordium formation. The new data and continuing studies on taste bud cell proliferation will be crucial for discerning core biological principles of taste bud formation.

Figure 7 Photomicrograhs of fungiform papilla and taste bud in a postnatal rat aged 10 days (A) and an adult rat (B). The nuclei of all cells are labeled green, whereas taste bud cells only are labeled red with an antibody directed against cytokeratin 19. The postnatal day 10 taste bud is smaller and has fewer cells than the adult bud. (Data from Hendricks and Hill, 1999.) (See color insert.)

G. Development of the Ganglion Cell Peripheral Receptive Field: Numbers of Fungiform Papillae and Taste Buds Per Ganglion Cell and Numbers of Ganglion Cells Per Papilla and Taste Bud The number and location of taste buds innervated by a single fiber or ganglion cell defines the receptive field for that neuron. Therefore, the functional unit for a primary afferent neuron relates to its receptive field. The development and organization of receptive fields were first examined in fetal and early postnatal sheep (Mistretta et al., 1988), in which an inverted “U” function described the relationship between age and the number of taste buds innervated by single chorda tympani neurons (discussion in Mistretta and Hill, 1995). The change from relatively small, to large, to small receptive fields indicates a highly dynamic system of neuron/target interactions. One of the implications of this morphological remodeling with age is that there are

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corresponding functional changes, addressed at length in a later section. By examining the innervation of single taste buds in rat, findings complementary to the receptive field results cited above have been provided. The approach in rat is different from that of quantifying the number of taste receptor elements per innervating fiber, because it determines the number of ganglion neurons that innervate a single taste bud (Fig. 8). In the latter approach, the location of the taste bud on the tongue surface does not predict how many geniculate ganglion cells innervate a fungiform taste bud (Krimm and Hill, 1998). However, there is a highly ordered and predictable relationship in mature rats, when the number of ganglion cells is related to the size of the taste bud. Beginning at postnatal day 40 and continuing through adulthood, the number of neurons that innervate each taste bud is directly related to taste bud size; the larger the taste bud, the greater the number of innervating ganglion cells (Fig. 9). By contrast, there is no relationship between the size of the taste bud and the number of innervating neurons in rats aged postnatal day 10 through postnatal day 30 (Fig. 10). Further studies on this age-related relationship demonstrated that the number of ganglion cells innervating a taste bud is established early in development, long before taste buds reach their mature size (Krimm and Hill, 1999). The mean number of ganglion cells that innervate single taste

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buds is similar from postnatal day 10 to postnatal day 40 in rat; only taste bud size increases with age (Krimm and Hill, 1999). Moreover, the majority of neurons that innervate a taste bud at 10 days postnatal continues to innervate it through 40 days. Surprisingly, the size of the taste bud in 40-day-old rats is predicted by the number of geniculate ganglion cells labeled at 10 days. These findings indicate that the neural “template” for the receptive field is determined early and becomes matched with taste bud size later in development. This match may involve interactions between taste buds and innervating neurons, most likely by way of diffusible factors. At least some of the interaction likely includes the neurotrophin family of molecules, discussed in a later section. These collective findings reveal the type and nature of nerve/target interactions during developmental regulation and maintenance of taste organ morphology, receptive fields, and taste function. For example, in rat, it is from late gestation through the second postnatal week that individual taste buds receive their mature complement of innervating neurons, which in turn establishes the size that the taste bud acquires. These processes must include interactions with molecules produced by target tissues (attractant and repellent) that act on innervating neurons and, reciprocally, are related to molecules that are produced by neurons that innervate taste receptor cells. In short, this reflects proper matching between neurons and targets and

Figure 8 (A) Photomicrograph of a coronal section through a fungiform papilla and taste bud in which Fluoro-Gold had been iontophoretically injected. (B) Photomicrograph of a section through the geniculate ganglion, illustrating a ganglion cell that was labeled with Fluoro-Gold after the papilla injection in A. The ganglion cell in B, therefore, innervated the taste bud illustrated in A. (From Krimm and Hill, 1998.) (See color insert.)

Taste System Development

Figure 9 Graph of the number of ganglion cells that innervate a single taste bud in fungiform papillae as a function of the respective taste bud volume in adult rat. Open dots denote data from taste buds on the tongue tip, and solid dots denote data from taste buds on the mid-region of the tongue. The regression line for the mid-tongue data is shown as a solid line, and dotted lines on either side show the 95% confidence limits. The correlation coefficient and regression equation are given in the lower right region of the figure. (From Krimm and Hill, 1998.)

actions of an ensemble of molecules that have varying functions during well-defined periods of development. One family of molecules that plays multiple roles and has had much recent experimental attention is the neurotrophins. H. Neurotrophin Molecules and Roles in Peripheral Taste Organ Development In addition to the role of neurotrophins in ganglion development and neuron survival (see Sec. II. A), these molecules also are critical in processes related to taste receptor organ development. These processes include early taste organ formation, growth, and maintenance and refinement of receptive field size (i.e., neuron/target matching). Furthermore, functional differentiation of ganglia that innervate the tongue is altered by neurotrophins (discussed in a later section). Thus, neurotrophins do not simply permit gustatory neurons to survive, but they play various roles in the complex processes of peripheral taste system development. As noted earlier, targeted gene deletion of the neurotrophin receptor, trkB, results in severe alterations in gustatory papillae in mice (Fritzsch et al., 1997). Furthermore, deletion of the gene for the trkB ligand, brain-derived neurotrophic factor (BDNF), results in as much as a 60% decrease in numbers of fungiform papillae

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(Mistretta et al., 1999; Nosrat et al., 1997). Interestingly, fungiform papillae in BDNF null mutants were disproportionately spared on the anterior tip of the tongue (Mistretta et al., 1999), demonstrating that not all papillae are affected similarly through the loss of this neurotrophin. Deletion of the gene for BDNF also affected development of the circumvallate papilla (Mistretta et al., 1999; Nosrat et al., 1997; Oakley et al., 1998), which was reduced in size by about 40% compared to the wild-type papilla (Fig. 11). Liebl et al. (1999) extended these findings by demonstrating that mice with targeted deletions of the gene for neurotrophin NT4/5, which also uses the trkB receptor, sustain a loss of fungiform papillae at birth. As may be predicted from the results listed above, deletions in the genes for trkB (Fritzsch et al., 1997), BDNF (Mistretta et al., 1999; Nosrat et al., 1997; Oakley et al., 1998), and NT4/5 (Liebl et al., 1999) all have led to significantly decreased taste bud numbers and size of remaining taste buds. In some cases, such as in circumvallate papilla in BDNF knockout mice, the magnitude of taste bud loss exceeds the magnitude of change in papilla size (Mistretta et al., 1999), indicating that these gene deletions may have an even greater effect on taste bud sustenance than on papilla development (Fig. 11). Regardless of the specific gene affected (i.e., trkB vs. BDNF) or the target (i.e., fungiform vs. circumvallate papillae and taste buds), the common effect is that there is an accompanying, significant loss of the respective ganglion cells (geniculate and petrosal). It is likely, therefore, that the morphological changes seen in the target organs are related to the loss of innervating neurons. Because there has been no study of taste papilla formation in embryonic BDNF or NT4/5/ mice, conclusions about early neurotrophin support from tongue papillae for geniculate or petrosal ganglia cannot be derived with these animals. Ganglia are dependent on neurotrophins in advance of taste papilla formation, so decreased numbers of ganglion neurons from early stages could then lead to loss of neural support for sustaining the early taste papillae, which form without intact innervation (Mistretta, 1998). What is clear from use of such models, however, is a direct association between ganglion cell loss and gustatory organ loss. An important conclusion emphasized by Mistretta et al. (1999) is that with loss of about 60% of the fungiform papillae (and thus resident taste buds) and of about 50% of neurons in the geniculate ganglion in BDNF/ mice, the remaining geniculate neurons do not extend neurites into the tongue to “rescue” papillae (and resident taste buds) that are lost due to reduced lingual innervation (Mistretta et al., 1999). This indicates that there is specificity in the nature of fungiform papilla innervation: all fungiform

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Figure 10 Relationship between the number of geniculate ganglion cells that innervate a single taste bud and the respective taste bud volume, shown for fungiform papillae in rats aged 10, 20, 30, and 40 days postnatal. Open symbols in A and B denote data from taste buds on the tongue tip, and solid dots in all graphs denote data from taste buds in the mid-tongue region. The dashed regression line in each graph is from data obtained from taste buds in the mid-tongue of adult rats. Dotted lines on either side of the regression line in D show the 95% confidence intervals corresponding to the adult data. The correlation coefficient and regression equation for postnatal day 40 rats are in the lower right region of panel D. (From Krimm and Hill, 1998.)

papillae are not uniformly dependent on a generalized innervation from any of a set of homogeneous ganglion cells. Furthermore, in the BDNF/ mice from the Reichardt laboratory used by Mistretta et al. (1999), only about 16% of trigeminal ganglion cells were lost, yet remaining trigeminal neurons did not “rescue” fungiform papillae from the tongue. In short, neither remaining geniculate nor trigeminal neurons substitute to rescue fungiform papillae and/or their resident taste buds. Some investigators have proposed that NT-3 sustains remaining papillae and taste buds in BDNF/ mice (Nosrat et al., 1997). Clearly multiple neurotrophins will participate in development of taste organs, and the nature and specifics of this participation are still emerging. Interesting data derive from examination of transgenic mice that are BDNF overexpressors (Krimm et al., 2001,

Nosrat et al., 2000), illustrating the complexity of interactions bewteen neurotrophins and lingual targets. In a somewhat unexpected result, in mice that overexpress BDNF there is a decrease in size and number of fungiform and circumvallate papillae and a decrease in number of taste buds (Krimm et al., 2000; Ringstedt et al., 1999). In one of the overexpressor models, the promoter sequence from the nestin gene resulted in a bolus of BDNF in the core of the tongue muscle and innervating fibers “stalled” at that location (Ringstedt et al., 1999). However, in an important and detailed study, Krimm et al. (2001) used a keratin 14 promoter to drive overexpression of either BDNF or NT4 in basal epithelial cells, so that fibers reached and innervated the lingual epithelium. Geniculate ganglion cells were increased in number by 93% (K14BDNF mice) and 140% (K14-NT4 mice). Neuron number

Taste System Development

Figure 11 Photomicrographs of histological sections of the circumvallate papilla and taste buds from the tongue of wild-type (/) and BDNF null mutant (/) mice at postnatal day 25. Sections were made parallel to the surface of the tongue and stained with hematoxylin and eosin. Compared with wild-type tongues, the circumvallate papilla in null mutants was smaller in diameter and length and lacked morphological integrity. Taste buds remained in / circumvallate papilla but were much reduced in number. (From Mistretta et al., 1999.)

in the trigeminal ganglion was unchanged (LeMaster et al., 1999). However, fungiform papillae and taste buds were reduced in number and size (Krimm et al., 2001). Papillae formed in normal numbers embryonically but were gradually lost postnatally. Careful analysis of innervation patterns in these tongues demonstrated that lingual innervation was directed to filiform papillae in preference to fungiform papillae. Although the lingual epithelium had generalized dense innervation from geniculate neurons, taste buds did not form in these extrapapilla areas, demonstrating again the necessity of papilla epithelium for lingual taste bud development (Mistretta, 1991, 1998). The “misdirection” of taste afferents into filiform papillae suggests that a specific localized distribution of BDNF and NT-4 is essential for target-nerve interactions that sustain gustatory organs (Krimm et al., 2001). Data on effects from loss of neurotrophin and associated receptors provide insight into normal developmental processes. The following is a working scheme of how taste

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bud development, after induction and initial differentiation stages, may be regulated in relation to neurotrophin functions (Fig. 12). In postnatal rat, the size of individual fungiform papillae and taste buds varies considerably, but systematically, across the tongue surface (e.g., Krimm and Hill, 1998). Once taste buds are induced, BDNF, which is expressed at the apex of developing fungiform papillae (Nosrat and Olson, 1995; Nosrat et al., 1996), could be made in disparate amounts in different papillae and taste buds, thereby determining the number of neurons that are sustained in synpatic relations with taste buds cells. The number of neurons sustained to innervate the taste bud would be proportional to the amount of BDNF produced by each taste bud. Therefore, the number of innervating gustatory neurons could be determined very early in development. In fact from the work by Krimm and Hill (2000) presented earlier (see Sec. III.E), the mature number of innervating neurons for each rat fungiform taste bud is determined before postnatal day 10. As seen earlier, the number of innervating neurons may then help determine the mature size of the taste bud achieved about 30 days later, when taste bud size is directly related to the number of innervating neurons (e.g., Krimm and Hill, 1998). That is, the maximal number of taste bud cells supported in each taste bud may be determined by the number of innervating neurons. Once the maximal number of cells is achieved, the rate of cell loss is matched by the rate of cell replacement. While the mechanism by which neurons direct taste bud growth is unknown, there is strong evidence that a factor(s) released from gustatory nerves support taste bud cells. This is best illustrated by the series of experiments in adult rodents showing that sectioning of gustatory nerves (Cheal and Oakley, 1977; Fujimoto and Murray, 1970; Guth, 1957; Von Vintschgau and Honigschmied, 1876) or blockage of axoplasmic transport by colchicine (Sloan et al., 1983) results in loss of taste buds. Neurotrophins could be one factor that participates in various aspects of taste bud cell development because neural transport of neurotrophins can proceed anterogradely as well as retrogradely (Tonra et al., 1998). IV. CENTRAL TARGETS OF GANGLIA: BRAINSTEM TRACTS AND NEURONS AND HIGHER ORDER RELAYS A.

Projections into the Nucleus of the Solitary Tract

In the rat, chorda tympani fibers begin synapse formation in the rostral pole of the nucleus of the solitary tract (NTS) as early as postnatal day 1 (Lasiter et al., 1989), even though

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Figure 12 Model of possible interactions between young, developing taste buds (after induction and initial stages of differentiation and innervation) and innervating neurons. Once taste buds are induced, BDNF could be made in disparate amounts in different papillae and taste buds, influencing the number of neurons sustained in synaptic relations with taste bud cells. See text for discussion of model.

some nerve fibers may arrive centrally much earlier (Altman and Bayer, 1982; Scott and Atkinson, 1998). However, the terminal field of the chorda tympani does not reach its full size until approximately postnatal day 25 (Lasiter, 1992). From their initial projections into the rostral pole of the NTS, chorda tympani axons migrate caudally and surround local neurons and the output neurons of the NTS. In contrast to terminations of the chorda tympani nerve in the rostral NTS, fibers of the glossopharyngeal nerve, which innervate taste receptors on the posterior tongue, do not enter the intermediate zone of the NTS until postnatal day 9–10 in rat (Lasiter, 1992). Moreover, the rostral-caudal expansion of this field is not complete until approximately postnatal day 45 (Lasiter, 1992). Preliminary findings (Sollars and Hill, unpublished data) indicate that the terminal field of the greater superficial petrosal nerve (GSP) is mature in size and topography long before the terminal fields of the chorda tympani and glossopharygeal nerves. This is especially noteworthy because the GSP and chorda tympani terminal fields overlap considerably at adulthood (Hamilton and Norgren, 1984). It is interesting to note that the sequence of primary afferent projection into the NTS follows the pattern of morphological and functional maturation noted for the respective receptor populations (see Sec. V). It is possible, therefore, that activity may play a role in terminal field

organization of gustatory afferents, similar to that seen in other sensory systems (see, e.g., Coleman, 1990). B.

Neurons in the Nucleus of the Solitary Tract

Neurons postsynaptic to chorda tympani fibers in the rat NTS also show dramatic changes in morphology, with dendritic lengths and dendritic branching increasing approximately threefold between P8 and P25 (Lasiter, 1992; Lasiter et al., 1989), a time window that approximates the developmental period when the terminal field of the chorda tympani nerve matures. Indeed, the collective increase in pre- and postsynaptic neurites in the NTS with age indicates that an increasingly large afferent message may influence circuit formation (e.g., Lasiter, 1992; Mistretta and Hill, 1995). These results are reinforced through findings in developing sheep, which, as noted earlier, have a significant prenatal gustatory development. Mistretta and Labyak (1994) demonstrated that the dendritic lengths and dendritic spine numbers of putative relay neurons in the NTS increased during the time when functional convergence occurred and when taste circuits matured. Therefore, in both sheep and rat, there appears to be a correspondence of maturation of function and structure in neurons that receive and then transmit gustatory information to more central structures.

Taste System Development

In contrast to the timing delay between terminal field and dendritic development in the rostral NTS, the period that separates these events in more caudally located NTS neurons is significantly shorter. However, maturation of pre- and postsynaptic elements occurs later in more caudally located NTS zones. Specifically, in rat only one week separates the morphological maturation of the pre- and postsynaptic elements in the NTS where the glosopharyngeal nerve projects (at bout P45) (Lasiter, 1992). C.

Higher-Order Gustatory Relays

The primary gustatory projection from the NTS is to the parabrachial nuclei in the pons (PBN). The development of this projection does not parallel that of the chorda tympani nerve to the NTS (Lasiter and Kachele, 1988, 1989). Specifically, second-order neuron projections from the NTS to the PBN do not begin until postnatal day 7 (about 1 week later than the chorda tympani to NTS projection) and the terminal field maturation is slower, attaining completion at about postnatal day 60 (Lasiter, 1992). There is an apparent serial development of these central gustatory projections; therefore, significant development of the PBN does not occur until some level of maturation is achieved in the NTS. Little information is available concerning the morphological development of the PBN and its more rostral relays. The delayed development of presynaptic elements (i.e., the terminal field) in the PBN is reflected in postsynaptic cells. Dendrites of PBN neurons arborize significantly between postnatal days 16 and 35 in rat (Lasiter and Kachele, 1988), the period during which projections to the next relay in the thalamic taste area occur and metabolic activity of PBN neurons increases (Lasiter and Kachele, 1988). These laterdeveloping morphological changes suggest that functional response maturation of the PBN may differ from those in the NTS and, as noted for terminal field development, may be serially dependent on NTS development.

V. FUNCTIONAL DEVELOPMENT OF THE GUSTATORY SYSTEM A.

Taste Buds and Peripheral Nerve Function

Large increases in responses to some, but not all, taste stimuli characterize the functional development of the peripheral gustatory system. The most notable change is in response to sodium salts. For example, the chorda tympani nerve in sheep (Mistretta and Bradley, 1983) and rat (Ferrell et al., 1981; Hill and Almli, 1980; Yamada, 1980) is poorly responsive to NaCl during early development. In

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sheep, in which there is a significant prenatal taste development (Bradley and Mistretta, 1973), low responses to NaCl occur from the last trimester and increase until adulthood (Mistretta and Bradley, 1983). In rats, in which taste development occurs almost entirely postnatally (Farbman, 1965; Mistretta, 1972), responses to sodium salts soon after birth are very low and then increase in magnitude through weaning (Ferrell et al., 1981; Hill and Almli, 1980; Yamada, 1980). In both species, sodium salts elicit the largest magnitude response of all stimuli in the chorda tympani at adulthood. By comparison, the developing gustatory system is highly responsive to some nonsodium salt stimuli as soon as it begins functioning. Citric acid and ammonium chloride (NH4Cl) produce vigorous responses in the chorda tympani nerve in rats aged 2 days postnatal (Hill and Almli, 1980). Therefore, the taste system responds with “mature” magnitudes to some stimuli early in development (Hill et al., 1982), unlike other sensory systems where peripheral neurons and receptor cells are “sluggish” to all stimuli (see Coleman, 1990). Developmentally, there is also a functional/morphological reorganization of receptive fields, as demonstrated in sheep (Nagai et al., 1988). Namely, during the period when the average receptive field size decreases in sheep (see Sec. III.G), there is an increase in the proportion of fibers that respond maximally to NaCl. Furthermore, the neurons with small receptive fields throughout development respond with higher responses to NaCl and fibers with larger receptive fields respond maximally to NH4Cl. B.

Epithelial Sodium Channels and Salt Taste Development

With the advent of amiloride, a drug used to block epithelial sodium channels, significant advances have been realized in identifying sodium taste transduction pathways and the development of these pathways. In adult rats, lingual application of amiloride attenuates responses to NaCl by 70–80% and eliminates responses to sodium acetate (Formaker and Hill, 1988). The difference in the amount of suppression relates to multiple or single transduction pathways for NaCl and sodium acetate, respectively, and appears to be dependent on the size of the anion (see Stewart et al., 1997). Developmental studies that have used the epithelial sodium channel blocker are summarized in the following paragraphs. Increased sensitivity to sodium occurs along with increased sensitivity to amiloride. The initial small taste responses to NaCl from the chorda tympani nerve in rats younger than 13 days are suppressed little by amiloride. However, the NaCl taste response becomes larger through development, as does the amount of response suppression

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by amiloride (Hill and Bour, 1985; Sollars and Bernstein, 1994). Thus, increased sensitivity to sodium appears to reflect an increase in functional amiloride-sensitive sodium channels (Hill and Bour, 1985). This relatively specific developmental change that may involve a single transduction pathway makes these rats interesting models for immunohistochemical study. With the use of a polyclonal antibody directed at the amiloride channel, immunopositive taste buds are unexpectedly seen on the tongue of rats aged one day postnatal (Stewart et al., 1995). This is unexpected because chorda tympani responses from early postnatal rats have small (or no) responses to sodium salts and little sensitivity to amiloride (Hill and Bour, 1985; Sollars and Bernstein, 1994). Since the antibody was polyclonal, it was proposed that at least some form of the channel is present in early developing taste buds and that the channels become functional later in development (Stewart et al., 1995). Alternatively, the channels may be functional in taste receptor cells of young rats, but not available to sodium ions that stimulate the apical domain (Stewart et al., 1995). That is, the channels may not be inserted into the membrane in the apical domain of taste receptor cells. Support for this alternative is found in whole cell patch clamp recordings of stimulus-induced conductances in postnatal rat (Kossel et al., 1997). Receptor cells from fungiform taste buds in P2 rats were as sensitive to amiloride as cells from P30 rats. Sodium-related conductances were suppressed in taste bud cells similarly throughout development. An important aspect of these studies is that apical and basolateral domains are not functionally separated in whole cell patch clamp recordings. Therefore, all stimulus-induced conductances are recorded, regardless of the location of the transduction site. Indeed, the relative lack of amiloride sensitivity in neural recordings (Hill and Bour, 1983; Sollars and Bernstein, 1995) suggests that whole cell recordings detect functional amiloride-sensitive channels that are not normally reached by chemical stimuli in intact tissue because of their location in the basolateral domain of taste receptor cells (Kossel et al., 1997). Further definition of the initial availability of functional sodium channels on the taste receptor membrane in early postnatal rats comes from biophysical analyses. Stimulusinduced ionic currents were recorded in vitro from dorsal lingual epithelia from P14 rats to adult, and sodium conductances reportedly increased during development with a corresponding increase in amiloride sensitivity (Settles and Mierson, 1993). Recently, these results were extended with an in vivo voltage clamp procedure. Briefly, voltage was applied across lingual epithelia in anesthetized rats in order to control the driving force of ions while recording

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neurophysiological taste responses from the chorda tympani nerve (Hendricks et al., 2000). With the use of biophysical modeling techniques, parameters such as channel densities (i.e., numbers/unit volume) and channel affinities (i.e., function) (Ye et al., 1993) were derived in developing rats. The number of functional sodium channels on the anterior tongue increased monotonically with age (Fig. 13), and furthermore, the channels also became more efficient (i.e., have higher affinities for sodium salts). Thus, an increase in channel densities and in channel function accounts for the dramatic increase in sodium salt responses during development. The apparently conflicting results between the whole cell patch recordings (Kossel et al., 1997) and results from the in vitro epithelia (Settles and Mierson, 1993) and in vivo neural recording (Hill and Bour, 1983) may be explained by translocation of the channel from the basolateral to the apical domain, where they are able to “sense” sodium stimuli. The translocation of functional amiloride channels in polarized epithelial cells has been demonstrated in a variety of tissues (see Garty and Benos, 1988; Garty and Palmer, 1997) and is not unique to gustatory tissue. Identification of factors and processes involved in channel trafficking and/or channel regulation during development would be a major advance in our understanding of salt taste development. Moreover, identification of the cellular and molecular events of channel production, including epithelial sodium channel subunit complementation, would serve to provide the underlying cellular mechanisms that are involved in this process.

Figure 13 Chorda tympani nerve response ratios versus NaCl electrochemical concentration for postnatal rats in four age groups. The data demonstrate a developmental increase in number and efficiency of sodium channels in taste bud cells. (From Hendricks et al., 2000.)

Taste System Development

In summary, these findings provide critical information regarding sodium response ontogeny. First, they point to the cellular locus in the development of sodium responses. Second, they establish a developing transduction pathway that is relatively well characterized in gustatory and nongustatory tissue (e.g., Avenet and Lindemann, 1988; DeSimone and Ferrell, 1985; Garty and Benos, 1988). Collectively, these results can be exploited to better understand the underlying mechanisms involved in sodium taste development. C.

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chorda tympani nerve was responsive (Bradley and Mistretta, 1980; Mistretta and Bradley, 1978a). This absence of NaCl responses was not seen in NTS in rats as young as postnatal day 14, which was the youngest age examined (Hill et al., 1983); however, it may be found in younger rats. Furthermore, in both rat (Hill et al., 1983) and sheep (Bradley and Mistretta, 1980; Mistretta and Bradley, 1978), mature NTS responses were achieved later in development compared to the chorda tympani (Bradley and Mistretta, 1973; Ferrell et al., 1981; Hill and Almli, 1980; Mistretta and Bradley, 1983; Yamada, 1980). For

Gustatory Ganglia

As seen above, there have been considerable advances in characterizing the developing peripheral gustatory system through neurophysiological studies of gustatory nerves and, more recently, taste receptor cells. However, virtually nothing is known about the functional development of cell bodies of the taste nerves in gustatory ganglia. It is likely, because of the recent attention devoted to the role that such ganglia have in the morphological induction and maintenance of taste buds, that more emphasis will be given to the functional characteristics of these cells. Indeed, in recent reports, investigators make use of ganglion explants from embryonic rat and whole cell recordings from the cultured ganglia. Numerous passive membrane and action potential properties are different in neurons from E16 trigeminal compared to geniculate ganglion (Fig. 14), indicating that at early stages in ganglion development, neurons have begun to differentiate functionally (Al-Hadlaq et al., 1999; Grigaliunas et al., 2002). Furthermore, explanted ganglion cells have altered membrane and action potential properties after exposure in culture to different neurotrophins (Al-Hadlaq et al., 2001, 2002). Because the neurotrophins used in these experiments are encountered by growing neurites in the tongue in vivo, the results indicate that neurotrophins could modulate ganglion cell function during outgrowth to gustatory target organs in the embryo. D.

Central Gustatory Nuclei

Recordings of taste responses from NTS neurons in rat (Hill et al., 1983) and sheep (Bradley and Mistretta, 1980; Mistretta and Bradley, 1978) demonstrated that central response development was similar in general sequence to chorda tympani nerve response development. Response magnitudes to sodium salts increased profoundly in NTS neurons during development. In contrast, NTS neuron responses to other stimuli, such as to NH4Cl, did not change with age. There was a complete lack of sensitivity in NTS neurons to sodium salts in fetal sheep, whereas the

Figure 14 Whole cell patch recordings in response to series of hyperpolarizing and depolarizing current pulses, shown at bottom, in neurons from trigeminal and geniculate ganglia. Ganglia were dissected and maintained in culture from embryonic rats at gestational day 16. Numerous passive membrane and action potential properties are different between trigeminal and geniculate cells even at this early embryonic stage. For example, as illustrated, at threshold currents trigeminal neurons generally produce a single action potential, whereas a substantial proportion of geniculate neurons produce mutiple action potentials. (Data from Grigaliunas et al., 2002.)

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example, in rats, mature taste responses to sodium salts in the chorda tympani occur at postnatal weeks 3–4. In the NTS, mature responses appear about one week later. Therefore, central taste response development is related not only to peripheral changes, but also to central synaptic changes (Bradley and Mistretta, 1980; Hill et al., 1983; Mistretta and Bradley, 1978). Synaptic organization or reorganization may occur during development in the NTS, which further delays development of taste responses. This probably results in a degraded neural signal across the first gustatory relay during the immature NTS period. Indeed, taste response development in sheep NTS neurons follows a structural refinement of receptive fields that can best be attributed to a remodeling of synapses from chorda tympani fibers onto NTS neurons (Vogt and Mistretta, 1990). Specifically, there is an increase in receptive field sizes during development in sheep NTS neurons that most likely reflects an increased convergence of chorda tympani fibers onto the central neurons with age. These findings, as well as increases in spontaneous rates and stimulus-elicited response frequencies (Bradley and Mistretta, 1980; Hill et al., 1983; Mistretta and Bradley, 1978), suggest that neural activity plays a role in the way that primary afferents synapse onto their central target neurons. The delay cannot be attributed to poorly developed NTS neurons because functional membrane characteristics are mature before the taste-elicited responses. In fact, the membrane parameters of resting membrane potential, action potential, and discharge properties change most between P5 and P15 in rats, with mature values reached by P20 (Bao et al., 1995). Spine density on these neurons increases from P5 to P10 and then decreases to adult stages. This provides further evidence that changes in the synapses and not the NTS neurons per se are what account for the prolonged functional changes postnatally. Similar developmental patterns are observed in the next synaptic relay, the PBN. Responses to taste stimuli that change developmentally at more peripheral levels also occur in the PBN (Hill, 1987). However, PBN neurons have increased response magnitudes to all stimuli from weaning to early adulthood, including responses to stimuli that are very effective in eliciting NTS responses during early development. Therefore, PBN neurons appear to be “sluggish” to all stimuli early in development, and then sensitivity gradually increases with age. The delayed functional maturation at successively higher synaptic levels suggests that central anatomical and/or neurochemical events are responsible for the unique developmental patterns at each neural relay.

Mistretta and Hill

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Mistretta and Hill Timmes, A., Nguyen, T., Lindsay, R. M., Acheson, A., and DiStefano, P. S. (1998). Axotomy upregultaes the anterograde transport and expression of brain-derived neurotrophic factor by sensory neurons. J. Neurosci. 18: 4374–4383. Tucker, K. L., Meyer, M., and Barde, Y. A. (2001). Neurotrophins are required for nerve growth during development. Nat. Neurosci. 4:29–37. Ulupinar, E., Jacquin, M., and Erzurumlu, R. S. (2000). Differential effects of NGF and NT-3 on embryonic trigeminal axon growth patterns. J. Comp. Neurol. 425:202–218. Von Vintschgau, M., and Honigschmied, J. (1877). Nervus glossopharyngeus and Schmeckbecher. Pflugers Arch. 14:443–448. Vogt, M. B., and Mistretta, C. M. (1990). Convergence in mammalian nucleus of solitary tract during development and functional differentiation of salt taste circuits. J. Neurosci. 10: 3148–3157. White, F. A., Chiaia, N. L., MacDonald, G. J., and Rhoades, R. W. (1995). Birth dates and survval after axotomy of neurochemically defined subsets of trigeminal ganglion cells. J. Comp Neurol. 352:308–320. Witt, M., and Reutter, K. (1997). Scanning electron microscopical studies of developing gustatory papillae in humans. Chem. Senses 22:601–612. Witt, M., and Reutter, K. (1998). Innervation of developing human taste buds. An immunohistochemical study. Histochem. Cell Biol. 109:281–291. Yamada, T. (1980). Chorda tympani responses to gustatory stimuli in developing rats. Jpn. J. Physiol. 30:631–643. Ye, Q., Heck, G. L. and DeSimone, J. A. (1993). Voltage dependence of the rat chorda tympani response to Na salts: Implications for the functional organization of taste receptor cells. J. Neurophysiol. 70:167–178. Zeng, Q., Kwan, A., and Oakley, B. (2000). Gustatory innervation and bax-dependent caspase-2: participants in the life and death pathways of mouse taste receptor cells. J. Comp. Neurol. 424:640–650. Zhang, C., Brandemihl, A., Lau, D., Lawton, A., and Oakley, B. (1997). BDNF is required for the normal development of taste neurons in vivo. NeuroReport 8:1013–1017.

37 Contemporary Measurement of Human Gustatory Function Marion E. Frank, Thomas P. Hettinger, Michael A. Barry University of Connecticut Health Center, Farmington, Connecticut, U.S.A.

Janneane F. Gent Yale University, New Haven, Connecticut, U.S.A.

Richard L. Doty University of Pennsylvania, Philadelphia, Pennsylvania, U.S.A.

I.

discrimination from human observers. Magnitude estimates and absolute detection thresholds for sweet, salty, sour, and bitter stimuli are measures of stimulus strength and are by far the most common taste data. Although tastequality categories are well defined, tests of taste-quality identification and discrimination are less well developed. The rationale for focusing on measures of taste intensity of prototypical stimuli representing four taste qualities is based on the concept that taste is truly a set of independent sensory systems (McBurney and Gent, 1979) that may be addressed separately (Bartoshuk, 1989a,b; Frank and Smith, 1991). In contrast, tests of odor identification (Cain, 1989; Doty et al., 1984) and discrimination (Wright, 1987) use a variety of odors without purporting to assess the gamut of odor qualities (Dravnieks, 1985). It is interesting that measures of threshold and identification for qualitatively distinct odors are highly correlated, suggesting that olfaction is a synthetic sensory system. Such is not the case for taste; correlations among measures of sweet, salty, sour, and bitter function are low (Cowart et al., 1997). Assessments of gustatory function that bypass human judgments include evoked potentials (electroencephalography) and magnetoencephalography (Kobayakawa et al., 1999; Plattig, 1991), as well as a number of brain imaging techniques (see below). Complicated computerized chemical stimulus–delivery devices are used because reliable timing of multiple stimulus repetitions is required. Activating a chemosensory system electrically greatly

INTRODUCTION

The chemosensory systems of taste, smell, and chemical irritation all discriminate among strengths and qualities of chemical stimuli. However, the three chemosensory systems utilize distinct neural pathways, have receptors that are distributed differently, and provide distinct information about chemical stimuli (Frank and Rabin, 1989). Strategies for measuring taste function reflect its special characteristics. The sense of taste, a special visceral chemical sense with receptors located within the oral cavity, discriminates chemical features that indicate nutrient or poison (Bartoshuk, 1991; Frank et al., 1992; Scott and Mark, 1987). It is generally accepted that most taste perceptions are sweet, salty, sour, or bitter. “Nontraditional” tastes (Hettinger et al., 1990) often are flavor sensations derived from retronasal stimulation of olfactory receptors (see Chapters 10, 22, and 44). The savory umami quality may be an exception (Hettinger et al., 1996; Yamaguchi, 1991). In contrast, smells are more diverse, defying consistent perceptual categorization (Beets, 1971), and irritations may form one perceptual category (Green and Lawless, 1991). Taste testing most frequently measures functional competence based on judgments of human subjects, an approach with origins in the late nineteenth century (Bartoshuk, 1978). Psychophysical techniques are designed to systematically obtain ratings of sensory intensity, sensory thresholds, quality identification, and quality 783

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simplifies these biological approaches; electric-taste stimuli are promising in this regard but do not separately activate taste subsystems (Frank and Smith, 1991). This chapter focuses on contemporary assessments of taste function commonly employed in applied settings and suggests possible future assessments. Other basic psychophysical paradigms, described for olfaction in Chapter 10, may also be applied to gustation. The reader is also referred to related chapters on the psychophysics of taste mixtures (Chapter 38) and the genetics of human taste perception (Chapter 40).

II. WHOLE-MOUTH AND REGIONAL TASTE TESTING There are two general approaches to taste testing with distinct objectives: whole-mouth testing and regional testing. As noted elsewhere in this volume (see Chapters 32, 35, and 44), taste is composed of several subsystems with distinct taste bud fields that are associated with distinct papillary structures [long known to contain taste-receptive elements (Bell, 1803; Haller, 1763; Horn, 1825; Müller, 1838)] and branches of cranial nerves (CN) (Frank et al., 1992). Branches of the facial nerve (CN VII), the chorda tympani and greater superficial petrosal nerves, innervate anterior tongue and palatal fields, respectively; in these fields taste buds are dispersed among many papillae (Imfeld and Schroeder, 1992; Miller and Bartoshuk, 1991). Lingual branches of the glossopharyngeal nerve (CN IX) innervate posterior tongue fields; in these fields many hundreds of taste buds are concentrated in several large circumvallate (Arey et al., 1935) and foliate papillae (HouJensen, 1933). Taste buds located in the caudal pharynx and larynx are innervated by the superior laryngeal branch of the vagus nerve (CN X) (Lalonde and Eglitis, 1961); they are sensitive to tonicity or pH and not thought to be involved in gustatory discrimination (Bradley, 2000). Functional differences of taste bud fields (Collings, 1974; Dunér-Engström et al., 1986; Sandick and Cardello, 1981) (see also below) and knowing that disease and injury may affect individual fields are rationales for regional taste testing. With regional testing, function in different taste fields is assessed separately by limiting stimuli to specific oral sites. Losses restricted to single taste bud fields due to a single malfunctioning nerve may go unnoticed (Bartoshuk, 1989b), perhaps because of mutual inhibition among the separate fields (Catalanotto et al., 1993; Lehman et al., 1995). In the more common whole-mouth testing, taste stimuli are not restricted spatially and may reach all taste bud fields, as in most real-life experiences. Whole-mouth testing is the norm in the food industry, where, for

Frank et al.

example, one may wish to evaluate preferences among product formulations with test panels made up of individuals without chemosensory dysfunction (Heymann and Lawless, 1997). Determination of a detection (absolute) threshold requires reliable identification of the presence of something, not a qualitatively discernable sensation, such as a sweet or salty taste. Classically, detection thresholds were determined directly by procedures such as the methods of constant stimuli and limits or indirectly by the method of adjustment (Fechner, 1860). (See below and Chapter 10 for more on classical psychophysical methods.) The method of adjustment was used to directly measure how perceived intensities of taste stimuli are adjusted with the passage of time or when presented as components of mixtures (e.g., Pangborn, 1984; Vanne et al., 1998; Woskow, 1967). However, because of the large numbers of trials required, the possibility of sensory adaptation, and/or subject fatigue, other direct methods have been developed that minimize trials and yet provide a reliable threshold estimate. In Sec. II.A and II.B popular whole-mouth and regional techniques for determining taste detection thresholds formulated in the mid-twentieth century are illustrated. They differ from each other in method of stimulus delivery and stringency of criterion performance. The whole-mouth techniques use 3 drops (Henkin et al., 1963) or 8 cups (Harris and Kalmus, 1949) to present taste solutions, a difference that affects the reliability at criterion performance as well as threshold values. The regional technique, electrogustometry, presents weak electrical currents via localized electrodes to stimulate “metallic” electric taste in the separate taste bud fields (Krarup, 1958; Mackenzie, 1955). A.

Three Drops or Eight Cups

The 3-drop and 8-cup techniques are two popular threshold procedures in the clinical taste literature (Gent et al., 1997; Hertz et al., 1975). The two techniques (Weiffenbach et al., 1983) define threshold by criterion performances that have quite different probabilities for false positives (Table 1). The probability of guessing correctly (PG) at the threshold concentration is 0.259 for the 3-drop and 0.014 for the 8-cup technique, but additional requirements at lower and higher concentrations (associated events) reduce probabilities for false-positive thresholds. In the 3-drop test (1/3), subjects are presented, on each trial, with three drops of liquid, one of which contains a taste stimulus; the other two contain pure water. Threshold is the concentration at which the subject chooses the drop containing the stimulus either three trials

Contemporary Measurement of Human Gustatory Function Table 1 Probabilities for False Positives in Threshold Tasks Events Task

Trials correct

Threshold

Associated

Combined

1/2 1/2 1/3 4/8

5 in row 4 reversals At least 2/3 1 errorless

0.0313 0.0156 0.259 0.0143

0.500 0.250 0.192 0.757

0.0156 0.00391 0.0498 0.0108

Each threshold test is identified by task (e.g., one of two stimuli contains taste: 1/2) and trials correct for threshold event (e.g., five in row); see text for additional information. The threshold event is the trial or series of trials on which the subject succeeds, and that stimulus is considered the threshold stimulus. Associated events are requirements on trials before and/or after the threshold trial.

in a row or in two of three trials (PG
1/33  6/33). The subject must also perform as well at the next higher concentration (probability
7/27) and make three incorrect choices or two of three incorrect choices on three trials at the next lower concentration (PG
8/27  12/27
20/27). The entire threshold sequence occurs by chance with a probability of (7/27) (7/27) (20/27), or about 0.05 (Table 1). In the 8-cup test (4/8), subjects are presented on each trial with 8 cups; 4 contain a taste stimulus and 4 contain pure water. The threshold event requires errorless performance; the subjects must divide the 8 cups into two groups, one containing the taste stimulus (PG
(4/8) (3/7) (2/6) (1/5)
1/70) (Table 1). The subjects must also make two or more errors at the next lower concentration (PG
53/70). The entire threshold sequence occurs by chance with a probability of about 0.01 (Table 1). Thus, five thresholds might be false positives with the 3-drop technique to each one false positive with the 8-cup technique. In a direct comparison, the three-drop technique yielded higher thresholds than the eight-cup procedure for both sucrose and NaCl (Weiffenbach et al., 1983) (Table 2), which is no surprise. The volume of stimulus sampled is either a drop (0.1 mL) or a sip (1 mL), yielding a big difference in numbers of taste buds contacted (Doty et al., 2001; Smith, 1971). Also, taste receptors are salivaadapted for the 3-drop (no water rinses) but water-adapted for the 8-cup (includes water rinses). Both stimulus volume and rinsing affect taste measurements (Lawless, 1987), and they may both affect NaCl detection more than sucrose detection (Weiffenbach et al., 1983). Choice of one of these two threshold techniques relies more on whether one wishes to test taste bud receptors in a saliva-adapted or water-adapted state (Rehnberg et al., 1992) than on differences in PG (Table 1). Temporal parameters of stimulation are important to consider in taste testing (Halpern, 1997). For example, sensory adaptation over time can have a profound

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influence on repeated or sustained testing of taste when intervening water rinses are not possible (see below). When a stimulus is applied constantly to the same lingual location, its perceived intensity will decrease markedly; in fact, under certain circumstances it disappears within a few minutes (Bujas et al., 1991a; Ganzevles and Kroeze, 1987a, b; Gent and McBurney, 1978; McBurney, 1976). Mouth movements tend to prevent complete adaptation (von Békésy, 1965). The spread of stimulus solution to tongue recesses untouched by the original tastant application and an increase in stimulus intensity at those sites may be at play. Recovery from adaptation occurs rapidly after replacing the adapting stimulus with water (Bujas et al., 1991a; Hahn, 1934). Both adaptation time and recovery time after rinsing vary with stimulus intensity and quality (Meiselman, 1968). Interestingly, a moderate degree of adaptation to 100 mM NaCl may actually enhance differential sensitivity (McBurney et al., 1967). The Weber fraction ( I/I), where I reflects the differential threshold from the standard (I), was 0.20 for subjects with unadapted tongues and 0.75 for subjects with adapted tongues. McBurney (1984) argues that adaptation may serve to adjust the taste system (as well as other sensory systems) to be maximally sensitive to changes in stimuli near a prevailing intensity. B.

Localized Electric Current

In electrogustometry, an electrical stimulus is applied to oral sites via a stimulus electrode, typically a small metal disk (for review, see Frank and Smith, 1991). Electrogustometers have the benefit of being light and portable and provide a well-defined stimulus to specific regions of the oral cavity. There is no need for chemical solutions, and the stimuli can be applied discretely in space and time. Detection thresholds for anodal stimuli are established, which—for 12.5 mm2 stainless steel electrodes— average 8 A for the anterior tongue, 13 A for the posterior tongue, and 20 A for the palate in normal Japanese subjects (Tomita et al., 1986). However, taste

Table 2 3-Drop and 8-Cup Detection Thresholds for 14 People Threshold in mM ( SEM) 3-drop NaCl Sucrose

38 (  7.2) 32 (  5.6)

8-cup 5.8 (  2.1) 12.9 (  1.9)

NaCl ( p , 0.001) and sucrose ( p , 0.01) mean thresholds (6 SEM) differ for the two methods. Source: Data from Weiffenbach et al., 1983.

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thresholds are sensitive to the procedure by which values are obtained (Weiffenbach et al., 1983), including the specific task and stringency of criterion performance. For example, average electrogustometric thresholds for the anterior tongue were 20 A when European subjects detected a current presented simultaneously with a tactile stimulus (Krarup, 1958) and 3 A when subjects detected a current added to an ongoing tactile stimulus (Fons, 1970). Standard psychophysical approaches are used to establish electric-taste thresholds. In a two-alternative forced-choice procedure (1/2) (Cain, 1989), paired current and blank are randomly and sequentially delivered through electrodes already positioned on the tongue until the subject chooses the current side five times in a row (Murphy et al., 1995; Sampson et al., 1993). In one study, threshold values were, on average, 4 A for 15 young subjects but 10 A for 17 seniors (Murphy et al., 1995). A correct two-alternative forced choice series (PG
1/25) must be preceded by an incorrect choice (PG
1/2), giving the threshold sequence a chance probability of 1/64 (Table 1). In staircase procedures (see below) peri-threshold stimuli are repeatedly sampled to increase reliability of threshold values (e.g., Miller et al., 2001). Electrogustometry efficiently verifies suspected chorda tympani nerve injury. Anodal currents of less than 40 A were undetected on the front of the tongue after nerve section (Grant et al., 1989), and a greater than 100-A side-to-side threshold difference is an accurate predictor of a unilateral denervation from Bell’s palsy (Groves and Gibson, 1974). Electrogustometry may be more powerful in a clinical setting (Ovesen et al., 1991; Stillman et al., 2000) than testing with chemicals (chemogustometry), which is more tedious and taxes patients’ already strained resources. In lung, breast, and ovarian cancer patients, for whom no differences were detected with chemogustometry, the average anterior-tongue anodal threshold was 30 A compared to 10 A in noncancer patients (Ovesen et al., 1991). Recently, an automated procedure for determining electrogustometric thresholds was developed and evaluated for test-retest reliability (Lobb et al., 2000; Stillman et al., 2000). Extending electrogustometry to assess supra-threshold taste function (Salata et al., 1991) with currents less than 100 A could be valuable for future psychophysical and biological clinical evaluation (evoked potential, functional brain imaging) (see sec. IV). In the next section, contemporary testing as practiced in clinics specializing in chemosensory disorders is described and evaluated.

Frank et al.

III. A.

CONTEMPORARY TASTE TESTING Assessment of Gustatory Function

Specialty clinics focusing on disorders of the chemical senses have in the last two decades devised and evaluated psychophysical protocols designed to effectively and efficiently assess taste function (Gent et al., 1997). Two measures of gustatory intensity processing (detection thresholds and intensity ratings) and one measure of gustatory quality processing (quality identification) are typically taken. Subjects sample chemical solutions from cups (“sip and spit”) and rinse with water between stimuli for whole-mouth testing. Electrical or chemical stimuli are restricted to the separate taste bud fields for regional testing. Throughout this section and Section IV, examples of measuring effects of age and taste disorders of common etiology on taste function are noted (see also Chapter 44). 1.

Whole-Mouth Taste Intensity

Contemporary testing of intensity processing by the gustatory system typically involves use of a single chemical compound to represent each of the taste qualities: sweet (e.g., sucrose), salty (e.g., NaCl), sour (e.g., citric acid), and bitter (e.g., quinine•HCl). Whole-mouth testing, in which taste solutions are sipped and moved throughout the oral cavity, assesses functioning of all taste bud fields simultaneously, either at threshold, which is “traversed” at the onset of any stimulation (Halpern, 1997), or across a range of effective stimulus concentrations. a. Taste Detection Thresholds. Detection thresholds have been defined by a two-alternative forced-choice (1/2) tracking (staircase) procedure (see above) as the average of concentrations at which the last four of five reversals occurred. A “reversal” is either an increase in presented concentration if one choice is incorrect, or decrease in concentration if two sequential choices are correct. A four-reversal threshold event (1wrong—2 right—1 wrong—2 right) has a PG of (1/2)(1/4)(1/2)(1/4)
1/64 and must be preceded by 2 correct responses; thus a “false positive” occurs with a probability of 1/256. Tracking is an order of magnitude more stringent than the 3-drop test described above (Table 1). Median “tracked” detection thresholds for 32 normal controls less than 36 years of age approximated 0.63 M for quinine sulfate and 1.6 mM for NaCl (Cowart, 1989). In comparison, average 8-cup NaCl threshold was 5.8 mM and average 3-drop NaCl threshold was 38 mM (Table 2). The rigorous tracking identified no age-related differences in whole-mouth detection thresholds for either citric acid or quinine sulfate (Cowart, 1989),

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yet citric acid is rated 22% and quinine is rated 40% lower in intensity by seniors at high suprathreshold levels (Frank et al., 1992; Weiffenbach et al., 1986). Whole-mouth detection thresholds may not be the best “clinical indicators” (Bartoshuk, 1989a), perhaps in part because of compensatory mechanisms among the several taste-bud fields (Bartoshuk, 2000; Catalanotto et al., 1993; Lehman et al., 1995; Matsuda and Doty, 1995).

magnitude matching protocol are used to normalize individualistic numerical ranges (Bartoshuk, 1989a, b). The ratio relations among intensities of the different stimuli are defined with magnitude matching, and responses are not confined to categories or limited by a visual analog scale, as they may be with category-scale procedures. In order to evaluate a patient’s level of function (normal or abnormal), comparisons are made between taste intensity ratings for a particular patient and those of appropriate normal controls. Taste intensity rating using either procedure has identified effects of age and clinical conditions that are associated with taste disorders. For example, to seniors, 18 mM citric acid and 180 M quinine sulfate had modest categorical taste intensities, whereas to young subjects they were strong (Table 3), (Cowart, 1989). Also, patients who had experienced head trauma rated citric acid concentrations numerically lower than other patients (Deems et al., 1991). A possible problem with these ratings is that individuals with a tendency for hyperbole/underestimation may use high/low values to describe perceptual experiences of many modalities (Cowart, 1989). The normalization to a modality incidental to taste with magnitude matching corrects for this problem; however, to be effective it requires subjects have normal hearing. Taste intensity ratings for sucrose, NaCl, citric acid, and quinine.HCl, normalized to loudness in a magnitude matching protocol, increased sixfold in young subjects but fourfold in seniors with a 10-fold increase in stimulus concentration (Bartoshuk, 1989b; Frank et al., 1992). This difference is consistent with seniors being unable to discriminate between two binary mixtures of sucrose and tartaric acid with mixture components differing slightly in intensity (Kaneda et al., 2000). Stimulus mixtures, which are the preponderance of stimuli encountered in natural situations, may prove effective in assessing taste dysfunction (Stevens, 1996; Stevens and Cain, 1993). Taste mixtures

b. Taste Intensity Rating Scales. Rating scales have been used to quantify responses to taste stimuli at suprathreshold intensity levels, which represent much of taste perceptual experience (Halpern, 1997). By the midtwentieth century, direct methods (e.g., Stevens, 1961), in which subjects make direct judgments of “subjective” magnitude, had replaced classical indirect methods (e.g., Fechner, 1860) (in which scales are derived from “objective” measures) as scaling methods of choice. With direct magnitude estimation, subjects may simply rate the intensity of subjective perceptions relative to a self-determined standard to produce a scale, whereas indirect scales are constructed from “just noticable difference” (JND) steps measured over the range of stimulus intensity. The multiple JNDs required may be measured directly or indirectly using the method of adjustment (see sec. II), also known as the method of average error. The standard deviation of the mean intensity match of a comparison stimulus to a standard is considered the JND. Ratings scales are often employed in industrial applications to establish the degree to which the taste of food is liked. For example, Pangborn and Giovanni (1984) had adults, whose intake of sweet foods was documented with a questionnaire, directly rate both the degree of liking and the perceived intensity of sweetness of lemonade containing different amounts of sucrose. In general, perceived taste intensity increased with sucrose concentration; however, for most individuals lemonade with intermediate levels of sucrose was liked most, yet there were some individuals who found either high or low sucrose levels more likeable. The subjects’ intake of sweet foods and their hedonic responses to the sweetness of lemonade were correlated. Numerical category and magnitude-matching procedures are the scaling methods (see Chapter 10 for more on methodological detail) used commonly in contemporary clinical applications. In the former procedure, patients rate taste intensities of concentration series within defined numerical categories (e.g., 0–12, labeled as “no taste” to “extremely strong taste”) (Cowart, 1989; Deems et al., 1991); in the latter, they concurrently rate the taste intensities and the loudness of a series of 1000 Hz tones on self-consistent ratio scales. The loudness ratings in the

Table 3 Mean Taste Intensities, 0 (No Taste) to 12 (Extreme Taste), of Citric Acid and Quinine Sulfate by Two Age Groups Intensity ratings Citric acid Quinine sulfate Age group

1.8 m

18 m

18 m

180 m

19–35 years 65–79 years

5.0 3.2

9.2 7.5

3.5 2.0

9.4 7.3

These data were a portion of the measurements made on 137 normal subjects from 10 to 79 years old. Statistical analysis of the entire data set indicated a significant effect of age on citric acid and quinine sulfate ratings. Source: Data from Cowart, 1989.

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have been used primarily to address mechanisms employed by the gustatory system to evaluate components of complex stimuli (e.g., Bartoshuk and Gent, 1985; Breslin and Beauchamp, 1995; Prescott et al., 2001). Chapter 38 presents information on how the gustatory system deals with stimulus mixtures, and the reader is referred to Chapter 10 for information on how the olfactory system deals with mixtures. An example of measurement of dysfunction with scaling procedures in those experiencing taste disorders comes from studies of taste function in women with burning mouth syndrome (BMS), a disorder discussed in Chapter 44. Magnitude-matched taste intensities of women with BMS were 12% lower than controls overall, with higher levels in NaCl and sucrose affected most (Formaker and Frank, 2000). Also, it is well known that many medications reversibly alter taste function (Frank et al., 1992; Mott et al., 1993; Schiffman et al., 1999, 2000). (The reader is also referred to Chapter 44 for more on this issue.) The resulting dysfunction of the gustatory system may be measured with scaling procedures. For example, treatment with 0.12% chlorhexidine gluconate, a bitter drug used to control accumulations of oral bacteria, results in frequent taste complaints and reduced patient compliance (Lang et al., 1988). Magnitude-matched intensities of midrange levels of NaCl and quinine •HCl were reduced 50% (Helms et al., 1995) following treatment, and similar effects were observed with a fixed-interval, 10-point category scale (Frank et al., 2001). Thus, direct ratings of subjective intensity permit across-subject comparisons and separate normal from abnormal function. Labeled magnitude scales (LMS) (Green et al., 1996) for taste have emerged recently as potential alternatives to direct magnitude estimation scales that require normalization (Bartoshuk, 1989a) or to labeled linear category scales (Cowart, 1989; Deems et al., 1991) that can suffer from ceiling effects (Bartoshuk, 2000; Lucchina et al., 1998). On one LMS, verbal labels of intensity are spaced quasilogarithmically rather than linearly. When subjects are instructed to treat the upper bound as “strongest imaginable oral sensation” or “strongest imaginable taste,” the LMS produces psychophysical functions equivalent to those produced by magnitude estimation (i.e., more likely to reflect an expansive continuum) (Green et al., 1996; Lawless et al., 2000). This particular LMS has proven to be a useful psychophysical tool to categorize a subject’s genetically determined taster status with intensity ratings of a concentration series of the bitter 6-n-propylthiouracil (PROP) (Lucchina et al., 1998). PROP “supertasters,” whose ratings accumulate at the ceiling of a fixed-interval category scale, are accommodated by the LMS (Bartoshuk, 2000).

Frank et al.

2.

Whole-Mouth Taste Quality

A straightforward test of taste is to have subjects report whether a tastant produces a sweet, salty, sour, or bitter sensation, although there is a bothersome semantic uncertainty in the use of taste quality names (Cowart et al., 1997; Hettinger et al., 1999; Meiselman and Dzendolet, 1967). Fractionated judgments, in which a subject rates the relative magnitude of the sweet, salty, sour, and bitter components, can also be made (e.g., Smith and McBurney, 1969; van der Klaauw and Smith, 1995), but such techniques may require selection of subjects (Kuznicki et al., 1983; Ossebaard et al., 1997) and/or training in order to increase reliability (e.g., Settle et al., 1986). In contemporary chemosensory clinics, patients are simply expected to identify the predominant taste quality of members of concentration series of prototypal sweet, salty, sour, and bitter solutions (Gent et al., 1997; Seiden et al., 1992). This involves identifying the taste quality of 15–20 solutions that are suprathreshold to normal controls (Cowart, 1989; Formaker and Frank, 2000). Such identification tasks are “objective” in the sense scoring is correctincorrect (Campbell, 1957; Weiffenbach, 1987). Although highly educated people perform at 90% correct—better than less educated people, who perform at 83% correct (Cowart, 1989)—age and medical conditions reported to affect taste (see Chapter 44) are reliably reflected in identification performance. Seniors more than 65 years of age correctly identified the taste quality of 87% of the solutions presented, whereas people 35 years of age or younger identified 95% (Cowart, 1989). Head trauma patients correctly identified the taste quality of 80% of the solutions, other patients identified 84% (Deems et al., 1991), and women with BMS identified 81% compared to 92% for controls (Formaker and Frank, 2000). Stimuli rated low in intensity are associated with more mistakes (Formaker and Frank, 2000; Frank et al., 2001), suggesting a redundancy in rating and identification data. Consistent misidentification patterns, however, may identify specific taste weaknesses (Gent et al., 1999) or distortions (Frank et al., 2001; Helms et al., 1995). 3.

Regional Taste Function

Measurement of taste function on the separate regions of the tongue may use either electrical (electrogustometry) or chemical (chemogustometry) stimuli (Frank and Smith, 1991). Electrogustometry, which is by nature a regional technique, was described above in the context of defining regional detection thresholds. In contrast to electrogustometry, regional chemogustometry can separately address tastes within each taste bud region, with measurements similar to those described above for whole-mouth testing.

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Contemporary specialty chemosensory clinics deliver sweet, salty, sour, and bitter chemical solutions to the separate taste bud fields via 5-mm-diameter filter paper discs (Tomita et al., 1986), cotton swabs (Bartoshuk, 1989a), or pipettes (Deems et al., 1991). It is possible to thicken stimulus solutions with cellulose to minimize stimulus spread (Kroger et al., 1997) or present chemical stimulants to circumscribed oral regions via gelatin cubes (Delwiche et al., 2000) or flow chambers attached to the lingual surface (e.g., Bujas et al., 1991a, b; Kelling and Halpern, 1988). A microprocessor-controlled gustometer that is capable of delivering chemical stimuli to small regions of the anterior tongue isolated by a vacuum seal (Hebhardt et al., 1999) may rival electrogustometry in stimulus control and sensitivity (Doty et al., 2001; Matsuda and Doty, 1995; Tunsuriyawong et al., 2000) in a clinical setting. Contemporary clinical chemogustometry compares either an individual patient’s “quality recognition” thresholds or quality identification and intensity ratings for suprathreshold stimuli to control values. Effects of aging on taste thresholds have been measured with the “filterpaper-disc” method (Tomita et al., 1986), in which concentration series are used to establish a threshold for a “clear taste quality,” which is named by the subject. A stimulus is presented once via a filter paper disc, and if no clear sensation (“no taste,” “undefined taste”) is indicated, a stronger stimulus is presented. “Recognition” thresholds determined with the filter paper discs showed seniors (60–92 years) generally had “low to moderate hypogeusia” for taste bud fields on the anterior tongue, posterior tongue, and palate. This conclusion was based on a doubling of average threshold concentration for sucrose, NaCl, and tartaric acid, and a quintupling of threshold concentration for quinine•HCl in the seniors compared to 11 to 29-year-olds (Tomita et al., 1986). This general increase in taste thresholds contrasts with specific effects of age on regional supra-threshold intensity ratings. Young people of 22 years average age rated citric acid sourness on the front of the tongue five times stronger than seniors over 74 years of age; however, the two age groups rated sucrose similarly (Bartoshuk, 1989b). The differences between regional ratings and quality thresholds cannot be explained by compensatory mechanisms among several taste-bud fields (Bartoshuk, 2000) alone. But recruitment mechanisms (Matsuda and Doty, 1995) may be more prominent for sweet taste than sour taste. If so, regional thresholds may be more sensitive “clinical indicators” than regional ratings. Results with signal-detection measures of taste sensitivity derived from judgments of certainty (Matsuda and Doty, 1995) are consistent with this suggestion. Many seniors were unable to detect high concentrations of NaCl

restricted to small lingual sites easily detected by younger individuals. Regional intensity ratings can identify dysfunction localized to specific taste bud fields not detected with whole-mouth ratings (Mott et al., 1994; Zuniga et al., 1994). For example, patients suffering from bulimia rated palatal taste stimuli less intense than controls, but lingual and whole-mouth ratings were normal (Rodin et al., 1990). Yet, following extraction of the four third molars, intensity ratings for whole mouth, palate and tongue were lower than controls (Shafer et al., 1999). Perhaps as the number of fields affected increases, whole-mouth function fails. Taste loss in specific oral regions, which may be accompanied by reports of taste phantoms and distortions (dysgeusia), has most often been associated with damage to taste nerves. (See also Chapter 44 for the kinds of nerve injury that may affect taste function and the effectiveness of nerve repair.) Regional taste deficits have been measured following neural damage stemming from middle-ear surgery/disease (Arnold, 1974; Bull, 1965; Chilla et al., 1982; Wiberg, 1971), facial/glossopharyngeal nerve disease/compression (Groves and Gibson, 1974; May and Schlaepfer, 1975; Taillibert et al., 1998; Todrank and Bartoshuk, 1991), oral surgery (Deems et al., 1991; Jacks et al., 1998; Mott et al., 1994; Zuniga et al., 1994, 1997), and head trauma (Bartoshuk et al., 1996; Costanzo and Zasler, 1991). Brain infarcts may also result in localized taste dysfunction (Fujikane et al., 1999). B.

Evaluation of Gustatory Assessments

Many of the tests devised by contemporary specialty clinics primarily address the processing of gustatory intensity perceptions with an expectation of establishing taste blindness (ageusia) or weakness (Gent et al., 1997). But subjective intensity, a quantitative perceptual variable, can be influenced adventitiously. Distinguishing between a genuine taste weakness and “normal” taste function requires data from specific controls precisely matched to patient characteristics, including their age, which had not, until recently, been considered so critical a variable (see above; see also Chapter 44) (Frank, 1994; Gilmore and Murphy, 1989; Murphy and Gilmore, 1989; Stevens et al., 1995). The range of normal taste function at any age is wide, perhaps reflecting in part varied numbers of taste buds present on individuals’ tongues (Doty et al., 2001; Miller and Bartoshuk, 1991; Zuniga et al., 1993) and genetic taster status (see below and also Chapter 40). Controls based on genetic status should account, in part, for the measured variability. Also, the disordered gustatory processing of taste quality manifest in those who experience dysgeusia has not been measured. Dysgeusia is considered the most

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crippling of taste disorders (Cowart et al., 1997; Deems et al., 1996; Mott et al., 1993; Seiden et al., 1992) 1. Aging and Taste Function Diagnosis of hypogeusia, alterations in perceptions of taste intensity, addressed with detection thresholds and magnitude ratings, requires comparison to age-matched controls. a. Thresholds. Detection-threshold concentrations, stimulus levels at which subjects reliably detect the presence of a stimulus, are “objective” assessments. Adult age extremes (averages: 24 and 83 year) show a 10-fold difference in average threshold concentration for sucrose, NaCl, citric acid, and quinine•HCl (Bartoshuk et al., 1986). For example, the median threshold for citric acid was 15 M for the younger subjects and 150 M for the older subjects. In fact, acid thresholds for only 3 of 18 young people overlapped values for the 18 seniors (Table 4). Agespecific distributions of normal thresholds are needed for specific protocols to diagnose hypogeusia.

identification of sweet, salty, sour, and bitter stimuli is routinely assessed (Gent et al., 1997), and recognition thresholds may be roughly determined (Tomita et al., 1986); however, precise taste-quality recognition thresholds (Collings, 1974), taste-quality discrimination, and taste-quality profiles (Hettinger et al., 1990; Schiffman, 1979) are not determined. Patients are asked to identify taste quality, but clinical data are reported simply as total “correct” (Cowart, 1989; Deems et al., 1991); even though patterns of errors could provide diagnostic information, as they do for smell (Cain, 1989). Their potential clinical value is suggested by frequent misidentifications of the salty prototype NaCl as bitter/sour following chlorhexidine treatment (Frank et al., 2001; Helms et al., 1995) (see also above). Taste-quality misidentification patterns could provide objective information to supplement self-reported quality distortions by patients with dysgeusia (Cowart et al., 1997; Gent et al., 1987, 1997; Mott et al., 1993). See the next section for promising future evaluations that address this problem. 3.

b. Ratings. Intensity ratings address suprathreshold function, not function at threshold (Bartoshuk, 1989a). For example, subjects rate intensities of 1.8–18 mM citric acid (Cowart, 1989) when threshold concentrations fall between 0.015 and 0.150 mM (Bartoshuk et al., 1986). Intensity ratings are “subjective,” but control distributions of ratings (Bartoshuk, 1989a,b) allow a patient to be described as falling at some percentile within normal function or outside of normal function. Because “normal” seniors rate suprathreshold taste stimuli less strong than do young subjects (Table 3) (Cowart, 1989), control distributions of ratings used to diagnose taste weakness should be age-matched to patients. 2. Assessment of Taste-Quality Processing Taste-quality discrimination is sparsely addressed currently in specialty chemosensory clinics. Taste-quality Table 4 Distribution of Detection Thresholds for Citric Acid for 18 Young Subjects and 18 Seniors 0.01 Young subjects Seniors

Molarity (mM) 0.011–0.05 0.051–0.1

0.11–0.5 0.5

4

11

3

0

0

0

0

6

9

3

There is a statistically significant difference in average threshold for the two age groups (Mann-Whitney U
13, p  0.001). Source: Data from Bartoshuk 1986.

Suitability for Clinical Setting

The dimensions of comprehensive taste testing could be daunting, given multiple taste substances and multiple bilateral taste fields. If possible, taste evaluations should be appropriate, concise, portable, faithfully administrable, and readily interpretable. a. Appropriateness. In existing specialty chemosensory clinics, triage limits comprehensive taste testing to selected subpopulations of patients (Cowart et al., 1997; Deems et al., 1991; Mott et al., 1993; Seiden et al., 1992). Justification for triage includes low frequencies of verified taste complaints, which relates to patient confusions between taste and retronasal smell (see also Chapter 10). Of 750 chemosensory patients evaluated at the University of Pennsylvania Smell and Taste Center (Deems et al., 1991), two thirds complained of taste loss, but 87% of these “taste complainers” also complained of smell loss. Test results verified taste loss in about 4% of all patients, about 6% of “taste complainers.” Verification of a complaint, however, depends upon the test procedure. (See sec. III for additional comments on the sensitivity of “clinical indicators.”) To dispel confusion between chemosenses (taste, smell, common chemical), patients may need to be asked to focus on sweet, salty, sour, or bitter tastes in an initial interview (Gent et al., 1987; Mott et al., 1993). Of 493 patients of the University of Connecticut (UConn) Taste and Smell Clinic, 42% complained about one, two, three, or four of the taste qualities. Distributions of “total” taste intensity

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ratings for thusly defined “taste complainers” and the 58% “noncomplainer controls” are shown in Table 5. “Total” taste is the sum of intensity ratings for sucrose, NaCl, citric acid, and quinine•HCl concentration series. There is a highly significant difference in the distributions (p  0.001; -square), suggesting that many patients do, as they report, perceive taste stimuli as less intense. Four percent of “noncomplainer” controls rated taste intensities at 200 or less, but 28% of taste complainers rated taste intensities this low. Thus, about one quarter of taste complainers had verified taste loss, and they may be the patients who would most benefit from comprehensive taste testing (see also below).

(Stillman et al., 2000; Tomita et al., 1986). However, anodal current, which is used in electrogustometry, elicits “metallic,” “sour-salty” (Tomita et al., 1986), or “sour” (Bujas, 1971; Bujas et al., 1986) perceptions, usually associated with salts and acids. In fact, anodal current may stimulate taste by driving cations (Na, H) through ion channels (Herness, 1985a; Ninomiya and Funakoshi, 1989). Electrogustometry does not use cathodal current; “cathode-on” (100A) elicits indistinct sweet-bitter tastes (Bujas, 1971) accompanied by somatosensory sensations and, at “cathode-off,” “metallic” sensations (Bujas, 1971; Herness, 1985b). Because regional weaknesses specific to any single taste quality—“dissociated” taste problems—are uncommon (Tomita and Horikawa, 1986), electrogustometry may be sufficient for evaluating most complaints of taste blindness or weakness in receptive fields for each of the taste nerves (e.g., Grant et al., 1989; Groves and Gibson, 1974) (see also above).

b. Management. The amount of time that the testing takes (conciseness), its portability, and the likelihood it will be faithfully administered in various clinical settings are important applied test-management considerations. i. Conciseness. Most taste-testing protocols used in contemporary specialty chemosensory clinics require at least one hour of patient-staff contact (Deems et al., 1991). The tests are primarily whole-mouth procedures that include presentations of multiple concentrations of compounds representing four taste qualities in order to determine detection thresholds and/or scale the subjective experience of sensory intensity (Gent et al., 1997) for each of the qualities. Sufficient data must have accumulated during the 20 years of specialty chemosensory clinic operation in the United States to support an evaluation of results aimed at establishing efficient tests of whole-mouth taste function. Comprehensive regional chemogustometry (six fields, four qualities) is composed of 24 subtests in a single replicate (Tomita et al., 1986); with 2 replicates this yields 48 trials with rinses after each. By collapsing the quality domain to one, electrogustometry (Frank and Smith, 1991) requires 6 subtests per replicate; and, because it does not require solutions and rinsing, it is a much faster procedure Table 5 Taste Intensity Ratings for “Taste Complainers” and “Controls” 0–200 Complainers Controls

“Total” taste intensity ratings 201–400 401–600

58 (28%) 11 (04%)

101 (49%) 160 (56%)

40 (20%) 99 (34%)

600 6 (04%) 18 (06%)

Entries are number of patients and, for each row, percentages in parentheses below. Frequency distributions for complainers and controls differ (-square
64.24, p  0.001). Source: Data from the UConn Taste and Smell Center.

ii. Portability. Portable, self-administered taste tests would facilitate longitudinal monitoring of taste function. Highly portable, self-administered smell tests utilizing microencapsulated “scratch-and-sniff” odor strips have been developed (Doty et al., 1984), and their results correlate quite well with clinically administered tests of smell identification and threshold (Cain and Rabin, 1989). Similar reliable taste tests are not yet available. Although electrogustometers designed for clinic use are portable (Frank and Smith, 1991), test self-administration, at home, would not be advisable with current designs. A practical means for easily assessing whole-mouth function is to have subjects chew pieces of filter paper impregnated with taste stimuli, a technique used to demonstrate the genetically based bimodal human sensitivity to phenylthiocarbamide (PTC) (Kalmus, 1971) and for PTC taste phenotyping in genetic studies (Reed et al., 1999). Similar techniques using paper sticks (Hummel et al., 2001), wafer, or tablet substrates (Hummel et al., 1997; Ahne et al., 2000) could be self-administered and would be sensitive to whole-mouth function, including saliva production. Unfortunately, clinical utility of such approaches is limited because regional testing may be required to establish the specific nerves that are compromised in some clinical situations. iii. Administration. Faithful administration of taste tests is essential for consistent and comparable outcomes. Many protocols used in specialty clinics require oversight by a psychophysicist, precluding easy technology transfer to clinical staff. Detection threshold protocols vary in complexity and acuteness (Cowart, 1989; Weiffenbach et al., 1983) (also see above), although acuteness may not corre-

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late with sensitivity to patient reports of taste disorders. The sensitivity of lengthy, complex taste testing and short, simpler testing is yet to be directly compared (see below). c. Interpretation. Divergent findings present in the taste literature such as those reported on the effects of aging (see above and Chapter 44) likely reflect differences in reliability and sensitivity of the various measurements. Unlike taste testing in studies designed to distinguish among groups, clinical test interpretation is for individual patients. Repeatability (or stability) of a measurement on retest (reliability) and validity of measurements for identifying taste disorders are key here. i. Reliability. Reliability for clinical tests is best studied for the range of function seen clinically to assess inherent measurement consistency. The correlation coefficients (Pearson’s r) between replicate measurements calculated to quantify reliability (see also Chapter 10) are very sensitive to the range of function represented. For example, replicate detection thresholds for n-butanol for 32 normal people showing a narrow range of smell function were more moderately correlated (r
0.68) (Cain and Gent, 1991) than replicate odor-identification scores (r
0.95) for 35 people with a wide range of smell function (Doty et al., 1985). Few studies of the reliability of taste measures have been conducted. Correlation coefficients were calculated for two replicate intensity ratings of sucrose, NaCl, citric acid, quinine•HCl, and n-propylthiouracil (PROP) concentration series obtained from patients of the UConn Taste and Smell Clinic (Table 6). The subjects included taste complainers and noncomplainers (see above). Measurement “reliability” for the intensity ratings ranged from r
0.54 for citric acid to r
0.74 for PROP. Sensitivity to this phenylthiourea-like compound is known to vary much among “tasters” and “nontasters” (see below). The 0.72 coefficient for sucrose ratings compares to a 0.86 value for detection thresholds in 12 normal subjects, retested on the same day (Mattes, 1988). The moderate coefficients for replicate ratings on a single occasion justify including duplicate ratings in “subjective” tests of suprathreshold taste function. There is a need for additional evaluation of measurement reliability for all clinical taste testing. ii. Validity. A test is valid if it accurately measures what it purports to measure. A clinical test has validity if it identifies genuine taste dysfunction. Consistent with clinical test validity are sensitivity, specificity, and positive predictive value for taste disorders (Doty, 1992). The proportion of persons with a taste disorder who test “positive” defines sensitivity. The proportion of persons without a

Frank et al. Table 6 Trial 1–2 Correlation for Whole-Mouth Taste Test Concentration series Sucrose NaCl Citric acid Quinine•HCl n-Propylthiouracil “Total taste”

r

n

0.72 0.56 0.54 0.66 0.74 0.64

843 843 839 690 838 678

Based on scatter plots, 0–2 outliers were identified and eliminated from each data set. Pearson’s correlation coefficients (r) are significant for each concentration series and “total taste” (see above) ( p  0.01); n is the number of patients. Source: Data from the UConn Taste and Smell Center.

taste disorder who test “negative” defines specificity. The proportion of all persons who test positive who actually have a taste disorder defines positive predictive value. Ideally, each indicator would equal 1.00. These measures of validity are yes-or-no proportions of specified populations; they do not address how well a test may document the severity of a disorder. Validity of a clinical taste test is most easily addressed if it is known that a disorder is present. Thus, we “created” a reversible dissociated taste disorder (bitter weakness) in 15 normal people by treatment with chlorhexidine (Frank et al., 2001; Gent et al., 1999; Helms et al., 1995). Quinine•HCl and citric acid (control) intensity ratings obtained from these people for concentration series were compared to control-population percentiles used for diagnosis by the UConn Taste and Smell Clinic. With weakness defined by a rating below the 15th percentile (Gent et al., 1997), 10 of the 15 people were positive for bitter weakness—a test sensitivity (“hit rate”) of 0.67. Thirteen of the 15 people were negative (greater than 15th percentile rating) for sour-weakness—a “correct rejection rate” of 0.87, about as expected for the criterion. When the criterion was set at the 5th percentile, the hit rate was 0.60, but the specificity was 0.93. This illustrates the trade-off between true and false positives. To calculate the “positive predictive value” for a clinical taste test, we assumed that UConn Taste and Smell Clinic patients who presented with a taste complaint (defined as above) (Table 5) had a taste disorder. For 288 “noncomplaining” controls, a “total” taste intensity rating (see above) of 270 falls at the 15th percentile, yielding 43 false positives. Of 205 complainers, 86 (42%) rated taste intensities at least this low: a test sensitivity of 0.42 for hypogeusia (total taste weakness). The proportion of complainers testing positive of all patients testing positive (complainers and noncomplainers) was 86/129, a

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0.67 positive predictive value. Thus, two thirds of 129 people who tested positive had a taste disorder, and one third did not have a taste disorder. If a 5th percentile criterion were adopted, test scores of less than 210 would indicate taste weakness, resulting in a hit rate of 0.30 and a positive-predictive value of 0.82. This illustrates the trade-off between positive predictive value and hit rate when test values for afflicted and normal populations overlap extensively. By identifying and segregating sources of taste score variability, it may be possible to identify more taste disorders without drastically reducing the positive predictive value. Because of the wide and overlapping range of taste function for young people and seniors or complainers and noncomplainers (e.g., Tables 4 and 5), it may be necessary to evaluate taste-test validity using different cutoffs for specific subpopulations. For example, for the measure of total taste weakness just addressed, a criterion of 5th percentile may be appropriate for noncomplainers but 15th percentile for complainers. This “good-faith” weighting of scores based on complainer status results in a sensitivity of 0.42, a specificity of 0.95, and a positive-predictive value of 0.87 for the distributions in Table 5. The measures of validity might improve if subpopulations were further divided on the basis of age or genetic taster status (see below). In the next section, promising future tests of taste function are described.

tions.” Chlorhexidine treatment resulted in a greater reduction in NaCl intensity ratings for low than for high concentrations (Lang et al., 1988) and an increased slope. In contrast, a slope flattening resulted from greater age-related decrements in taste intensity ratings for high than for low stimulus concentrations (Bartoshuk et al., 1986; Frank et al., 1992; Schiffman and Clark, 1980; Schiffman et al., 1981, 1981a), an effect also seen in women suffering from oral burning (Formaker and Frank, 2000). Just as distinct slope changes were observed in chorda-tympani nerve responses for NaCl concentration series in desalivated (decrease) and sodium-deprived (increase) rats (Catalanotto et al., 1986), distinct psychophysical function signatures may designate distinct biological conditions. If slope differences reflect differences in how well intensities are discriminated, as they may (Kaneda et al. 2000), intensity discrimination tests for pairs of low and high stimulus levels could provide “objective” diagnostics for specific taste disorders.

IV.

POTENTIAL FUTURE TESTING

Psychophysical measures of taste stimulus discrimination, biological assessments of gustatory CNS structure/ function, and the identification of genetic taster status hold promise for future clinical use. A.

Taste Stimulus Discrimination

“Objective” intensity discrimination, quality recognition threshold, and taste stimulus identification paradigms may have value in the diagnosis of cases of hypoguesia and dysgeusia. 1.

Intensity Discrimination

Intensity ratings from low to high stimulus levels may indirectly address intensity discrimination. The slope of the psychophysical function illustrates how sharply “subjective” intensity grows with concentration and may correlate with how well subjects “objectively” discriminate between concentration levels. Magnitude estimate data on chlorhexidine treatment, aging, and burning mouth suggest that the “slopes” may be affected distinctly by different “condi-

2.

Recognition Thresholds

Recognition thresholds are the lowest concentrations for which stimulus quality can be identified. Unfortunately, taste stimuli may have several qualities, especially at weaker intensities (Bartoshuk et al., 1978; Halpern, 1997). Nonetheless, quality recognition thresholds can be measured “objectively” if a “correct” quality is defined (Weiffenbach, 1983). For example, subjects may be asked to select which drop (two water, one sucrose) has a taste and to name its taste quality (Henkin and Christiansen, 1966). If they choose the sucrose, they are correct in choosing the taste (detection threshold); if they label it sweet, they have “correctly” recognized its taste quality. Choosing sweet, salty, sour, and bitter decreases “false positives” for the 3-drop task (Table 1) by one quarter to 0.0125. If the number of labels from which subjects choose is not specified, probabilities for “false positives” (Schiffman et al., 1990) cannot be calculated. Two distributions of detection and recognition thresholds for sucrose (Henkin and Christiansen, 1967) are presented in Table 7. Before the tongue was anesthetized, median recognition threshold (30 mM) was higher than detection threshold (6 mM); with the tongue anesthetized, recognition and detection thresholds were at about the same high value (150 mM). Apparently, 150 mM sucrose could be detected with little lingual sensory input. A triad threshold procedure, used to compare thresholds of many chemical compounds for young and senior subjects, defines the detection threshold event as correct selection of the stimulus (presented in one of three cups) on three consecutive trials (Schiffman et al., 1979). A variant

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Table 7 Distribution of Detection and Recognition Thresholds for Sucrose for 11 Subjects Before and After Lingual Anesthesia Molarity (mM)

Normal Detect Recognize Anesthetized Detect Recognize

3

6

12

30

60

150

300

2 0

7 1

2 3

0 7

0 0

0 0

0 0

0 0

0 0

0 0

2 0

3 0

4 8

2 3

There is a significant difference in the detection and recognition threshold medians in normal (p  0.01) but not anesthetized states (Wilcoxon tests). The numbers of subjects at the median values are italicized. Source: Data from Henkin and Christiansen, 1967.

acute for the foliate papillae, with mean threshold falling at 0.7 mM, and quinine•HCl recognition was most acute on the soft palate, with mean threshold falling at 13 M. Adapting this Sw-Sa-So-Bi recognition threshold protocol to the clinical setting, either in a whole-mouth or a regional form, would be a worthwhile achievement. It should be possible to correct for unequal individualistic guessing rates (Weiffenbach, 1983) by having a computer keep frequency counts and adjusting reversal requirements. Recognition thresholds, like detection thresholds, are “objective,” but quality recognition may assess more closely the “real-world” taste domain and become a reliable and valid addition to a test battery. 3.

of this procedure has been employed for years in industry and is known as the “triangle test.” On each trial the concentration is increased. “False-positive” probability is (1/3) (1/3) (1/3) (2/3)
2/81 (0.0247) (compare to Table 1): three correct choices preceded by an incorrect choice. A similar three-correct criterion was used for recognition threshold but no quality-label set was specified. Under these “open-label” conditions, about half the subjects had not recognized NaCl as “salty” even at 64 times the detection threshold concentration. However, with a closed set of four labels (sweet, salty, sour, bitter), salty recognition for NaCl occurred, on average, at twice the detection concentration, with an individual maximum of 10 times (Henkin and Christiansen, 1967). Thus, it is practical and efficient to restrict label choice in quality-recognition testing. Recognition thresholds for stimuli of four distinct taste qualities (sweet, salty, sour, bitter: Sw-Sa-So-Bi) have been measured simultaneously, with random presentation of four solutions (Collings, 1974; Collings et al., 1976; McBurney and Moskat, 1975). If the stimulus was sucrose, the correct response was “sweet,” if NaCl, “salty,” if acid, “sour”; and if quinine, “bitter”. Solution-soaked, 4-mmdiameter filter paper disks were used to measure regional recognition thresholds (Collings, 1974) with a forcedchoice, six-reversal tracking procedure. “False-positive” probability for a six-reversal threshold sequence (one wrong—one right—one wrong—one right—one wrong— one right) combined with an initial reversal is (3/4) (1/4) (3/4) (1/4) (3/4) (1/4)
27/4096 (1/4)
0.00164. Recognition of sweet sucrose and salty NaCl were most acute for fungiform papillae, mean thresholds approximating 20 mM. Although few taste receptors were stimulated and intensity ratings increase with numbers of receptors stimulated (Smith, 1971), the value is similar to 3-drop, whole-mouth recognition thresholds (Henkin and Christiansen, 1966). Sour citric acid recognition was most

Quality or Stimulus Identification

As noted in detail in Chapter 38, intensity ratings may be obtained for a “predominant quality” of a stimulus (Bartoshuk, 1989b; Tennissen and McCutcheon, 1996) or alternately for its Sw-Sa-So-Bi components (McBurney and Bartoshuk, 1973; Sandick and Cardello, 1981; Smith and McBurney, 1969; Smith and van der Klaauw, 1995). The result for the latter procedure is a Sw-Sa-So-Bi profile of perceptual intensity for each subject, which may accurately identify effects of disorders or experimental manipulations (Ossebaard et al., 1997) on taste quality processing. Another way to profile taste quality is to have a group of subjects choose quality labels for stimuli and calculate frequencies across subjects. Like Sw-Sa-So-Bi intensity profiles, Sw-SaSo-Bi frequency profiles for NaCl differ for the front and back of the tongue. NaCl is most frequently “salty” for the front and “sour” for the back; congruently, its salty intensity is rated highest on the front and its sour intensity rated highest on the back (Sandick and Cardello, 1981). Profiling is employed in industrial settings to determine the subcomponents of an overall perceptual effect (Heymann and Lawless, 1997). Subjects given additional quality labels, such as “soapy,” “sulfurous,” or “metallic” (Hettinger et al., 1990), generate more detailed frequency profiles, especially if stimuli have nongustatory aspects. Table 8 presents profile frequencies for Polycose® (Ross Laboratories, Columbus, OH), a dietary supplement composed of a mixture of glucose-containing oligosaccharides (Hettinger et al., 1996) that may have a unique polysaccharide taste to rats (Feigin et al., 1987; Sclafani, 1987). At 3.2% (w/v) Polycose had a prominent “sweet” quality with the nose open but not with the nose clamped. Vanillin, benzaldehyde, isoamyl acetate, and cinnamaldehyde also have “sweet” odors (Dravnieks, 1985). At higher concentrations, Polycose had a sweet taste and some other olfactory quality.

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The above work emphasizes the need to control olfaction in taste-profiling studies. It is wise to profile taste stimuli with the nose occluded to minimize olfactory influences. Taste stimuli may also be irritants at high concentrations (Green and Lawless, 1991), but proper selection of taste stimulus concentration may minimize such confounds (Frank et al., 2001; Gilmore and Green, 1993; Portmann et al., 2001). Clearly, subjects integrate nongustatory sensations into ratings for concordant taste stimuli (Clark and Lawless, 1994; Frank and Archambo, 1986; Frank et al., 1989; 1993; Hettinger et al, 1990; Schiffman et al., 1981, 1981b). Taste-quality frequency profiles might be useful for clinical tests if patients were presented with selected stimuli numerous times. Patient profiles (or “taste spaces” derived from multivariate analyses) could be compared with profiles (or spaces) for control subjects (e.g., Schiffman, 1979; Schiffman and Dackis, 1975). Because of the serious impact of dysgeusia on patients’ lives (Deems et al., 1996), such comprehensive evaluation of taste function, including hedonic ratings (Frank and Archambo, 1986) for taste stimuli, may be warranted. Measuring the confusion among taste stimuli in a task in which stimuli are presented for identification multiple times is a promising “objective” procedure for documenting sensory dysfunction. Such an approach in the form of a confusion matrix for odors (Köster, 1975; Wright, 1987) was used as part of a clinical olfactory test battery (Wright et al., 1991) (see also Chapter 10). More recently, the confusion matrix paradigm was applied to taste (Gent et al., 1999, 2002; Hettinger et al., 1999). For taste confusion matrix (TCM) studies, a number of taste stimuli (e.g., 10) are presented repeatedly (10 times each) for identification from a list of stimulus labels. Stimulus names, not quality names, are used to avoid limitations imposed by the typical use of four qualities and to directly parallel Wright

et al. (1991). Performance measures calculated from the matrix formed from the correct and incorrect responses (Table 9) include percent correct and two measures taken from information theory (calculated in bits of information transferred) (Attneave, 1959) that quantify response consistency (T10, for a 10-stimulus test) and pairwise stimulus discrimination (T2). Confusion matrix methodology detected effects of taste-altering compounds such as gymnemic acid (Gent et al., 1999) and chlorhexidine gluconate (Gent et al., 2002), demonstrating its potential utility as an “objective” clinical test of taste function. Gymnemic acid treatment imparts a specific deficit for the detection of sweet stimuli (Frank et al., 1992); chlorhexidine treatment imparts taste distortions along with deficits in the detection of salty and bitter stimuli, but sweet stimuli are unaffected (Frank et al., 2001). Comparison of average TCMs for gymnemic acidtreated to water-treated subjects is analogous to comparing performance of a dichromat to a trichromat on a test of color deficiency (e.g., Ishihara plates) and can likewise provide an objective result. Comparison of Ts for particular pairs of stimuli may distinguish among different kinds of dysgeusia, analogous to testing for protanopia and deuteranopia with color matching. Although taste weakness may be more common among dysgeusic patients (18%) than controls (7%), the need for a test that clearly differentiates them is well recognized (Cowart et al., 1997). B.

Biological Correlates of Taste Function

In previous sections we focused on psychophysical measurements that have a direct association to psychological sensations and perceptions. They accurately and quantita-

Table 8 Chemosensory Quality Profiles for Polycose® Quality names Sweet

SA/SO/BI

SP/SU/ME

Other

None

Smell





















3.2% 10% 32% Total

5 8 9 22

0 8 10 18

1 0 0 1

2 0 0 2

3 1 0 4

0 0 0 0

3 4 3 10

0 1 0 1

0 0 0 0

8 2 0 10

The category SA/SO/BI includes salty, sour, and bitter; SP/SU/ME includes soapy, sulfurous, and metallic. The 3 concentrations of Polycose elicit different patterns of response (p  0.001), the pattern for 3.2% Polycose differs for nose-open (smell ) vs. nose-clamped (smell ) ( p  0.001), and total frequencies across categories differ for nose-open vs. nose-clamped ( p  0.001). Data are for 10 subjects with one or two responses each. Source: Data from Hettinger et al., 1996.

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Table 9 Calculation of Information Transferred, T10, for an Experimental Subject from the subject’s Taste Confusion Matrix Response label Stimulus

Salt

Salt substitute

MSG

Quinine

Acid

Sugar

Artificial sweetener

Saltsugar

Acid sugar

Quininesugar

Row sum (X)

0 0 0 2 0 1 0 0 0 2

0 1 0 8 0 1 1 0 0 4

0 0 0 0 8 1 2 0 7 0

0 0 0 0 0 0 1 0 0 0

0 0 1 0 0 4 4 0 0 1

2 0 2 0 0 0 0 4 0 1

0 0 1 0 1 1 2 0 3 0

0 0 0 0 1 1 0 0 0 2

10 10 10 10 10 10 10 10 10 10

14

5

15

18

1

10

9

8

4

100

Salt substitute

MSG

Quinine

Acid

Sugar

Artificial sweetener

Saltsugar

Acid sugar

Quininesugar

px•log2(1/px)

0.00 0.00 0.07 0.00 0.00 0.19 0.19 0.00 0.00 0.07 0.33

0.11 0.00 0.11 0.00 0.00 0.00 0.00 0.19 0.00 0.07 0.31

0.00 0.00 0.07 0.00 0.07 0.07 0.11 0.00 0.15 0.00 0.29

0.00 0.00 0.00 0.00 0.07 0.07 0.00 0.00 0.00 0.11 0.19

A. Matrix of Response Frequencies NaCl 8 0 KCl 2 7 MSG 0 6 Quinine HCl 0 0 Citric Acid 0 0 Sucrose 0 1 Aspartame 0 0 NaCl-sucrose 6 0 Acid-sucrose 0 0 Quinine-sucrose 0 0 Column sums(Y)

Stimulus

16

Salt

B. Matrix of Information [pxy•log2(1/pxy)], where pxy
Cell Frequency/100 NaCl KCl MSG Quinine HCl Citric Acid Sucrose Aspartame NaCl-sucrose Acid-sucrose Quinine-sucrose py•log2(1/py)

0.29 0.11 0.00 0.00 0.00 0.00 0.00 0.24 0.00 0.00 0.42

0.00 0.27 0.24 0.00 0.00 0.07 0.00 0.00 0.00 0.00 0.40

0.00 0.00 0.00 0.11 0.00 0.07 0.00 0.00 0.00 0.11 0.22

0.00 0.07 0.00 0.29 0.00 0.07 0.07 0.00 0.00 0.19 0.41

0.00 0.00 0.00 0.00 0.29 0.07 0.11 0.00 0.27 0.00 0.45

0.00 0.00 0.00 0.00 0.00 0.00 0.07 0.00 0.00 0.00 0.07

0.33 0.33 0.33 0.33 0.33 0.33 0.33 0.33 0.33 0.33

C. Calculation of T T
Hx  Hy  Hxy
1.71, where Hx
[px•log2(1/px)]
3.32; Hy
[py•log2(1/py)]
3.08; and Hxy
[pxy•log2(1/pxy)]
4.69 Source: Data from Hettinger et al., 1999.

tively assess the functioning of the gustatory system and have provided the basis for much of our current understanding of human gustatory function. Combined with stimulus specification from psychophysical measures, biological correlates of taste function such as evoked potentials and brain images reveal patterns of central nervous system activation resulting from gustatory stimuli in normal subjects and, more recently, in patients (see Chapters 11 and 12 for olfactory applications). Functional magnetic resonance imaging (fMRI) and positron emission tomography (PET) reveal primary and secondary cortices involved in gustatory processing with

excellent spatial but poor temporal resolution. Of these, fMRI is preferred because of resolution, cost, availability, and noninvasiveness. Use of evoked potentials (electroencephalography: EEG) and magnetoencephalography (MEG) can provide excellent temporal but poor spatial resolution of taste-related events, but they are in the early stages of development. The use of multichannel systems, particularly for MEG, improves spatial resolution, but EEG is preferred due to cost and availability. Taste has been at a disadvantage compared to hearing, sight, or touch with regard to the detection of sensoryevoked potentials or functional images such as PET, MEG,

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and fMRI. This is in part due to the relatively deep position and mixed modality of the primary gustatory cortex, which makes signals difficult to detect and localize. The location of the human primary gustatory cortex, the largest potential source of pure gustatory, cerebral cortical-evoked responses, is the superior part of the insula in humans (Small et al., 1999). The insula is a deeper structure than primary visual, auditory, or somatosensory cerebral cortices, and, compared to other primary sensory cortices, more nongustatory responses occur and, conversely, a smaller percentage of neurons are taste responsive (PlataSalaman et al., 1992). Furthermore, it is difficult to present precisely timed taste stimuli at high frequency, and taste stimuli are usually accompanied by somatosensory cues, which complicate determination of the taste component. Nonetheless, advances in stimulus presentation techniques and in the resolution of signal versus noise now make taste imaging studies feasible and useful. Taste stimuli can be delivered with semi-precise timing to restricted regions of the tongue either in aqueous solution (Kobayakawa et al., 1999; Plattig, 1991) or as gases (e.g., acetic acid) (Genow et al., 1998; Kobal, 1985). Gaseous stimuli can be applied to a broader area of the tongue compared to regionally applied aqueous solutions. Although lower concentrations are effective with a larger stimululated area (Smith, 1971), few taste stimuli are gaseous. Electric taste stimuli can be delivered with precise timing (see above), and regardless of mechanism, electric taste sensitivity accurately parallels spatial chemical taste sensitivity in patients with taste loss (Frank and Smith, 1991). Thus, in these patients electric taste is a promising tool for measurement of evoked cortical responses. Sensory adaptation (see above) limits the presentation frequency of electric taste and many chemical taste stimuli to about 4 Hz, a frequency sufficient for measuring cortical responses. However, bitter or irritant chemicals such as capsaicin are more problemmatic because their effects may linger for minutes (Breslin and Tharp, 2001; Green, 1991; McBurney et al., 1972, 1997). Delivery of taste stimuli without nontaste (somatosensory) cues requires careful attention. Chemical taste stimuli can be delivered in a constant flow of carrier (water or artificial saliva) or in a gaseous form with minimal somatosensory cues. Electrial stimuli can be presented with few accompanying somatosensory cues if the electrode is held in position during control and stimulus periods (Barry et al., 2001). Nonetheless, it is still possible for a subject to associate the touch of stimulator probes with taste stimuli, and the resulting memory may affect CNS responses. The high levels of chemical and electrical stimuli required when using small stimulus probes with patients may stimulate common chemical receptors of

distinct trigeminal or general visceral systems (Gilmore and Green, 1993). Water itself may activate CNS regions that respond to taste stimuli (Zald and Prado, 2000), and important activation may be obscured if the response to water is subtracted from the response (Frey and Petrides, 1999). The use of artificial saliva may reduce such “water” effects (O’Doherty et al., 2001). Remarkable recent progress has been made in imaging human gustatory areas. Cortical activation in response to chemical and electric-taste stimuli has been localized to the insula and overlying operculi with PET, MEG, and fMRI (Barry et al., 2001; Faurion et al., 1999; Frey and Petrides, 1999; Kobayakawa et al., 1999; O’Doherty et al., 2001; Small et al., 1999). Multiple insular areas have been found to be activated. Co-activation of the inferior parietal cortex may reflect somatosensory stimulus components, although this is not known for sure. Interestingly, co-activation occurs in the amygdala and orbitofrontal cortex, two secondary gustatory cortical areas that in nonhuman primates respond to motivational state (Rolls, 2000; Yan and Scott, 1996). Although there is little evidence for spatial separation of cortical sweet, salty, sour, and bitter representations, aversive and pleasant tastes may be separately represented within the orbitofrontal cortex (O’Doherty et al., 2001), and aversive tastes may differentially activate the pregenual cingulate gyrus (Zald et al., 1998). These cortical representations may have relevance to brain imaging of the dysgeusic patient who perceives taste phantoms as unpleasant. Hedonic ratings (Frank and Archambo, 1986) of taste stimuli and phantoms might also be undertaken in these cases to help interpret results. Based on taste-quality recognition deficits in subjects with temporal lobectomy and preferential activation of the right anterior temporal lobe (including the amygdala) by taste stimuli in normal subjects, the right anterior temporal lobe has been implicated in taste-quality recognition (Small et al., 1997). Furthermore, taste stimuli restricted to either the left or right side of the tongue preferentially activate the right insular cortex (Barry et al., 2001), and a small ventral insular region is primarily activated in the dominant hemisphere (Faurion et al., 1999). These findings suggest that patterns of hemispheric dominance influence gustatory processing. Central representations of taste phantoms (dysgeusias that occur without taste stimulation) have not been identified. However, CNS activation of many of the cortical areas noted above occurred when patients recalled a taste phantom, and effective treatment of the phantom resulted in less evoked activity (Henkin et al., 2000). In summary, new imaging technologies show promise for revealing human gustatory system function. With attention to the limitations of techniques and stimulation

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paradigms, these imaging technologies will become increasingly useful in the clinical setting. C.

Genetic Taster Status

As noted above, identification of a patient’s taster status may help account for normal variability in taste measurements and thus increase the positive predictive value of a clinical test. The idea is to have controls specific for PTC/PROP taster status subpopulations. Also, associations of PROP tasting phenotypes with disease states, food choice, and taste perception (Bartoshuk, 2000; Bartoshuk et al., 1996; Drewnowski et al., 2001; Guo and Reed, 2001) (see also Chapter 40), make it appropriate to ascertain genetic taster status in patients with taste complaints. The ability to taste thioureas such as phenylthiocarbamide and related compounds such as n-propylthiouracil is genetically determined; “tasters” can detect and recognize as bitter PROP solutions of 0.1 mM or less, whereas “nontasters” cannot. One interpretation of the phenotype is that PROP tasting is a dominant trait due to a single autosomal gene located on 5p15 (Reed et al., 1999) that may be associated with a member of a family of taste receptors (Adler et al., 2000; Matsunami et al., 2000). This has important implications for mechanisms of bitter perception. PROP is preferred for phenotyping because PTC has a sulfurous odor that may be detected retronasally. The proportion of nontasters in Caucasian Europeans, about 30%, is higher than for most other racial groups; some American Indian populations may be exclusively tasters of PTC (Kalmus, 1971). A consequence of the existence of PTC-PROP tasters and nontasters in the preponderance of population studies (Guo and Reed, 2001) is the occurrence of bimodal distributions of thresholds and intensity ratings in most populations. Compared to nontasters, PROP tasters perceive other substances, especially other bitter stimuli, to be more intense compared to nontasters (Bartoshuk, 2000; Delwiche et al., 2001). Although even sweet stimuli (Gent and Bartoshuk, 1983), qualitatively similar to sucrose in tasters and nontasters alike (Rankin and Marks, 1992), were rated less intense by nontasters (Bartoshuk, 1979), taster-nontaster intensity differences for stimuli not structurally related to phenylthiourea are quantitatively much smaller than PTC or PROP differences. It is possible that specific phenotyping for PROP/PTC tasting is obscured by a general taste sensibility (Frank and Korchmar, 1985; Guo and Reed, 2001), which could be corrected by appropriate normalization (Gent and Bartoshuk, 1983).

PROP tasting status has been determined in a number of ways, including measuring detection thresholds, which have been very important in establishing genetic models (Guo and Reed, 2001; Reed et al., 1995), and perceptual scaling. Magnitude estimates of a series of PROP solutions from 0.056 to 1.8 mM [concentrations surrounding the antimode of the bimodal PROP threshold distribution (Reed et al., 1995)], normalized to tones, have also been used to phenotype individuals (Bartoshuk, 1989a). A simpler test uses filter paper that has been impregnated with saturated PROP and dried (Luchina et al., 1998; Reed et al., 1999). Thresholds and ratings may be differentially associated with two genetic loci (Guo and Reed, 2001).

V.

SUMMARY AND CONCLUSIONS

Current taste testing in specialty clinics comprehensively addresses alterations in perceptual intensity. Evaluation of the extensive patient and control data on taste weakness and taste-quality misidentification should suggest streamlined testing that is both reliable and valid. Additional attention to objective measures of taste discrimination and CNS processing will promote appreciation of consequences and causes of all taste disorders, including dysgeusia.

ACKNOWLEDGMENTS This work was supported by the National Institute of Deafness and other Communicative Disorders at NIH (P50 DC00168) and the University of Connecticut Health Center (UCHC). We gratefully acknowledge the help of Dr. Jonathan M. Clive (UCHC Office of Biostatistical Consultation) with probability calculations and statistical comparisons. REFERENCES Adler, E., Hoon, M. A., Mueller, K. L., Chandrashekar, J., Ryba, N. J., and Zuker, C. S. (2000). A novel family of mammalian taste receptors. Cell 100:693–702. Ahne, G., Erras, A., Hummel, T., and Kobal, G. (2000). Assessment of gustatory function by means of tasting tablets. Laryngoscope 110:1396–1401. Arey, L. B., Tremaine, M. J., and Monzingo, F. L. (1935). The numerical and topographical relations of taste buds in human circumvallate papillae throughout the life span. Anat. Rec. 64:9–25.

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38 Human Perception of Taste Mixtures Hendrik N. J. Schifferstein Delft University of Technology, Delft, The Netherlands

I. INTRODUCTION

Despite differences in approach, taste research by both groups has mainly focused on quality and intensity. Because of this, the current chapter is restricted to these two dimensions of taste sensations. A number of pioneering studies performed in psychological laboratories in Germany in the late nineteenth century focused specifically on the role of taste quality in the perception of taste mixtures. Indeed, a classic debate developed between Kiesow (1894, 1896) and Öhrwall (1891) with regard to the nature of the percept elicited by taste mixtures. Kiesow argued that sweet, sour, salty, and bitter were four qualities within one modality, whereas Öhrwall argued that they constituted four separate modalities. Von Skramlik (1926) supported Kiesow’s perspective by suggesting that mixtures of substances representing the four basic tastes could duplicate the complex taste experience elicited by any inorganic salt. He developed a set of “mixture equations” consisting of the concentration levels of quinine hydrochloride, sodium chloride, tartaric acid, and glucose that produced a mixture with taste properties similar to a particular inorganic salt concentration. As we shall see later in this chapter, the analytic versus synthetic debate continues until the present day. In taste intensity research, early workers initially employed simple stimuli (e.g., solutions of sucrose in water) to determine the lowest concentration that could be reliably discerned, i.e., the detection threshold (e.g., Von Skramlik, 1926) (see also Chapter 37). After having studied the taste properties of single substances, researchers subsequently began to investigate how taste sensitivity

During the consumption of a food product, the human gustatory system is stimulated by a large number of different chemicals. Perceiving such a chemically complex stimulus requires a reception mechanism, a transduction mechanism, and an integration mechanism. After specific chemicals have bound to receptor sites, the receptor cells activate the afferent neural system. The perceptual system then integrates the signals released by the taste receptor cells. During the perceptual process many substances, or the signals they elicit, interact. The sensation elicited by an unmixed component, therefore, usually differs from the sensation elicited by that same component as part of a complex stimulus. The present chapter focuses on the ways in which mixture components interact and the methods by which these taste interactions can be determined. Research on taste interactions has evolved from two streams of research: experimental psychology and food technology.* Experimental psychologists try to unravel the mechanisms by which humans perceive the world surrounding them. In perceptual research they have found taste sensations to be characterized by the dimensions of quality, intensity, duration, and location (Külpe, 1893; Boring, 1942). Food technologists, on the other hand, strive to develop foods that are optimally palatable.

*For

more extensive historical overviews, the reader is referred to Bartoshuk et al. (1985) and De Graaf (1989). 805

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changes when a second substance is added (e.g., Hahn and Ulbrich, 1948). Unfortunately, detection threshold measurements only inform about whether a subject can detect a stimulus concentration or not. In and of itself, a threshold gives no information about the dynamic range of sensory functioning. Hence, research attention shifted from determining absolute sensitivity to assessing the relationship between physical concentration and suprathreshold intensity, namely, the psychophysical function. For taste mixture research, this implied that researchers investigated how the perceived intensity of a substance was affected when a second substance was added. Most of this research was done after the Second World War, largely in response to complaints of American soldiers about the sensory properties of the food rations they had received. This increased the need to understand the determinants of flavor and food acceptance and stimulated research on taste perception in the United States (Peryam, 1990). As foods can be regarded as highly complex mixtures of tastants, food technologists became particularly interested in taste and smell interactions. The first psychophysical functions relating physical concentration to perceived taste intensity for a single substance were probably derived from measures of absolute and differential sensitivity using Fechner’s (1860) law. However, this indirect scaling approach has rarely been used in taste research (Meiselman, 1972), largely because it is very laborious. In mixture investigations, a different indirect scaling procedure became popular: taste interactions were determined by relating the intensities of mixed and unmixed stimuli to equi-intense concentrations of a reference substance (e.g., Fabian and Blum, 1943; Yamaguchi et al., 1970). For example, in sweetener mixture research each stimulus was matched in sweetness intensity to a sucrose concentration. In such a “scalingby-matching” procedure, the form of the psychophysical function for the reference substance partly determines whether the mixture components interact or not. For example, Cameron (1947) found that sugar mixture intensity often exceeded the sum of the intensities of its components when sweetness intensity was expressed in

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sucrose concentrations. When sweetness intensity was expressed in glucose units, however, mixture intensity was approximately equal to the sum of the intensities of the unmixed components. More recently, investigators have tried to assess perceived intensity by the use of category rating or magnitude estimation, the so-called direct scaling methods. In direct scaling, the subject is instructed to give a direct estimate of perceived intensity by choosing a response category, putting a mark on a line scale, or giving a numerical response. Direct scaling experiments can usually be performed much faster than discrimination or matching experiments. However, the investigator is unsure whether the direct rating is a good representation of the perceived intensity. According to an S-O-R (stimulus-organismresponse) view of single stimulus intensity judgment, three different stages can be distinguished in direct judgment of a single stimulus (Fig. 1). First, a psychophysical function transforms the physical stimulus into a subjective experience, a percept. The percept, temporarily stored in the sensory buffer, is subsequently encoded in order to enable storage in memory and judgmental processing. During encoding, attentive processes enable the subject to focus upon the sensation attribute required by the experimental task. The subject’s conception of what is meant by the requested attribute (e.g., “sweetness intensity”) affects stimulus processing at this stage. After encoding, the stimulus can be represented on an internal, subjective continuum. The coded sensation is no longer a sensation, but a cognition. Any sensory scale derived from rated intensities necessarily represents coded sensations. After encoding, the third processing stage transfers the values of the coded sensations on the subjective continuum into responses on the response scale provided. This process reflects the judgment function or response output function (Frijters, 1993). For direct scaling methods, the perceived (coded) intensity of a single stimulus can only be derived from the obtained responses by assuming that the judgment function implies a linear transformation (interval scale values) or a multiplication with a constant (ratio scale values). It is possible to test the validity of this assumption

Figure 1 Conceptual outline of a direct scaling procedure using single stimulus presentation. The relationship between physical stimulus and perceived sensation is given by the psychophysical function. The sensation is subsequently encoded and transformed into a response. (Adapted from Schifferstein, 1994.)

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in appropriately designed experiments (Klitzner, 1975; De Graaf et al., 1987). Direct scaling in taste mixture research started with descriptions of taste interactions in mixtures with similar or dissimilar tastes (e.g., Kamen et al., 1961). From Pangborn’s investigations on taste interactions in aqueous solutions and foods (Pangborn, 1960, 1961, 1962, 1965; Pangborn and Chrisp, 1964; Pangborn and Trabue, 1964), the idea appeared that dissimilar tasting substances generally suppress each other’s specific taste intensity. After the taste interactions were described, mathematical models were developed (e.g., Moskowitz, 1974; Bartoshuk and Cleveland, 1977; Frijters and Oude Ophuis, 1983) to predict and understand taste mixture interactions. The diversity of these models is large. This is not surprising if we realize that the locus of the mechanism responsible for the interaction between two substances A and B may, in principle, reside anywhere in the pathway from aqueous solution to the overt behavior (response). The substances may react chemically, forming new compounds, or physicochemically, forming complex structures. Furthermore, they may interact biophysically in the periphery of the sensory system, on their way to the receptor or in competing for receptor sites (peri-receptor events) (e.g., Birch, 1980; Carr et al., 1989). In addition, they may interact at the level of the receptor cell, in the afferent nerve bundles, in the central nervous system or at the level of conscious experience, i.e., the percept (Kroeze, 1978, 1979; Lawless, 1979; De Graaf and Frijters, 1989). For the development of mixture models, assumptions have to be made concerning the locus of the mixture interactions and the mechanisms responsible for those interactions. Thus, there are mixture models that describe interaction on the basis of competition for receptor sites or transducer mechanisms (e.g., Beidler, 1971; Frijters and Oude Ophuis, 1983; Ennis, 1989, 1991). Another set of models focuses on interaction in the afferent neural system (e.g., Moskowitz, 1974; Carr et al., 1989; McBride, 1989), whereas others focus upon the interactions between perceived sensations at the central perceptual level (e.g., Berglund et al., 1973; McBride, 1989; Schifferstein and Frijters, 1993). In this chapter I discuss taste interactions at both threshold and supra-threshold intensity levels. With respect to gustatory quality, I distinguish between mixtures of similar and dissimilar tasting substances. In the first mixture type, all components elicit similar taste qualities. If such a mixture is tasted, it leads to the formation of a homogeneous percept, consisting of only one taste sensation. For example, if a subject tastes a mixture of sucrose and aspartame, only one sweet sensation is perceived (assuming that both substances do not elicit side tastes). In the second

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mixture type, dissimilar tasting substances are mixed, leading to the formation of a heterogeneous percept in which several taste qualities can be identified. For example, a sucrose/citric acid mixture elicits a sweet and a sour taste. Subjects may be requested to judge the sweetness, sourness, or total taste intensity of such a mixture. Total taste intensity then refers to the overall strength of the percept, irrespective of taste quality. In the present chapter, the discussion of mixture models is kept to a minimum. The interested reader is referred to reviews by Frijters (1987) and Schifferstein and Frijters (1993). The next sections of this chapter focus on the perception of mixtures of similar or dissimilar tasting substances. In each section, a methodological section is followed by an overview of interactions that occur at near-threshold and supra-threshold concentration levels. In addition, the section on mixtures of dissimilar tasting substances discusses qualitative changes, perceived complexity, and the role of cognitive factors in mixture perception.

II. MIXTURES OF SIMILAR TASTING SUBSTANCES A.

Determination of Mixture Interactions

In binary mixtures of similar tasting substances (often referred to as homogeneous mixtures), both compounds contribute to the intensity of the sensation perceived, and interactions between substances can be described by terms like hypoadditivity, additivity, and hyperadditivity (Berglund et al., 1976). The absence of interaction (additivity) is best defined as the case where the intensity of a mixture equals the intensity that can be expected on the basis of the psychophysical functions for the individual components (Berenbaum, 1989; Sühnel, 1993). Since a psychophysical function is usually nonlinear, doubling the intensity elicited by concentration x differs from the intensity elicited by concentration 2x. Therefore, the intensities elicited by unmixed components cannot be added, but must be corrected for the nonlinearity in the form of the psychophysical functions of the mixture components (Bartoshuk, 1975; De Graaf and Frijters, 1988). If a scaling-by-matching procedure is used in which each mixed and unmixed stimulus is matched in intensity to a reference substance (see sec. I), the degree of mixture interaction is corrected for nonlinearity in the psychophysical function of the reference substance. It is preferable, however, to derive what is meant by “additivity” from the psychophysical functions of the two mixture components and not from the psychophysical function of an arbitrary reference substance.

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1. Comparison Rules Starting from a Fixed Intensity Level Starting with concentration x of substance A, a concentration y of a similar tasting substance B can be found equal in intensity to x. If A and B do not interact, the definition of additivity given above implies that a linear combination of x and y must result in a mixture equal in intensity to x and y. If the psychophysical response is used as a direct measure of intensity, additivity thus implies that: Rpxqy
Rx
Ry

if p  q
1

R(pxqy)
Rx
Ry

symbols used here are different from those used by De Graaf and Frijters (1986) and Nahon et al. (1996).

with p  q
1 and   1 (4)

In other words, hyperadditivity is demonstrated if: Rpxqy  Rx with Rx
Ry and p  q
1

(1)

where Rpxqy is the response to the mixture, Rx is the response to concentration x of A, Ry is the response to concentration y of B, and p and q are the proportions of x and y in the mixture. According to De Graaf and Frijters (1986), this iso-intensity comparison can be theoretically deduced from Beidler’s (1971) mixture model under the assumption that a linear relationship exists between the electrophysiological response to a mixture and the intensity of the sensation elicited by that mixture. The Beidler model assumes that the mixture components compete for adsorption at the same receptor sites. Additivity could thus be regarded as equivalent to competition for receptor sites. Under additivity, the relationship between a concentration of A and a concentration of B in a binary mixture is given by (De Graaf and Frijters, 1986): A
x  xy B if Rx
Ry (2) This equation implies that if the concentration of B is plotted as a function of the concentration of A, under additivity all equi-intense mixtures should lie on a straight line connecting concentration x on the x-axis with y on the y-axis. For multicomponent mixtures, the previous equation can be extended.* For example, if a third component C is added to the mixture, the iso-intensity comparison predicts that: A
x  xy B  xz C if Rx
Ry
Rz (3) where z is the concentration of C that is equi-intense to x and y (Nahon et al., 1996). Binary mixture series can be constructed with constant A/B ratios that vary in solute concentrations, the so-called equiratio mixtures (Frijters and Oude Ophuis, 1983). For each equiratio mixture series, a concentration level [(pxqy)] can be determined that elicits a sensation, equi-intense to the sensations elicited by x and y. Using these data, an iso-intensity curve can be constructed by connecting all equi-intense mixtures. If the curve lies *The

below the additivity line (  1), the mixture components exhibit hyperadditivity. In this case, the concentrations in an equi-intense mixture are lower than expected under additivity. If the iso-intensity curves lie above the additivity-line (  1), A and B behave hypoadditively. In formulas, hyperadditivity is found when:

(5)

Figure 2 shows the results De Graaf and Frijters (1986) obtained when they used the iso-intensity comparison to investigate the taste interaction between glucose and fructose. The binary mixtures equisweet to a fixed glucose level tend to lie below the lines connecting the glucose levels to the equisweet fructose levels. Figure 2 thus leads to the conclusion that glucose and fructose exhibit hyperadditivity: lower concentrations of glucose and fructose are needed to obtain a certain sweetness intensity than expected under additivity. 2.

Comparison Rules Starting from Fixed Stimulus Levels

If fixed concentration levels are used in the construction of series of mixtures and the intensities of these mixtures are determined, three different comparison rules can be used

Figure 2 Iso-intensity comparison for glucose/fructose mixtures. Dotted lines connect the concentration levels of glucose/fructose mixtures predicted to be equisweet under additivity. The drawn lines connect concentration levels that were found to be equisweet. (Data from De Graaf and Frijters, 1986; figure adapted from Ennis, 1989).

Human Perception of Taste Mixtures

to determine whether taste interaction occurs. These comparison rules assume that the perceived intensities of all mixed and unmixed stimuli are known. According to Figure 1, however, the response to a stimulus is related to its perceived intensity by a response output function. Therefore, in order to use these comparison rules, assumptions have to be made concerning the form of the response output function. Some comparison rules require intensities assessed on an interval level (linear response output function), whereas others assume ratio scale values (linear response output function with fixed zero point). a. Summated Response Comparison. In the summated response comparison, the response to a mixture (Rxy) is compared to the sum of the responses to the unmixed components (Rx  Ry). Since the summated response comparison implies an addition of responses, the taste intensities must be assessed at the ratio level. If the intensity of a mixture is plotted against the sum of the intensities of the unmixed components, usually a curvilinear relationship is found. Since additivity according to the summated response comparison implies Rxy
Rx  Ry, the part of the curve lying above the diagonal suggests hyperadditivity, whereas the part below the diagonal suggests hypoadditivity. As already noted in sec. II.A, however, each comparison rule has to incorporate the fact that psychophysical functions are usually nonlinear. If concentrations x of A and y of B are equi-intense, the intensity of a mixture containing x  y should be compared to the intensity of 2x or 2y and not to the sum of the intensity of x plus the intensity of y. The latter comparison is exactly what the summated response comparison does. Therefore, the interactions found with the summated response comparison could better be referred to as apparent taste interactions. The only way in which the summated response comparison can make a useful contribution to taste interaction research is by comparing the observed mixture interaction to the apparent within-substance interactions of the unmixed substances. For each unmixed component, a curve can be constructed for a set of hypothetical mixtures of the unmixed substance with itself. Constructing such a curve is possible if a solution containing concentration 2x is conceived of as a mixture of x with x, or as a mixture of 1/2 x with 11/2 x, and so on. Subsequently, the intensity of the hypothetical mixture (S2x) can be plotted as a function of the sum of the intensities of the unmixed components (SxSx or S1/2 xS11/2 x). In this way, a curve can be constructed for each of the unmixed substances, and these two curves can be compared to the mixture curve. Figure 3 shows the results of the summated response comparison for the perceived intensities of 50/50

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glucose/fructose (GluFru) mixtures (De Graaf et al., 1987). In the construction of this figure, psychophysical functions for fructose, glucose, and the GluFru 50/50 equiratio mixture type were approximated using secondorder polynomials in which the natural logarithm of the perceived sweetness intensity was used as the dependent variable, and the natural logarithm of the concentration and its squared value were used as the independent variables (R2  0.999). These functions were used to interpolate the sweetness intensities that were not determined experimentally. For simplicity, the curves for unmixed glucose and unmixed fructose were constructed under the assumption that they were based on 50/50 selfmixtures. No rules exist as to whether the mixture curve should be compared to the fructose curve, the glucose curve or to some compromise between these two. Therefore, an additivity curve was constructed based on the predictions of the iso-intensity comparison. Figure 3 shows that all curves are negatively accelerating. The GluFru 50/50 mixture curve lies above the fructose curve and the additivity curve. It crosses the glucose curve at low intensity levels. These results indicate that glucose/fructose mixtures exhibit hyperadditivity. b. Factorial Plot Comparison. In the factorial plot comparison, mixture intensity is plotted as a function of

Figure 3 Summated response comparison for glucose/fructose, glucose/glucose, and fructose/fructose 50/50 mixtures. The intensity of a mixture is plotted as a function of the sum of the intensities of its unmixed components. The additivity curve shows the predictions of the iso-intensity comparison. (Data from De Graaf et al., 1987.)

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one mixture component’s intensity. For each concentration of the second component, a separate curve is drawn. According to this comparison rule, apparent additivity implies that the effect of adding a certain concentration y of component B to a solution of component A is constant for all levels of A. The factorial plot then shows a set of parallel curves. In the case of apparent hypoadditivity, the curves converge; in the case of apparent hyperadditivity they diverge. Similar to the summated response comparison, the original factorial plot comparison rule does not take into account that psychophysical functions are not linear. Therefore, mainly apparent taste interactions are revealed by this rule. In order to reveal the “real” degree of taste interaction, again the apparent mixture interactions have to be compared to the apparent within-substance interactions. Since the psychophysical functions for tastants are usually negatively accelerating, the apparent within-substance interactions mostly exhibit a converging pattern (De Graaf and Frijters, 1988; Schifferstein and Frijters, 1990). If the degree of convergence in the mixture plot is greater than the degrees of convergence in the plots for the unmixed substances, A and B behave hypoadditively. If the degree of convergence is smaller for the mixtures, the components behave hyperadditively. No method is yet available to test whether the degrees of convergence differ significantly. Using the definition of additivity from the iso-intensity comparison, a factorial plot can be constructed showing the pattern of apparent interactions under conditions of additivity. Figure 4 shows the expected mixture intensity as a function of the sweetness of unmixed fructose if glucose and fructose behave additively. Psychophysical functions for the unmixed substances were estimated using second-order polynomials (see above). Similar secondorder polynomials were estimated relating glucose concentration to the sweetness intensity of glucose/fructose mixtures for each fixed level of fructose. The experimentally determined sweetness intensities of the four GluFru 50/50 mixtures in Figure 4 are higher than the predicted intensities. Apparently the experimental data indicate less convergence than the additive case. These results, therefore, point at hyperadditivity between fructose and glucose. c. Equimolar Comparison. In the equimolar comparison, the perceived intensities of the unmixed components are compared to the mixture intensity at one total molarity. For example, the intensity of a mixture of 0.5 M fructose and 0.5 M glucose is compared to the intensities elicited by 1.0 M fructose and by 1.0 M glucose. The equimolar comparison is the only comparison rule that has incorporated the form of the psychophysical function in its comparison.

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By determining psychophysical functions for equiratio mixture series, the position of each mixture curve relative to the curves for the unmixed substances can be established (Fig. 5). However, there are no strict definitions of hyper- and hypoadditivity within the equimolar comparison. Only if the mixture curves lie above or below both component curves is taste interaction evident.

B. Describing and Predicting Mixture Interactions In the previous sections, methods were described that determine the nature and the degree of interaction between two substances. If we want to use the outcomes of this type of research to develop a general model that describes and predicts mixture interactions, the comparison rules that start out from fixed intensity levels or from fixed stimulus concentrations both have their limitations. If we determine the degrees of interaction for several intensity levels, we still need a model relating physical concentrations to perceived intensity levels. On the other hand, if we start out from fixed stimulus concentrations, we need to calculate

Figure 4 Factorial plot comparison for glucose/fructose mixtures. The intensity of each mixture is plotted as a function of the intensity of each concentration of fructose with a separate curve for each level of glucose. The sweetness of unmixed fructose was assessed for solutions containing 0, 0.125, 0.25, 0.5, and 1.0 M fructose. The factorial plot was obtained by calculating the sweetness of these fructose levels mixed with each of five levels of glucose expected under additivity. The filled circles show the perceived intensities of GluFru 50/50 mixtures obtained experimentally. (Data from De Graaf et al., 1987.)

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mixture model. This model starts out from the psychophysical functions of the two unmixed substances. These functions are approximated using the formula of S. S. Stevens’s (1975) power law: R
kC n (6) where R is the group average intensity response, C is the concentration level in molarity, and k and n are constants. Subsequently, Schifferstein (1996) determined a concentration level at which the two substances yield the same average response. This concentration level is called the intensity unit (IU) and the response corresponding to one IU is called Req. The revised mixture model uses equally intense concentration levels as the unit of physical measurement, instead of molarity. If concentration C is expressed in IU, the psychophysical functions for substances A and B read: Figure 5 Equimolar comparison for glucose, fructose, and three equiratio mixture types. (Adapted from De Graaf et al., 1987.)

the intensity levels expected under additivity to determine whether the mixture components interact. In an attempt to construct a psychophysical mixture model that can be universally applied to predict the reported intensity for all binary combinations of similar tasting substances, Schifferstein (1996) proposed a revised version of Frijters and Oude Ophuis’s (1983) equiratio

nA

nB

RAi
ReqCAi and RBj
ReqCBj

(7)

respectively. For a set of mixtures in which the relative proportion of IUs for substance A is p and for substance B is q
1  p, the revised equiratio mixture model predicts a mixture function by: pn qnB

A RABpqij
ReqCABpqij

(8)

In this equation, CABpqij represents the total mixture concentration level in IU. Analogous to the iso-intensity comparison, a mixture with CABpqij
1 is predicted to

Figure 6 The sweetness of aspartame, acesulfame-K, and aspartame/acesulfame-K equiratio mixtures in 3.3 mM citric acid. (A) Geometric mean of the observed sweetness responses as a function of the total sweetener concentration of the stimulus. (B) Predictions of the equiratio mixture model presented in Schifferstein (1995). (C) Predictions of the equiratio mixture model as revised by Schifferstein (1996). (Parts of this figure were adapted from Schifferstein, 1996.)

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yield the same response as the unmixed components (Req) (Schifferstein, 1995). Figure 6 shows the predictions of this revised equiratio mixture model for the sweetness of aspartame/acesulfame-K mixtures in 3.3 mM citric acid. Figure 6A shows the observed magnitude estimates for the sweetness of unmixed aspartame, unmixed acesulfame-K, and three AspAcK equiratio mixture series. The concentration ratios in molarity in these three series are 75/25, 50/50, and 25/75, respectively. Figure 6B gives the approximations of the psychophysical functions for the two unmixed substances obtained by regression analysis using Eq. (7) after log-transformation of responses and concentration levels. Furthermore, Figure 6B shows the predictions of the equiratio mixture model using concentration levels expressed in IU [Eq. (8)]. The predictions of the sweetness responses to the mixtures in Figure 6B are low compared to the observed responses. These deviations are mainly due to mixture interactions. To account for the hyperadditivity and to increase the predictive validity of the equiratio mixture model, Schifferstein (1996) suggested replacing the constant Req by an empirical estimate: Req
Req  I 兹苶 pq

(9)

where I is an interaction index. Furthermore, predictive validity can be increased by correcting departures from the power law by a nonlinear response output transformation. These deviations are apparent when the observed psychophysical functions for aspartame and acesulfame-K (Fig. 6A) are compared to their estimated counterparts (Fig. 6B). If different output transformation functions are used for different scaling methods, the equiratio mixture model can be applied to psychophysical data gathered with any scaling method. Figure 6C shows the improvement in predictions after including the interaction index (I
14.85) and correcting for deviations from the power law (see Schifferstein, 1996). For reference, the regression functions of the unmixed substances (Fig. 6B) have also been transformed using the nonlinear transformation (Fig. 6C). The revised equiratio mixture model has been shown to be very successful in predicting the intensity response to mixtures of sweeteners and mixtures of acids (Schifferstein, 1996). C.

Near-Threshold Interactions

Hahn and Ulbrich (1948) determined taste thresholds for many combinations of similar tasting substances. Two substances A and B were defined to behave additively if the threshold concentration for AB mixtures deviated less than 5% from the concentration predicted by combining p times

the threshold concentration of A with 1-p times the threshold concentration of B [0  p  1]. This line of reasoning is similar to the iso-intensity comparison with the detection threshold as the fixed intensity level. For about 200 combinations of bitter-, salty-, sweet-, and sour-tasting substances, the mixture components were found to behave additively. Analogously, J. C. Stevens (1997) found that the detection threshold concentrations in a mixture of three sweeteners did not differ significantly from one third times the threshold concentrations for the unmixed substances. In a mixture of six sweeteners, the threshold concentrations did not differ significantly from one sixth times the concentrations for the unmixed substances. D.

Suprathreshold Interactions

Many studies investigating suprathreshold interactions between similar tasting substances have suffered from confusion concerning the way in which mixture interactions should be assessed. Most of the interactions reported as mixture interactions were contaminated with apparent withinsubstance interactions. For the present overview, studies were selected that used the iso-intensity comparison or that separated apparent interactions from mixture interactions. Most research on mixtures of similar tasting substances has been carried out using sweet-tasting substances. Hyperadditivity was found in mixtures of fructose/glucose (De Graaf and Frijters, 1986), glucose/sucrose (Frijters et al., 1990), fructose/sucrose (De Graaf and Frijters, 1988), and aspartame/acesulfame (Schifferstein, 1996). Frank et al. (1989) reported that out of 31 binary combinations of sugars, sugar alcohols, and artificial sweeteners, 18 mixture types showed hyperadditivity, 2 showed hypoadditivity, and 11 behaved approximately additively. A striking example of hyperadditivity outside sweetener mixture research is found for the so-called taste enhancers. For this type of taste interaction to occur, monosodium glutamate (MSG) or glutamic acid is mixed with a 5-ribonucleotide (Yamaguchi, 1967; Rifkin and Bartoshuk, 1980). For these substances, combining two virtually tasteless solutions results in a mixture with an easily perceptible umami taste (Fig. 7). Apparently, mixtures of similar tasting substances mostly show additivity or hyperadditivity. De Graaf and Frijters (1986) noted that the degree of hyperadditivity increases with increasing concentration levels (see Fig. 2). Additivity corresponds with the predictions of the Beidler (1971) mixture equation, which is based on competition for one set of receptors. Therefore, De Graaf and Frijters (1986) argued that glucose and fructose show complete competition at low sweetness levels. With increasing sweetness levels, however, the degree of competition

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tures of artificial sweeteners (aspartame, saccharin, acesulfame, and cyclamate mixtures). This observation suggests that substances with similar chemical structures (sugars, sugar alcohols) show less hyperadditivity than substances with more variation in chemical structures (artificial sweeteners). Such an inference makes sense if multiple sweetener receptors exist (Faurion et al., 1980) (see Chapter 35). Alternatively, the observed hyperadditivity can be described by a model stating that different numbers of molecules react with one receptor or by a model stating that independent substance-receptor complexes compete for separate transducers (Ennis, 1989, 1991).

Figure 7 The taste interaction between monosodium glutamate (MSG) and disodium 5-inosinate (IMP). The total taste intensity of a solution is plotted as a function of the proportion of IMP in the mixture. The total amount of substance was kept at 0.05 g/dL. (Adapted from Yamaguchi, 1967.)

decreases because one or both sweeteners have additional binding sites, next to a large number of common sites. These results are in line with the finding of asymmetrical cross-adaptation between substances (Schiffman et al., 1981; Lawless and Stevens, 1983). Frank et al. (1989) observed the largest degrees of hyperadditivity for mix-

III. MIXTURES OF DISSIMILAR TASTING SUBSTANCES A.

Determination of Mixture Interactions

In mixtures of dissimilar tasting substances (often called heterogeneous mixtures), one component does not contribute to the intensity of the sensation elicited by the other component. The phenomenon that the intensities of the component sensations within and outside the mixture are equal is called independence. If the intensity in the mixture percept is higher than the intensity outside the mixture, this is called synergism or mixture enhancement. The converse

Figure 8 (A) The sweetness intensity of sucrose, citric acid, and citric acid/sucrose mixtures as a function of the sweetness of unmixed sucrose with a separate curve for each citric acid concentration. (B) The sourness of citric acid, sucrose, and citric acid/sucrose mixtures as a function of the sourness of unmixed citric acid with a separate curve for each sucrose concentration. (Adapted from Schifferstein and Frijters, 1990.)

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is called antagonism or mixture suppression (Berglund et al., 1976; Frijters, 1987). Investigating taste interactions in mixtures of dissimilar tasting substances by studying the specific taste intensities (sourness, sweetness, saltiness, and bitterness) of the components inside and outside the mixture is fairly simple and straightforward. If the substances under investigation elicit only one taste quality, the effect of substance A on the taste intensity of substance B can be assessed by comparing the intensity of unmixed B to the intensity of B in an AB mixture. For this type of investigation, only ordinal information is needed to conclude whether suppression or enhancement occurs. Figure 8 shows the results of a study on taste interaction in sucrose/citric acid mixtures (Schifferstein and Frijters, 1990). In Figure 8A, the sweetness of the mixtures is given as a function of the sweetness of unmixed sucrose. The sweetness intensity of all mixtures lies below the diagonal, implying mixture suppression. Figure 8B shows the sourness of the mixtures as a function of the sourness of unmixed citric acid. Similar to Figure 8A, all mixtures lie below the diagonal, implying sourness suppression by sucrose. In addition, Figure 8B clearly shows that the degree of suppression increases with increasing sucrose concentration. If taste interaction is studied using total taste intensity estimates of mixed and unmixed stimuli, both mixture components contribute to the intensity studied. Since taste quality has now become an irrelevant stimulus attribute,

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the research methodology is similar to that used for studying mixtures of similar tasting substances (see sec. II.A). De Graaf and Frijters (1989) developed a conceptual framework describing the interrelationships among the physical and psychological intensity variables that play a role in the perception of mixtures of dissimilar tasting substances at supra-threshold concentration levels (Fig. 9). The physical concentration of an unmixed stimulus is denoted by  and the physical concentration of a component in a mixture by . The taste intensities of single substances outside the mixture are denoted by  and the taste intensities of the mixture or its components within the mixture are denoted by !. The Roman subscripts A and B refer to two dissimilar tasting chemicals, while the Greek subscripts  and  refer to the qualities of the specific taste sensations elicited by these two substances. The Greek subscript  refers to total taste intensity. The subscripts i and j represent particular concentrations of the chemicals A and B in mol/L. In Figure 9, relation 1 describes the physical mixing of i mol A (Ai) and j mol B (Bj) to obtain the mixture ABij. The lines connecting Ai and i (2A) and Bj and j (2B) represent the psychophysical functions relating the physical concentrations of the unmixed substances to their corresponding specific taste intensities (e.g., sucrose concentration and sweetness intensity). The psychophysical functions relating stimulus concentrations (Ai and Bj) to

Figure 9 Outline of interrelationships among concentration levels, perceived specific intensities, and total taste intensities when two qualitatively dissimilar tasting substances are mixed. (Adapted from De Graaf and Frijters, 1989.)

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total taste intensities (i and j) are given by relation 3. Relation 4 relates the specific taste intensity elicited by a stimulus to its total taste intensity. If the substances under investigation elicit no side tastes, this relationship can be described by an identity function (j
j). Each physical mixture ABij evokes a total taste intensity !ij (5). In addition, the mixture elicits two specific taste sensations, !i and !j (6). Relationship 7 gives the connection between the specific taste sensations inside (!j) and outside (j) the mixture. This is the relationship where mixture interactions (suppression/enhancement) are most evident. The total taste intensity of the mixture percept may be related to the total taste intensities (8: i and j) or specific taste intensities (9: i and j) of the unmixed components, or to the specific taste intensities of the component sensations within the mixture percept (10: !i and !j). When the conceptual framework specified in Figure 9 is used, the intensity of each sensation evoked by the mixture (!i, !j, and !ij) is measured as an attribute of the mixture percept. Relation 10 investigates how the overall intensity of the mixture is related to the intensities of its component sensations. The specification of this relationship implies that the specific taste sensations are regarded as the elements of a larger construct, the percept. The percepts are considered sensory in nature, and a structuralistic view is adopted in which specific taste sensations and total taste intensity are all regarded as attributes of the mixture percept. Taste mixture interaction patterns depend mainly on the relative ratios of the perceived component intensities and not on the absolute intensities (Schifferstein and Kleykers, 1996). Consequently, the interaction pattern can be characterized by plotting the relative ratio of the intensities in the mixture RM
!i / (!i  !j)

(10)

as a function of the relative intensities of the unmixed components RU
i /(i  j)

(11)

(Olsson, 1993). The latter index was originally proposed under the name  by Patte and Laffort (1979). Figure 10 shows the results of these calculations for the sucrose/citric acid mixtures presented in Figure 8 after correcting these data for differences in the size of scale units on the sweetness and the sourness scale (Schifferstein and Frijters, 1990). This analysis shows that mixture suppression in citric acid/sucrose mixtures is asymmetrical: when equi-intense components (RU
0.50) are mixed, the mixture tastes primarily sweet (RM
0.70). For a mixture in which both components taste equally strong (RM
0.50),

Figure 10 Relative ratio of the intensities in the mixture RM
!sweet/!sweet  !sour as a function of the relative intensities of the unmixed components RU
sweet/sweet  sour for sucrose/citric acid mixtures. (Data from Schifferstein and Frijters, 1990. Adapted from Schifferstein and Kleykers, 1996.)

more citric acid should be added (RU
0.39). Asymmetrical suppression is frequently observed for mixture components eliciting different taste qualities (Schifferstein and Kleykers, 1996). B.

Qualitative Changes

For two dissimilar tasting substances A and B that are mixed in successively different concentration ratios, Hambloch and Püschel (1928) described five stages of the perceptual experience. Beginning with a mixture where the quality elicited by substance A has a high intensity, while the concentration of B is hardly perceptible, the quality elicited by B is totally suppressed. This can be the case even though the concentration of B is detected when presented unmixed. When, subsequently, the concentration of A is decreased while that of B is increased, the quality of B may not yet be recognizable but the presence of B changes the character of the mixture percept. The mixture percept seems to remain homogeneous in this second stage. However, the mixture can be discriminated from a solution of unmixed A on the basis of taste quality. In the third stage, both components can be discriminated and can be attended to. When both mixture components are easily perceptible, the sensations in the mixture percept differ qualitatively from the sensations elicited by the unmixed components. Kuznicki and Ashbaugh (1979) have shown that the size of the quality shift is directly related to the concentration of the added component. For example, if sucrose is added to NaCl, the qualitative difference in salti-

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ness quality between the mixture and unmixed NaCl increases with increasing sucrose level. After a further decrease of the A/B ratio, the quality elicited by A is no longer recognizable, but the quality of the mixture differs from that of a solution of unmixed B. Finally, in the fifth stage, the taste of A is fully suppressed by the presence of substance B. The extensions of the five stages discussed above depend upon the two substances that are investigated (Hambloch and Püschel, 1928). Some of these qualitative changes in the mixture percept become apparent when subjects are asked to report whether a mixed stimulus consists of component A, component B, or an AB mixture. Figure 11 gives the proportion of subjects who thought a sucrose/citric acid mixture consisted of only sweetener, only acid, or a mixture of these two substances as a function of the relative intensity of the unmixed components (RU). The probability of identifying the mixture as one of the components increases as the relative taste intensity of that component increases. The probability that the stimulus is identified as a mixture is largest when the probability that it is identified as a sweetener equals the probability that it is identified as an acid. These data confirm Hambloch and Püschel’s (1928) idea that intensity and quality are two attributes that are heavily interdependent, and that both attributes depend upon both solute concentrations: the probability that a stimulus is identified as a mixture is highest when the component intensities in the mixture are about equal (RM
0.50 in Fig. 10). C.

Complexity of Mixture Perception

The sensations  and  elicited by two dissimilar tasting substances may be perceived separately (analysis) or may combine into one new mixture sensation  (synthesis). If the taste sensations synthesize into a new, homogeneous

sensation !ij, it should be impossible to scale the specific taste intensities of the component sensations within the mixture percept (!i and !j), since no separate sensations can be distinguished within a homogeneous percept. As noted earlier in this chapter, the controversy between those who argue that the taste modality functions analytically and those who state that it is a synthetic sense goes back to the nineteenth century (Öhrwall, 1891; Kiesow, 1894). More recently, McBurney and colleagues (McBurney, 1974; McBurney and Gent, 1979) have defended the analytical position, whereas Schiffman and Erickson (1971, 1980) have argued in favor of the synthetic view (see Chapter 37). According to Kubovy’s (1981) theory of indispensable attributes, the perceived numerosity (heterogeneity) of a discrete stimulus depends on whether the stimulus elements vary on an indispensable attribute or not. Without detectable variation on such an indispensable attribute, the reported perceived numerosity deviates from the physical numerosity. Similar to all other senses, event time is an indispensable attribute for taste: if two taste sensations are perceived one after the other, subjects are likely to conclude that the overall percept consists of two elements. Taste quality could be the second indispensable attribute for taste. However, taste quality has no corresponding entity in the physical world. Chemically entirely different substances may elicit similar taste sensations (e.g., sucrose and aspartame), whereas chemically more similar substances may elicit entirely different taste sensations (e.g., HCl and NaCl). Although the complexity of a mixture percept varies with the number of physical components and with the ratio and the absolute concentration levels of the components (O’Mahony et al., 1983), unmixed stimuli are not consistently judged as singular (homogeneous percept) and mixtures are not consistently perceived as more-thanone (heterogeneous percept) (Erickson and Covey, 1980; Erickson, 1982) (Fig. 11).

Figure 11 The proportion of cases in which a sucrose/citric acid mixture was identified as unmixed sweetener, unmixed acid, a mixture, or water as a function of the relative intensities of the unmixed components RU
sweet/sweet  sour. (Adapted from Schifferstein and Kleykers, 1996.)

Human Perception of Taste Mixtures

An important issue that has been overlooked in the analytic-synthetic discussion is that even though taste stimuli may be automatically processed holistically, they can be analyzed if time and mental resources are available. When performing tasks under time constraints, subjects are incapable of selectively attending to one individual taste sensation in a mixture percept without noticing the other sensation(s) in the percept. In addition, the presence of nontarget tastes results in a quality shift in the target sensation (Kuznicki and Ashbaugh, 1979, 1982). These findings show that the sensory system primarily integrates information in order to include all stimulus information into the percept (Kroeze, 1990). The degree of dissimilarity between the sensations is likely to affect the perceived complexity of the mixture percept. If component sensations are easily confused, the percept is likely to be more homogeneous than if they are not. For example, sweetness is easily discriminated from other sensations (Schiffman and Erickson, 1971), whereas subjects often confuse sourness and bitterness (Gregson and Baker, 1973). Therefore, sweet-sour percepts are probably more heterogeneous than sour-bitter percepts. When subjects are not forced to make judgments under time constraints, cognitive processes gain importance. Stimulus dimensions that are originally perceived as integral can be analyzed into their component sensations, as more processing time becomes available (Lockhead, 1966; Garner, 1981). During the analysis of a pattern of taste sensations, attention shifts between the different parts of the percept. In analyzing the mixture percept, subjects select the sensation they are expected to judge without considering the other sensations. The effort required in analysis depends heavily upon the outcomes of the primary perceptual processes: the complexity of the percept, the degree of organization, and the locus of the integration process (Kroeze, 1990). D.

Total Taste Intensity

Schifferstein and Frijters (1993) investigated what happens when subjects are instructed to integrate all stimulus information to judge the total taste intensity of a mixture. In past research, the total intensity of a mixture (!ij) has been related either to the total taste intensities of the unmixed components (i and j) (relation 8 in Fig. 9) or to the specific taste intensities of the unmixed components within the mixture percept (!i and !j) (relation 10 in Fig. 9). The first type of models relates the mixture total intensity to the total intensities of the unmixed components. As a consequence, these models need to account for mixture interactions. In addition they have to predict how total intensity is derived from the characteristics of the mixture percept. Although the predictive validity of some of these models may

817

be good, they tend to oversimplify the perceptual process. As a result, they are unable to account for several empirical phenomena, such as asymmetrical mixture interaction (Schifferstein and Frijters, 1993). Unlike the first type of models, models that predict mixture total intensity from the specific taste intensities in the mixture do not have to account for mixture interactions, because they use the specific sensations elicited by the mixture as input. Implicit in the latter models is a serial processing view: first, the mixture components interact, and, subsequently, the resulting specific taste sensations yield the total taste intensity of the mixture. This type of model can only be applied to heterogeneous mixture percepts, since no specific taste sensations can be distinguished within a homogeneous percept. De Graaf and Frijters (1989) suggested that the total taste intensity of a mixture percept follows from a weighted sum of the specific taste sensations elicited by that mixture. Since empirical studies yielded weights near unity (De Graaf and Frijters, 1989; Schifferstein and Frijters, 1990; Schifferstein and Frijters, 1992), this model was simplified to the (unweighted) sum of sensations model: (12) !ij
!i  !j This model yields accurate predictions, and it can handle all empirical mixture phenomena (Schifferstein and Frijters, 1993). E.

Cognitive Factors

Mixture interactions are mostly regarded as the outcome of psychophysical and psychosensory processes. However, mixture interactions as observed at the level of the obtained responses are affected by changes in stimulus set and task requirements. Different tasks require different types of decision making and will, therefore, yield different results. Consequently, every result has to be interpreted in relation to the method by which it was obtained. In an experiment on quinine•HCl/citric acid mixtures, Frank et al. (1993) found the interaction pattern to vary with response task. If subjects were instructed to judge only the bitterness of the samples, the responses to the mixtures exhibited enhancement. If they judged the total intensity of the stimulus and subsequently broke this rating up into eight component ratings, the bitterness responses exhibited suppression. Mixtures of quinine with sucrose exhibited mixture suppression under all task instructions (Fig. 12). Frank et al. (1993) argued that the discrepancy between the two conditions stems from a difference in the conceptual definitions used under the different task instructions. Due to confusion of sourness and bitterness, sourness intensity is (partly) included in the bitterness

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Figure 12 The effects of 0.25 M sucrose and 2.5 mM citric acid on the bitterness of quinine hydrochloride (QHCl). (A) Bitterness ratings when subjects were instructed to judge only the bitterness intensity of each solution. (B) Bitterness ratings when subjects were instructed to judge total taste intensity and break this rating up into eight component ratings (sweet, salty, sour, bitter, almond, lemon, fruity, other). (Adapted from Frank et al., 1993.)

judgments during the first task, but not during the second task. The sensations of sweetness and bitterness are qualitatively dissimilar and thus unlikely to be confused. Consequently, they show a similar interaction pattern under both task instructions. Some experimental results suggest that characteristics of the stimulus set, such as the proportion of mixtures in the stimulus set (Kroeze, 1982a; Schifferstein, 1994) and the taste qualities perceived (Rankin and Marks, 1991), differentially affect ratings for mixed and unmixed stimuli. In these cases, the investigator is inclined to conclude that the degree of mixture interaction depends on stimulus set composition. This effect of stimulus set composition is probably mediated by the expected properties for a target stimulus as evoked by the preceding stimuli (Cardello et al., 1993). Human subjects can produce a fairly good estimate of the intensity they expect for an imaginary mixture, of which they have only tasted the unmixed components (Schifferstein, 1997; Stevenson and Prescott, 1997). Figure 13 shows a graph in which the perceived sourness of sucrose/citric acid mixtures (panel A) can be compared to the expected sourness of an imaginary sucrose/citric acid mixture (panel B). In the latter case, the subjects did not actually taste the mixture—they only tasted the components separately. Despite differences, the two plots

are strikingly similar. Analogous to expectations derived from stimulus contexts, expectations based on preconceived ideas about stimulus interactions could affect subjects’ responses to taste mixtures.

F.

Near-Threshold Interactions

J. C. Stevens (1995) investigated whether dissimilar tasting components behave additively at detection threshold level. Following his line of reasoning, additivity implies that the threshold concentration of each component in a mixture of n components should equal 1/n times its threshold concentration measured separately. J. C. Stevens found no significant deviations from the additivity rule for mixtures of sucrose, NaCl, citric acid, and quinine • HCl. In more complex mixtures with up to 24 components representing the four taste qualities, however, the detection concentration levels tended to be higher than those expected under conditions of additivity (Stevens, 1997). These findings suggest that some hypoadditivity occurs, even when all components are present below their detection threshold level. Hahn and Ulbrich (1948) performed a series of experiments in which the threshold concentration for substance A was determined in the presence of a subthreshold

Human Perception of Taste Mixtures

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Figure 13 Factorial plot of the mean sourness intensity ratings for perceived (A) and imaginary (B) mixtures of sucrose and citric acid. The citric acid concentrations were 0.0, 1.25, 2.5, 5.0, and 10.0 mM. (Adapted from Schifferstein, 1997.)

concentration of a dissimilar tasting substance B. For 93 out of 123 combinations tested, they found thresholds equal to the thresholds for unmixed A. For the other 30 binary combinations, the threshold concentration of A in the mixture was lower than the concentration for unmixed A. This decrease in threshold concentration was partly accounted for by the fact that component B produced a side taste equal in quality to the taste elicited by A. Threshold concentrations generally increase if a suprathreshold concentration of a dissimilar tasting component is added (Heymans, 1899). The size of the increase in threshold concentration is highly substance specific. J. C. Stevens (1996) reported that a high concentration of NaCl raised the threshold concentration of citric acid nearly 13-fold, whereas those for sucrose and quinine•HCl increased only 3-fold. The effects of a masking component on the detection threshold of a dissimilar tasting substance seem to be symmetrical: for all combinations of sucrose, NaCl, citric acid, and quinine•HCl, Stevens (1996) found that the effect of a proportional increase in the concentration of masker A on the threshold concentration for B was similar to the effect of a proportional increase in the level of masker B on the threshold concentration of A. When two supra-threshold maskers are combined, their effects on the detection threshold of a third substance seem to add up (Stevens and Traverzo, 1997).

G.

Suprathreshold Interactions

As noted in previous sections, the experimental results of a mixture experiment depend upon the way in which the experiment is carried out. Mixture components may elicit taste sensations that are, to some degree, similar or confusable. In this case, instructing subjects to judge only one specific taste intensity may result in finding mixture enhancement (Frank et al., 1993). The degree of enhancement is positively correlated with the reported intensity of the components’ side tastes (Kroeze, 1982b). For example, the enhancement of the sweet taste of sucrose by NaCl depends upon the intensity of the sweetness of NaCl. However, if subjects are requested to judge multiple stimulus attributes or if they are instructed to judge the difference in intensity between a mixed and an unmixed stimulus, mixture suppression is found (Pangborn, 1961; Frank et al., 1993). Since mixture suppression can occur under all different types of instructions, but enhancement occurs only for some of them, mixture suppression seems to be sensory in nature, whereas enhancement seems primarily cognitive. Mixture suppression has been demonstrated for many different substance combinations (Pangborn, 1960). The degree of suppression depends upon the method used, the taste qualities involved, the substances used, and their concentration levels. It would be confusing and tedious to

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summarize all mixture interaction patterns reported in the existing literature. Instead, a number of phenomena have been observed in mixture research that give an impression of the complexity of this field (see below). First, mixture interaction patterns depend mainly on the relative ratios of the perceived component intensities and not on the absolute intensities (Schifferstein and Kleykers, 1996). In addition, the suppressive effect of one taste quality on another quality may be similar for various substances. For example, Schifferstein and Frijters (1991) found that equisweet sweetener concentrations suppressed the sourness of citric acid to the same degree. Further research is needed to demonstrate whether this result is a general finding for taste interactions or whether it is quality specific. However, both these principles indicate that perceived intensity is more important in determining the degree of mixture suppression than the chemical structures or the concentration levels of the tastants involved. Second, mixture suppression is often asymmetrical when both mixture components are present at supra-threshold levels (Schifferstein and Kleykers, 1996). At equi-intense levels of the unmixed components, one sensation may be suppressed more than the other sensation (Fig. 10). In quinine/NaCl mixtures, for example, the bitterness of quinine is suppressed to a large degree when NaCl is added to quinine, whereas the saltiness elicited by NaCl remains almost unaffected (Schifferstein and Frijters, 1993). Third, the total taste intensity of a binary mixture is well predicted by the sum of the specific taste intensities in the mixture percept (De Graaf and Frijters, 1989; Schifferstein and Frijters, 1993). Consequently, the total intensity of a mixture can be higher or lower than the intensity of one of its unmixed components, depending on the degree of suppression in the mixture. IV.

SUMMARY

Most food products can be conceptualized as extremely complex mixtures of substances that can stimulate one or more of the senses. Taste mixture research teaches us that even the interaction between two components that elicit gustatory sensations only is already quite complex. At detection threshold level, many substances behave additively. However, threshold concentrations increase in the presence of a dissimilar tasting supra-threshold component. If two components are present at supra-threshold level, mixtures of similar tasting substances behave additively or hyperadditively, whereas dissimilar tasting substances exhibit suppression. Reports of mixture enhancement in heterogeneous mixture types are probably due to side tastes of the substances used or to method-dependent changes in the subjects’ conceptual definitions. Possibly, the degree

of interaction for similar tasting substances is related to differences in the components’ chemical structures. In interactions between dissimilar tasting substances, not the chemical properties but the relative intensities of the unmixed components appear to be a primary determinant of the degree of interaction. Unfortunately, the current state of knowledge does not permit predictions of the degree of mixture suppression and the direction and size of its asymmetry from knowledge about the physical and chemical characteristics of the mixture components only. ACKNOWLEDGMENTS The author thanks R. A. Frank, J. E. R. Frijters, and M. J. M. Theunissen for their useful comments and suggestions on an earlier draft of this chapter. REFERENCES Bartoshuk, L. M. (1975). Taste mixtures: Is mixture suppression related to compression? Physiol. Behav. 14:643–649. Bartoshuk, L. M., and Cleveland, C. T. (1977). Mixtures of substances with similar tastes: A test of a psychophysical model of taste mixture interactions. Sensory Proc. 1:177–186. Beidler, L. M. (1971). Taste receptor stimulation with salts and acids. In Handbook of Sensory Physiology, Vol. 4, Chemical Senses, Part 2, Taste, L. M. Beidler (Ed.). Springer-Verlag, New York, pp. 200–220. Berenbaum, M. C. (1989). What is synergy? Pharmacol. Rev. 41:93–141. Berglund, B., Berglund, U., Lindvall, T., and Svensson, L. T. (1973). A quantitative principle of perceived intensity summation in odor mixtures. J. Exp. Psychol. 100:29–38. Berglund, B., Berglund, U., and Lindvall, T. (1976). Psychological processing of odor mixtures. Psychol. Rev. 83:432–441. Birch, G. G. (1980). Theory of sweetness. In Carbohydrate Sweeteners in Food and Nutrition, P. Koivistoinen, and L. Hyvönen (Eds.). Academic Press, London, pp. 61–75. Boring, E. G. (1942). Sensation and Perception in the History of Experimental Psychology. Appleton-Century-Crofts, New York. Cameron, A. T. (1947). The Taste Sense and the Relative Sweetness of Sugars and Other Sweet Substances. Scientific Report Series No. 9, Sugar Research Foundation, New York. Cardello, A. V., Melnick, S. M., and Rowan, P. A. (1993). Expectation as a mediating variable in context effects. In Proceedings of the Food Preservation 2000 Conference, Natick, MA. Carr, W. E. S., Trapido-Rosenthal, H. G. and Gleeson, R. A. (1989). Stimulants of feeding behavior in marine organisms: receptor and perireceptor events provide insight into mechanisms of mixture interactions. In Perception of Complex Smells and Tastes, D. G. Laing, W. S. Cain, R. L.

Human Perception of Taste Mixtures McBride, and B. W. Ache (Eds.). Academic Press, Sydney, pp. 27–45. De Graaf, C. (1989). Psychophysical Studies of Mixtures of Tastants. Doctoral dissertation, Wageningen Agricultural University, Wageningen, the Netherlands. De Graaf, C., and Frijters, J. E. R. (1986). A psychophysical investigation of Beidler’s mixture equation. Chem. Senses 11:295–314. De Graaf, C., and Frijters, J. E. R. (1988). Assessment of the taste interaction between two qualitatively similar-tasting substances: a comparison between comparison rules. J. Exp Psychol. Human Percept. Perform. 14:526–538. De Graaf, C., and Frijters, J. E. R. (1989) Interrelationships among sweetness, saltiness and total taste intensity of sucrose, NaCl and sucrose/NaCl mixtures. Chem. Senses 14:81–102. De Graaf, C., Frijters, J. E. R., and van Trijp, H. C. M. (1987). Taste interaction between glucose and fructose assessed by functional measurement. Percept. Psychophys. 41:383–392. Ennis, D. M. (1989). A receptor model for binary mixtures applied to the sweetness of fructose and glucose: De Graaf and Frijters revisited. Chem. Senses 14:597–604. Ennis, D. M. (1991). Molecular mixture models based on competitive and non-competitive agonism. Chem. Senses 16:1–17. Erickson, R. P. (1982). Studies on the perception of taste: Do primaries exist? Physiol. Behav. 28:57–62. Erickson, R. P., and Covey, E. (1980). On the singularity of taste sensations: What is a taste primary? Physiol. Behav. 25: 527–533. Fabian, F. W., and Blum, H. B. (1943). Relative taste potency of some basic food constituents and their competitive and compensatory action. Food Res. 8:179–183. Faurion, A., Saito, S., and MacLeod, P. (1980). Sweet taste involves several distinct receptor mechanisms. Chem. Senses 5:107–121. Fechner, G. T. (1860). Elemente der Psychophysik. Leipzig: Breitkopf und Härtel. Frank, R. A., Mize, S. J. S., and Carter, R (1989). An assessment of binary mixture interactions for nine sweeteners. Chem. Senses 14:621–632. Frank, R. A., van der Klaauw, N. J., and Schifferstein, H. N. J. (1993). Both perceptual and conceptual factors influence taste-odor and taste-taste interactions. Percept. Psychophys. 54:343–354. Frijters, J. E. R. (1987). Psychophysical models for mixtures of tastants and mixtures of odorants. In Olfaction and Taste IX, S. D. Roper, and J. Atema (Eds.). The New York Academy of Sciences, New York, pp. 67–78. Frijters, J. E. R. (1993). Functional measurement in the study of mixture percepts. Chem. Senses 18:93–100. Frijters, J. E. R., and Oude Ophuis, P. A. M. (1983). The construction and prediction of psychophysical power functions for the sweetness of equiratio sugar mixtures. Perception 12:753–767. Frijters, J. E. R., van der Klaauw, N. J. B. M., and Kranen, B. (1990). The use of molal concentrations in a psychophysical investigation of Beidler’s mixture equation. Chem. Senses 15:659–669.

821 Garner, W. R. (1981). The analysis of unanalyzed perceptions. In Perceptual Organization, M. Kubovy, and J. R. Pomerantz (Eds.). Erlbaum, Hillsdale, NJ, pp. 119–139. Gregson, R. A. M., and Baker, A. F. H. (1973). Sourness and bitterness: confusions over sequences of taste judgments. Br. J. Psychol. 64:71–76. Hahn, H., and Ulbrich, L. (1948). Eine systematische Untersuchung der Geschmacksschwellen. III. Abschliessende Mitteilung. Pflügers Arch. Gesammte Physiol. Menschen Tiere 250:357–384. Hambloch, H., and Püschel, J. (1928). Über die sinnlichen Erfolge bei Darbietung von Geschmacksmischungen. Zeitschrift Sinnesphysiol. 58:136–150. Heymans, G. (1899) Untersuchungen über psychische Hemmung. Zentralbl. Physiol. 21:321–359. Kamen, J. M, Pilgrim, F. J., Gutman, N. J., and Kroll, B. J. (1961). Interactions of suprathreshold taste stimuli. J. Exp. Psychol. 62:348–356. Kiesow, F. (1894). Beiträge zur physiologischen Psychologie des Geschmackssinnes. Philosoph. Studien 10:523–561. Kiesow, F. (1896). Beiträge zur physiologischen Psychologie des Geschmackssinnes. Philosoph. Studien 12:255–278. Klitzner, M. D. (1975). Hedonic integration: test of a linear model. Percept. Psychophys. 18:49–54. Kroeze, J. H. A. (1978). The taste of sodium chloride: Masking and adaptation. Chem. Senses Flavour 3:443–449. Kroeze, J. H. A. (1979). Masking and adaptation of sugar sweetness intensity. Physiol. Behav. 22:347–351. Kroeze, J. H. A. (1982a). The influence of relative frequencies of pure and mixed stimuli on mixture suppression in taste. Percept. Psychophys. 31:276–278. Kroeze, J. H. A. (1982b). The relationship between the side tastes of masking stimuli and masking in binary mixtures. Chem. Senses 7:23–37. Kroeze, J. H. A. (1990). The perception of complex taste stimuli. In Psychological Basis of Sensory Evaluation, R. L. McBride, and H. J. H. MacFie (Eds.). Elsevier, London, pp. 41–68. Kubovy, M. (1981). Concurrent-pitch segregation and the theory of indispensable attributes. In Perceptual Organization, M. Kubovy, and J. R. Pomerantz (Eds.). Erlbaum, Hillsdale, NJ, pp. 55–98. Külpe, O. (1893). Grundriss der Psychologie. Engelmann, Leipzig, pp. 30–33. Kuznicki, J. T., and Ashbaugh, N. (1979). Taste quality differences within the sweet and salty taste categories. Sensory Proc. 3:157–182. Kuznicki, J. T., and Ashbaugh, N. (1982). Space and time separation of taste mixture components. Chem. Senses 7:39–62. Lawless, H. T. (1979). Evidence for neural inhibition in bittersweet taste mixtures. J. Compar. Physiol. Psychol. 93:538–547. Lawless, H. T., and Stevens, D. A. (1983). Cross adaptation of sucrose and intensive sweeteners, Chem. Senses 7:309–315. Lockhead, G. R. (1966). Effects of dimensional redundancy on visual discrimination. J. Exp. Psychol. 72:95–104.

822 McBride, R. L. (1989). Three models for taste mixtures. In Perception of Complex Smells and Tastes, D. G. Laing, W. S. Cain, R. L. McBride, and B. W. Ache (Eds.). Academic Press, Sydney, pp. 265–282. McBurney, D. H. (1974). Are there primary tastes for man? Chem. Senses Flavour 1:17–28. McBurney, D. H., and Gent, J. F. (1979). On the nature of taste qualities. Psychol. Bull. 86:151–167. Meiselman, H. L. (1972). Human taste perception. CRC Crit. Rev. Food Technol. 3:89–119. Moskowitz, H. R. (1974). Models of additivity for sugar sweetness. In Sensation and Measurement, H. R. Moskowitz, B. Scharf, and J. C. Stevens (Eds.) Reidel, Dordrecht, The Netherlands, pp. 379–388. Nahon, D. F., Roozen, J. P., and De Graaf, C. (1996). Sweetness flavour interactions in soft drinks. Food Chem. 56(3):283–289. Öhrwall, H. (1891). Untersuchungen über den Geschmackssinn. Skand. Arch. Physiol. 2:1–69. Olsson, M. J. (1993). The perception of odors in interaction. Doctoral dissertation, department of psychology, University of Stockholm, Sweden. O’Mahony, M., Atassi-Sheldon, S., Rothman, L., and MurphyEllison, T. (1983). Relative singularity/mixedness judgements for selected taste stimuli. Physiol. Behav. 31:749–755. Pangborn, R. M. (1960). Taste interrelationships. Food Res. 25:245–256. Pangborn, R. M. (1961). Taste interrelationships. II. Suprathreshold solutions of sucrose and citric acid. J. Food Sci. 26:648–655. Pangborn, R. M. (1962). Taste interrelationships. III. Suprathreshold solutions of sucrose and sodium chloride. J. Food Sci. 27:495–500. Pangborn, R. M. (1965). Taste interrelationships of organic acids and selected sugars. In Food Science and Technology. Vol. III. Quality Analysis and Composition of Foods, J. M. Leitsch (Ed.). Gordon and Breach, London, pp. 291–305. Pangborn, R. M., and Chrisp, R. B. (1964). Taste interrelationships. VI. Sucrose, sodium chloride, and citric acid in canned tomato juice. J. Food Sci. 29:490–498. Pangborn, R. M., and Trabue, I. M. (1964). Taste interrelationships. V. Sucrose, sodium chloride, and citric acid in lima bean purée. J. Food Sci. 29:233–240. Patte, F., and Laffort, P. (1979). An alternative model of olfactory quantitative interaction in binary mixtures. Chem. Senses Flavour. 4:267–274. Peryam, D. R. (1990). Sensory evaluation—the early days. Food Technol. 44(1):86–91. Rankin, K. M., and Marks, L. E. (1991). Differential context effects in taste perception. Chem. Senses 16:617–629. Rifkin, B., and Bartoshuk, L. M. (1980). Taste synergism between monosodium glutamate and disodium 5-guanylate. Physiol. Behav. 24:1169–1172. Schifferstein, H. N. J. (1994). Sweetness suppression in fructose/citric acid mixtures: a study of contextual effects. Percept. Psychophys. 56:227–237.

Schifferstein Schifferstein, H. N. J. (1995). Prediction of sweetness intensity for equiratio aspartame/sucrose mixtures. Chem. Senses 20: 211–219. Schifferstein, H. N. J. (1996). An equiratio mixture model for non-additive components: A case study for aspartame/acesulfame-K mixtures. Chem. Senses 21:1–11. Schifferstein, H. N. J. (1997). Perceptual and imaginary mixtures in chemosensation. J. Exp. Psychol. Human Percept. Perform. 23:278–288. Schifferstein, H. N. J., and Frijters, J. E. R. (1990). Sensory integration in citric acid/sucrose mixtures. Chem. Senses 15: 87–109. Schifferstein, H. N. J., and Frijters, J. E. R. (1991). The effectiveness of different sweeteners in suppressing citric acid sourness. Percept. Psychophys. 49:1–9. Schifferstein, H. N. J., and Frijters, J. E. R. (1992). Two-stimulus versus one-stimulus procedure in the framework of functional measurement: a comparative investigation using quinineHCl/NaCl mixtures. Chem. Senses 17:127–150. Schifferstein, H. N. J., and Frijters, J. E. R. (1993). Perceptual integration in heterogeneous taste percepts. J. Exp. Psychol. Human Percept. Perform. 19:661–675. Schifferstein, H. N. J. and Kleykers, R. W. G. (1996). An empirical test of Olsson’s interaction model using mixtures of tastants. Chem. Senses 21:283–291 Schiffman, S. S., Cahn, H., and Lindley, M. G. (1981). Multiple receptor sites mediate sweetness: evidence from cross adaptation. Pharmacol. Biochem. Behav. 15:377–388. Schiffman, S. S., and Erickson, R. P. (1971). A theoretical review: a psychophysical model for gustatory quality. Physiol. Behav. 7:617–633. Schiffman, S. S., and Erickson, R. P. (1980). The issue of primary tastes versus a taste continuum. Neurosci. Biobehav. Rev. 4:109–117. Stevens, J. C. (1995). Detection of heteroquality taste mixtures. Percept. Psychophys. 57:18–26. Stevens, J. C. (1996). Detection of tastes in mixture with other tastes: issues of masking and aging. Chem. Senses 21: 211–221. Stevens, J. C. (1997). Detection of very complex taste mixtures: generous integration across constituent compounds. Physiol. Behav. 62:1137–1143. Stevens, J. C., and Traverzo, A. (1997). Detection of a target taste in a complex masker. Chem. Senses 22:529–534. Stevens, S. S. (1975). Psychophysics. Wiley, New York. Stevenson, R. J., and Prescott, J. (1997). Judgments of chemosensory mixtures in memory. Acta Psychol. 95:195–214. Sühnel, J. (1993). Evaluation of interaction in olfactory and taste mixtures. Chem. Senses 18:131–149. Von Skramlik, E. (1926). Handbuch der Physiologie der Niedrigen Sinne. Vol. 1. Die Physiologie des Geruchs- und Geschmackssinnes. Georg Thieme, Leipzig. Yamaguchi, S. (1967). The synergistic taste effect of mono sodium glutamate and disodium 5-inosinate. J. Food Sci. 32: 473–478. Yamaguchi, S., Yoshikawa, T., Ikeda, S., and Ninomiya, T. (1970). Studies on the taste of some sweet substances. Part II. Interrelationships among them. Agric. Biol. Chem. 34:187–197.

39 The Ontogeny of Human Flavor Perception Judith R. Ganchrow The Hebrew University–Hadassah School of Dental Medicine Founded by the Alpha Omega Fraternity, Jerusalem, Israel

Julie A. Mennella Monell Chemical Senses Center, Philadelphia, Pennsylvania, U.S.A.

I.

INTRODUCTION

Retronasal olfaction contributes significantly to the complexity of flavor. This is clearly noted by head cold sufferers who lose the ability to discriminate common foods when olfactory receptors are blocked. Similarly, foods often “taste” better after a person quits smoking, perhaps because their sense of smell has improved (Frye et al., 1990), allowing them to detect more subtleties of flavor. The role of smell in flavor is critical in distinguishing the flavor of strawberry from cherry and in enjoying foods like licorice, vanilla, and citrus, which are experienced through the sense of smell. It should be noted that other properties of food (e.g., texture, temperature, irritation) are also very important to its perceived flavor. Because little experimental work has been done in this area of infant flavor perception, this article focuses on human taste and olfactory development, alone and in unison, as it relates to flavor. Further discussion of the ontogeny of olfactory perception as it relates to social behaviors can be found in Chapter 15. Contemporary research on the development of taste and smell is based on more than a century-long legacy examining receptor morphogenesis and determining functional maturity. Early studies attempted to determine the onset of function and how it related to mature perception. However, research on preverbal infants obviated the use of traditional threshold and suprathreshold psychophysical measures, and when such methods are applied in early childhood,

Flavor, a powerful determinant of human consummatory behavior throughout the life span, is a product of several sensory systems, most notably those of taste and smell. The perceptions arising from these two senses are often confused and misappropriated, with such sensations as vanilla, meat sauce, fish, chocolate, and coffee being erroneously attributed to the taste system per se. In fact, taste sensations, mediated by taste buds distributed throughout the oral cavity (see Chapter 32), are largely those of sweet, sour, bitter, salty, and perhaps metallic and savory (e.g., the “umami” taste of monosodium glutamate). Smell sensations, on the other hand, encompass thousands of diverse qualities, some of which are noted above. The receptors for the olfactory system, located high in the nasal chambers, are stimulated not only during inhalation (orthonasal route), but during suckling in infants and deglutition in adults, when molecules reach the receptors by passing from the oral cavity through the nasal pharynx (retronasal route) (Fig. 1). It is this retronasal stimulation arising from the molecules of foodstuffs that leads to the predominant flavor sensations. As noted in detail in several chapters of this volume (e.g., Chapters 2–6), olfactory sensations result from the activation of 1000 or more distinct types of chemical receptors located on millions of receptor cells lining the upper recesses of the nasal cavity. 823

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Figure 1 Sagittal section of infant’s head demonstrating retronasal and orthonasal routes of olfaction.

cognitive factors, such as attention span, often make testing difficult. One solution to such problems was to focus on reflex-like responses (e.g., salivation, patterned facial motor reactions including sucking). However, because touch by itself can initiate sucking behavior, it could be mistakenly concluded that an infant doesn’t respond to a liquid tastant if its concentration is not sufficient to override touch responses. This may explain why several early studies failed to demonstrate taste discrimination in infants (see Lichtenstein, 1893/94). Furthermore, gustatory modulation of some of these response patterns appears to have a very limited developmental time span, so the same measures cannot be performed at all ages (see below). As an alternative to the study of reflex motor coordination, consummatory behavior has also been used for cross-age comparisons. This, too, can be problematic because such behavior assumes uniformity in the ability to control and modulate ingestion as a function of taste or flavor feedback irrespective of age and potentially confounds sensory measurement with preference measurement (indifference does not imply not tasting). In addition, fluctuations in factors mediating thirst and/or postingestional responses may override the effect of flavor components in controlling intake volume. Moreover, not all chemicals utilized in adult taste perception studies can be offered to infants for ingested volume measures (e.g., see Crook, 1981). Lastly, consummatory behavioral studies may be highly variable if basic stimulus (e.g., concentration, temperature, volume, and type of diluent) and methodological (e.g., degree of satiation and experience) parameters are not controlled. However, many of these issues can be resolved by using a within-subject design that controls for these variables.

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These difficulties notwithstanding, dramatic strides have been taken in the past few years, especially in the measurement of outcomes of early experience, in potentially modifying the hedonic value of oral stimuli during development. Both preference measures of ingestion and accompanying motor displays in infants reflect the hedonic aspect of chemosensory stimuli (e.g., Berridge, 2000) and have provided a key for understanding developmental preferences and aversions. Available data support the thesis that behaviors associated with gustatory and olfactory function are robust during early development and contribute significantly to adaptation, if not survival, in the developing infant’s world. At the same time, flavor perception and the accompanying expression of consummatory-related behaviors are influenced by postnatal maturational and both physiological and environmental events and are exquisitely vulnerable to modification throughout the life span. The focus of this chapter is on the ontogeny of human utilization of the senses of taste and smell, often in the context of flavor. A brief description of taste and olfactory receptor development pre- and perinatally will serve as a foundation for assessing the behavioral findings.

II. ONTOGENY OF TASTE AND OLFACTORY RECEPTORS A.

Taste

Early studies, often based on very limited observations (e.g., Hellman, 1922; Hoffmann, 1875; Tuckerman, 1890), suggested that taste buds and their positions in taste papillae appear in an adult-like form prenatally. More detailed recent studies indicate that fungiform, foliate, and circumvallate papillae are all present by about week 10 of gestational age (Bradley and Stern, 1967; Hersch and Ganchrow, 1980; Piras and Mazzarello, 1985; Witt and Reutter, 1997). Originally, the distribution of fungiform papillae was believed to shift from a uniform scattering across the anterior two thirds of the tongue in infants to more lateral and distal concentrations in children and adults. Goldschmidt (1927) refuted this concept by mapping the tongues of 80 living infants and children ranging in age from 1 week to 13 years. He found both types of papillar distributions evident across this age range, with no progressive tendency favoring either. Indeed, the number and distribution of late gestational fetal papillae appear to be roughly similar to those throughout childhood and adulthood. Serial section analysis of fetal tongues by Goldschmidt (1927) revealed that early fungiform papillae (about 4 months) contained only single apical taste buds. More

Ontogeny of Human Flavor Perception

recent studies have identified taste pores in fetal fungiform papillae before the end of the 4th month (e.g., Bradley, 1972; Hersch and Ganchrow, 1980; Witt and Reutter, 1996) (see also Chapter 32). Taste pores provide access for gustatory stimuli to interact with taste bud receptor cells and are generally considered to mark functional maturity (Mistretta, 1972), although taste bud cells continue differentiating after the pores open (Witt and Reutter, 1997). Between weeks 8 and 13, taste cell synaptogenesis is increasingly apparent (Witt and Reutter, 1996). Clearly the newborn infant is endowed with a rich population of gustatory receptors. For example, extralingually, 2583 taste buds were reported to be dispersed over the soft palate, epiglottis, pharynx, and larynx (Lalonde and Eglitis, 1961). In addition, each circumvallate papilla (of which there are usually 9–12) is estimated to already contain around 250 taste buds between the ages of 0 and 11 months (Heiderich, 1906); each foliate papilla around 1500 buds (Tuckerman, 1888); and each fungiform papillae 0–12 taste buds (Arvidson, 1979). In general, there is no linkage between age and number of taste buds per fungiform papilla (e.g., Arvidson, 1979). In children, the number of taste buds per circumvallate papilla was reported by Heiderich (1906) to remain constant from 1–3 years (242  18, N
20) through 4–20 years (252  74, N
9). Other work suggests there may be a slight increase in number of foliate taste buds from birth to 60 years (see Cowart, 1981, for review). Due to large interindividual variability in adult papillary taste bud numbers (e.g., Miller, 1988), longitudinal developmental studies would be required to assess whether there is an intraindividual progression in bud numbers. Unfortunately, this is a technically difficult, if not impossible, task. However, it is doubtful that taste recognition would be significantly affected, even if there was an increase in bud numbers during childhood development, since (1) a single fungiform papilla contains the taste receptor population sufficient to discriminate between different taste stimuli (e.g., Harper et al., 1966) and (2) adults can recognize salt, sweet, sour, and bitter using only one taste bud (Arvidson and Friberg, 1980). Using videomicroscopy, it has recently been reported that while the number of taste pores/fungiform papilla is similar in young adults and 8-year-olds, the latter have a higher density of papillae (Hutchinson et al., 2000). This could give children an advantage for both threshold performance and suprathreshold magnitude estimates if only a small area of tongue is stimulated. This advantage should disappear with tongue growth increasing the space between papillae (see Temple et al., 2002). Overall, the gustatory system is remarkably redundant in its receptor endowment. Assuming the taste bud complement is complete at birth, any major improvement

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in discrimination, or taste perception in general, might be expected to depend on central rather than peripheral processing changes during development, although some taste cell receptor membrane features could be dependent upon experience or ontogeny. B.

Olfaction

As noted in detail in Chapters 2, 6, and 15, the human olfactory system and the formation of the nasal structures are also well developed prior to birth. Primary olfactory receptors are present by the 8th week of gestation (see Schaal, 1988; Schaal et al., 1995b, for review). Furthermore, histochemical analyses of human fetal olfactory tissue have noted the presence of olfactory marker protein, a biochemical correlate of olfactory receptor function, as early as the 24th week of gestation (Chuah and Zheng, 1987; Johnson et al., 1995). By the 6th month of gestation, the epithelial plugs that obstruct the external nares have resolved, and these open airway passages then become continuously bathed in amniotic fluid (Schaffer, 1910). During the later stages of gestation, the fetus swallows significant amounts of amniotic fluid, inhaling more than twice the volume it swallows (Pritchard, 1965; Schaffer, 1910). As will be discussed below, increasing evidence suggests that the fetus may be exposed to a unique chemosensory environment prior to birth. In other words, amniotic fluid may represent the first exposure to flavors (both tastes and retronasal odors) that will subsequently be provided by mother’s milk and then the foods of the table.

III. ONTOGENY OF BEHAVIORAL RESPONSES: TASTE A.

Fetus

Although the response of human fetuses to tastants has never been directly investigated, indirect evidence suggests that prenatal monitoring of taste stimuli dissolved in amniotic fluid is possible in late gestation. Thus, chemosensory stimuli may modulate the intrauterine tendency to swallow (see Beauchamp et al., 1991; Mistretta and Bradley, 1975, for comment and review). In this context, increased swallowing following sweet, and decreased swallowing following bitter, could be interpreted as an early preference and aversion, respectively. In addition, consequences of physiological challenges (e.g., electrolyte imbalances) to the fetus during pregnancy, in terms of postnatal preferences (see below), could be partially mediated by the accompanying fetal taste experience.

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B. Premature Infant Taste-induced behavioral responses have been reported in premature infants (6–9 months gestational age) using a variety of chemicals, stimulation techniques, and recording methodologies. From early in the 6th gestational month, sweet stimuli (represented by lactose, glucose, sucrose, and/or saccharin solutions) were found to consistently modulate the sucking reflex whether via an artificial nipple for electrophysiological measurement or via stimulus-soaked absorbent cotton for direct observation of motor responses (Eckstein, 1927; Maone et al., 1990; Martin du Pan 1955; Stirnimann, 1935; Tatzer et al., 1985). While some of these studies were not done blinded, the authors usually interpreted the response as a “strong positive” or “acceptance” behavior. These data suggest that the gustatory system is functioning and interacting with systems controlling affect. Taste reactivity, as measured by orofacial expressive motor patterns at any age, may be subject to hierarchical brain modulation such that the final product carries more of a hedonic or palatability message, rather than straight stimulus identification (see Berridge, 2000, for review). Infants born around 8 weeks earlier than term (1.1–2.8 kg) cease crying transiently when repeatedly receiving 0.1 ml of 12% sucrose or 10% glucose intraorally, but the response is much less durable than is seen for preterm (born 36–37 weeks) or term-born infants (Smith and Blass, 1996). Water under the same conditions is ineffective. In addition, palatable (e.g., sweet) taste stimuli promote “calming,” analgesic-like reactions in preterm as well as more mature infants during heel lance and other invasive procedures (see below), suggesting possible early functional connections with limbic forebrain and periaqueductal gray matter (e.g., Pomonis et al., 2000; see Berridge, 2000, for review of brain substrates for palatability). In addition, a drop of pure lemon juice is commonly found to increase reflex salivation in premature infants (weighing 1.15–1.83 kg), accompanied by apparent increases in sucking vigor and sometimes retching (Eckstein, 1927). The bitter stimulus quinine (0.0005, 0.005, and 0.05 M) typically retards sucking in such infants. Individual differences in facial expressions have been noted in premature infants and appear to be most marked to “salty” stimuli. For example, in one study of 20 premature infants (1.2–2.9 kg) presented with 0.9% NaCl, more than 50% responded with a rejecting grimace, although 4 readily accepted this solution (Martin du Pan, 1955). In another study (Eckstein, 1927), NaCl (1% solution) produced indifference in two thirds of the infants, and rejection in the other third, during the final trimester of gestation.

Ganchrow and Mennella

Thus, on the basis of these studies it may be concluded that taste buds are capable of conveying gustatory information to the central nervous system by the 6th gestational month and that information is available to systems organizing changes in salivation, sucking, facially expressive, and other affective behaviors.

C.

Newborn Infant

1.

Skeletal and Autonomic Reactions to Taste Stimuli

Facial expression has long been recognized as a potential tool for studying gustatory sensation in newborn infants, although late nineteenth- and early twentieth-century research yielded inconsistent results (see Büssem, 1895; Kussmaul, 1894; Peiper, 1928; Preyer, 1912; Stirnimann, 1935). More recently, J. E. Steiner pioneered the use of videoanalysis in the study of taste-mediated facial expressions in infants, and results have been cross-culturally confirmed, indicating neonates are capable of robust and differentiable taste experiences in the first postnatal hours or days (e.g., Bergamasco and Berlado, 1990; Ganchrow et al., 1983; Kalmus, 1976; Rosenstein and Oster, 1988; Steiner, 1973, 1974, 1977, 1979a, b, 1987). Taste stimuli eliciting these behaviors have included chemical solutions labeled by adults as sweet, sour, bitter, and umami (“delicious”). As in premature infants, the definition of the response to “salty” (NaCl) remains elusive, but generally tends towards the indifferent-to-aversive (grimace, crying) end of the spectrum (e.g., Lichtenstein, 1893/1894; Maekawa et al., 1991; Rosenstein and Oster, 1988; Stirnimann, 1935). Figure 2 presents examples of facial features elicited by taste stimuli. In Steiner’s laboratory, hundreds of full-term infants (with Apgar scores ranging from 8 to 10 and weighing at least 2.9 kg) have been examined before their first hospital postnatal feeding experience. In brief, the face of the relaxed and reclining (tilted up to about 35°) newborn infant is videotaped for at least 60 seconds (unstimulated condition), and then 0.2–0.5 ml of sterile taste solution or distilled water are inserted into the mouth via a disposable graduated pipette (stimulated condition). Water rinses or 3- to 5-minute time intervals intercede between stimulus presentations. Later, specific facial movements are tabulated by trained and/or untrained observers in a blind procedure. The type, degree, frequency, and duration of complexes of facial motor movements are related to the different taste stimuli and their intensity (e.g., Ganchrow et al., 1983; Steiner, 1987). In brief, sweet and umami stimuli [e.g., 0.1–0.5% monosodium- or potassium-glutamate (MSG or PG) dissolved in clear vegetable soup] are frequently associated

Ontogeny of Human Flavor Perception

827

Figure 2 Examples of facial reactions to “sweet” (0.04 M sucrose), “sour” (0.24 M citric acid), “bitter” (0.00007 M quinine sulfate), and “umami” (0.5% monosodium or potassium glutamate) as compared to “neutral” (distilled water) expressed in term-born neonates during their first postnatal hours. Single-frame pictures photographed from previously videotaped materials. (Courtesy of J. E. Steiner, The Hebrew University, Jerusalem.)

with lip licking, (e.g., sweet: Fig. 2, rows 2 and 3; umami: Fig. 2, rows 2 and 3), accompanied by rhythmic sucking (often with a distinct smacking sound) (e.g., sweet: Fig. 2, rows 1 and 4; umami: Fig. 2, row 5), followed by relaxed face/smiling (e.g., sweet: Fig. 2, row 5; umami: row 4). The constellation of these patterned lip and tongue movements are sometimes referred to as “mouthing” and used as a reliable measure of sweet or “palatable” in infant studies (e.g., Barr et al., 1999). Bitter stimuli elicit mouth corner depression (Fig. 2, rows 1 and 2), gaping (Fig. 2, rows 3–5), tight eye closure (Fig. 2, rows 2–5), flat tongue protrusion (Fig. 2, rows 3 and 4), head turning/shaking, and drooling. Similar but diminished responses are produced by sour stimuli (e.g., mouth corners down: Fig. 2, row 1; gaping: Fig. 2, row 5), with the addition of sustained and/or rhythmic lip

pursing (Fig. 2, rows 2–4) and nose wrinkling. Water, on the other hand, usually elicits only transient alerting and a minimum of mouth movements associated with swallowing, while responses to the unseasoned clear soup diluent are described as slightly to moderately aversive (Steiner, 1987). Observers unaware of stimulus parameters regularly interpret these videotaped responses to sweet and umami as hedonically positive (acceptance) and to bitter as hedonically negative (rejection) (e.g., Ganchrow et al., 1983; Steiner, 1987). While sour is generally considered to be a somewhat aversive taste, in one study (Rosenstein and Oster, 1988), 0.12 M citric acid has been categorized as eliciting neutral-to-hedonically positive reactions mirroring results obtained in newborn rats and rabbits (Ganchrow et al., 1979, 1986). This could be an interpretation error due

828

to an apparent overlap of some sour- and sweet-induced features (e.g., lip pursing/sucking), or an immaturity of sour-taste receptor mechanisms, or a lack of learned hedonic response associated with these stimuli. This ambiguity was not found in precocial hatchling chicks under similar experimental conditions and using the same citric acid concentrations: Observers classified the chicks’ responses to citric acid as hedonically negative (Ganchrow et al., 1990). That facial response is reflex-like is supported both by its appearance in anencephalics (Steiner, 1973, 1979b) and by the fact that facial motor features elicited by sucrose can be classically conditioned to a tactile stimulus in newborns 2–48 hours of age (Blass et al., 1984). In addition, single response components such as tongue movements can be reliably elicited in newborns by tastants in a concentration-dependent manner (e.g., Nowlis and Kessen 1977; Weiffenbach, 1977). Further support for its root reflexive nature comes from studies demonstrating similar taste-induced oral motor behaviors in decerebrate and intact rats (Grill and Norgren, 1978). As in premature infants, newborn infants show individual differences in taste-induced orofacial reactivity (e.g., Bergamasco and Beraldo, 1990; Ganchrow et al., 1983; Maekawa et al., 1991; Stirnimann, 1935), possibly reflecting either genetic differences in the predisposition for facial emotional expression or in taste receptor endowment. For example, human taste bud density can vary as much as 100-fold between individuals irrespective of age (Miller, 1988), affecting the perceived intensity of a tastant and the spectrum of expressive movements that is elicited. Thus, one would expect that the weaker the stimulus, the more the individual variation in response emission. Prenatal chemosensory experience might also contribute to this variability (see below). Sweet taste solutions of sucrose and aspartame (0.12%) also reliably elicit hand-mouth contacts perinatally (e.g., Barr et al., 1994, 1999; Blass et al., 1989; Smith and Blass, 1996; Zeifman et al., 1996), and these could be interpreted as precursors to later feeding-related behaviors. This tasteelicited behavior is stimulus bound to the presence of the sweet taste and declines during the first 2 weeks postpartum. It is noteworthy that ongoing crying is diminished and eliminated for some time after the gentle insertion of droplets (as little as 0.1 ml) of sucrose or aspartame into the oral cavity. The ability of the sweet-perceptive system to regulate ongoing crying is most apparent perinatally and appears to decrease over the first 6 weeks (e.g., Barr et al., 1994; Smith and Blass, 1996; Zeifman et al., 1996), often requiring larger volumes of stimulus to obtain the effect at all. Bitter tastants (0.25% quinine) presented under similar

Ganchrow and Mennella

conditions also produce a very transient calming (Graillon et al., 1997), possibly due to attentional factors, and indicative again of a broadly functional taste system at birth. A functioning affective component to the newborn’s response to taste stimuli is also suggested by taste-induced autonomic responses (e.g., Ashmead et al., 1980; Blass and Watt, 1999; Crook and Lipsitt, 1976; Haouari et al., 1995; Herschel et al., 1998; Lipsitt et al., 1976; Lipsitt, 1977; Rao et al., 1997). Experimental conditions seem to dictate the direction of change. For example, heart rate increased proportionally with the introduction of 5% and 15% sucrose onto ongoing sucking from an artificial nipple in a situation where increased sucking amplitude did not yield larger volumes. Conversely, heart rate decreased when sweet was introduced during agitation, producing an overall calm (e.g., Blass and Watt, 1999). Furthermore, Fox and Davidson (1986) reported a taste-induced asymmetry of brain electrical activity, which is usually associated with hedonically positive emotional reactions, although the response had not yet achieved a mature form. Specifically, they observed left-sided frontal activation (a correlative sign of affectively positive or approach behavior) in response to sucrose as compared with water in 2- to 3-day-old newborns. 2.

Sucking and Intake

Direct measures of intake in the first postnatal days also suggest the neonate has a strong, positive hedonic response to sweet-tasting stimuli. Sucrose, as well as MSG solutions (the latter dissolved in clear soup), are consistently ingested in larger volumes compared to bitter, sour, salty, and/or neutral stimuli (e.g., Beauchamp and Moran, 1982; Beauchamp and Pearson, 1991; Desor et al., 1973; Maller and Desor, 1973). In addition, heavier infants tend to consume greater absolute volumes of sweetened solutions than those with less weight (e.g., Nisbett and Gurwitz, 1970; Desor et al., 1973; Kajiura et al., 1992). For umami, the possibility exists that glutamates (e.g., MSG) in clear broths may interact with odor and/or viscosity (Maga and Lorenz, 1982), affecting, in turn, intake and/or flavorinduced facial expressions. To date this issue has not been behaviorally addressed in infants. Newborns can also discriminate among different concentrations of sweet stimuli as determined by sucking rates (e.g., Engen et al., 1978) and measures of the temporal organization of sucking (e.g., duration of sucking bursts, interburst intervals, and within-burst pace of sucking) (e.g., Crook, 1977; Crook and Lipsitt, 1976; Eckstein, 1927). For instance, increasing the sucrose concentration (0.0612–0.5 M) produces corresponding increases in the number of sucks regardless of birthweight (Grinker et al.,

Ontogeny of Human Flavor Perception

1986). However, these measures are not always sensitive to differences among nonsweet substances, in contrast to facial reaction measures. For example, in one study infants from birth to 6 days of age did not express rejection of 0.12–0.24 M urea compared to the 0.07 M sucrose diluent on several measures of intake and sucking behavior (Kajiura et al., 1992). This could imply immaturity of cellular receptor processing specific to urea. However, the impact of the sweet diluent at this young age may also have been important since earlier studies using quinine dissolved in water did report a clear disruption of newborn sucking behavior (e.g., Desor et al., 1975b; Eckstein 1927) as well as a tendency for a damped response to sour and salt stimuli (Crook, 1978; Desor et al., 1975b; Eckstein 1927). Indeed, a significant depression of sucking for 0.2–0.4 M salt solutions has been reported (Beauchamp et al., 1994), consistent with previous data (Maller and Desor, 1973) revealing a depressed intake of 0.2 M NaCl compared to water. In summary, by all measures newborns perceive and respond positively to sweet stimuli. For other tastes, testing conditions seem especially important. Although under certain circumstances relatively concentrated bitter, sour, and salt stimuli elicit definitive behavioral responses, the diluent, the stimulus and its concentration, and the dependent measure may influence the results. Also, “negative” tastes experienced may not always be able to override and inhibit an ongoing sucking response. Preference (“wanting”), measured as amount ingested, and orofacial hedonic reactivity (“liking”) are sometimes independent and probably mediated by separate neural mechanisms (see Berridge, 2000, for review), whose emerging expression could be developmentally dependent. 3.

Palatability and Pain Measures

In addition to initiating neonatal responses supporting consummatory behavior, sweet taste sensibility has recently been shown to diminish pain reactivity, with an accompanying calming effect and decreased heart rate (see above) in preterm and term-born infants, extending comparable findings in rats (see Blass and Watt, 1999, for review). Behavioral measures have included facial expression, visual analog scale estimates of distress, incidence and duration of crying, or some combination of these. The effect is generally sustained for at least 4 minutes after cessation of the gustatory stimulus. Two ml of 12% (0.33 M) sucrose was usually sufficient to consistently produce a significant analgesic response (see Stevens et al., 1997, for systematic review), although 30–75% sucrose or glucose are more commonly used in clinical trials.

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Although more detailed investigation is needed, it would appear that, within limits, variations in sweet molecule type, suprathreshold stimulus concentration, solution volume, and presentation as a flow or on a dipped pacifier do not differentially affect the calming- or pain-reducing effects of sweet-tasting stimuli (e.g., Barr et al., 1999; Blass and Shah, 1995; Blass and Watt, 1999; Carbajal et al., 1999; Ramenghi et al., 1996; see also Haouari et al., 1995). This suggests the possibility of an “on-off” rather than a graded relation between the gustatory afferent and the presumed opioid-mediated efferent response (Blass and Shah, 1995; see also Lieblich et al., 1984; Pomonis et al., 2000). Exceptionally, lactose failed to reduce distress in the concentration range of 0.17–0.51 M in both humans (Blass, 1997; Blass and Smith, 1992) and rats (Blass and Shide, 1994). However, isocontour matching of sweetness across various sugars in adults (Cameron, 1947) would require a 0.8 M lactose concentration to match the optimal 0.33 M (12%) sucrose, suggesting the concentration of lactose used during testing was probably below threshold for producing the effect. The analgesic effect appears to be due to taste, per se, since the same sweet solution is ineffective in producing analgesia when administered intragastrically in preterm (32–36 weeks) infants (Ramenghi et al., 1999). Preterm infants as young as 25 weeks postconception are able to express the pain-relieving sweet response (Johnston et al., 1997). The calming effect is induced within seconds of sucrose delivery, well in advance of stomach clearance or absorption. Overall, in these perinatal ages, gender, gestational age, and immediate postnatal age are noninfluencial factors (e.g., Overgaard and Knudsen, 1999). Other flavor and orosensory components are also sufficient to produce the above effects and may act synergistically with sucrose. These include milk flavor and some of its constituents (fat, protein) (e.g., Barr et al., 1996; Blass, 1997), but not colostrum (E. M. Blass and L. B. WattMiller, personal communication). Orotactile components, such as a pacifier (Blass and Hoffmeyer, 1991; Blass and Watt, 1999; Carbajal et al., 1999), also produce the analgesic effect but are transient compared to sweet stimulation (Smith et al., 1990) and are not accompanied by consonant significant drops in heart rate (e.g., Blass and Watt, 1999). It is interesting to note that besides oro-tactile stimulation (e.g., Rolls et al., 1999), fatty acids may depolarize taste cells (see Gilbertson, 1998). Taste “salience” has been suggested to play a role in human infants since another lipid, corn oil, did not elicit similar calming effects (Graillon et al., 1997).

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D.

Ganchrow and Mennella

The First Year

1. Facial Reactions and Sucking Behavioral responses to taste stimuli shift towards producing more voluntary consummatory consequences in the preverbal first year. Spontaneous facial expressions to taste stimuli remain fairly stable over the first postnatal month and then gradually decrease, being replaced by more noticeable intentional behaviors such as refusal to open the mouth or pushing the spoon away with the hands (Lichtenstein, 1893/94). Still, comparing infants in the first 6 postnatal days to those 0.5–6 months of age, Kajiura et al. (1992) demonstrated that the balance of facial expressions and body movements indicative of rejection of urea (0.12–0.24 M) shifted away from indifference and towards rejection. This was confirmed by a more even decrease in intake in older infants, suggesting a change in urea bitter sensitivity. Changes in sucking parameters are less evident early on, and until 1.5 months sucking responses are similar to those of younger infants (Eckstein, 1927). Still, Maekawa et al. (1991) argue that a discriminative sucking response to taste stimuli has a decremental tendency, especially around 3–5 months, such that 0.25% NaCl and 0.1% tartrate are progressively less likely to induce a sucking reaction of rejection. Therefore, it becomes useful to discuss facial expression and sucking in conjunction with intake under the heading of “preference,” i.e., willingness to consume a taste substance. 2. Preference Using several different acceptance measures, it has been found that responses to sweet and umami stimuli (the latter in soup or salt solutions) throughout the first postnatal year ranges from preferred to indifferent, with no age trends evident after the first week (e.g., Beauchamp and Pearson, 1991; Beauchamp et al., 1984, 1998; Büssem, 1895; Lichtenstein, 1893/1894; Vasquez et al., 1982). In general, bitter and sour stimuli are rejected at concentrations well above threshold (e.g., Lichtenstein 1893/1894; Neuman, 1896; Vasquez et al., 1982; see also Desor et al., 1975b). Futhermore, when the bitter tastant was presented in one study as urea dissolved in 0.07 M sucrose, older (2-week- to 6-month-old) infants tended to reject all urea concentrations (0.12–0.24 M) (Kajiura et al., 1992). This is in contrast to newborns (see above), whose sucking responses were more controlled by order of stimulus presentation than by stimulus taste. These results suggest that for some bitter substances, developmental factors, presumably at the receptor level, may govern the ability to actively reject “bitter” presented within a mildly “sweet” context (see also Beauchamp et al., 1991).

Responses to sodium chloride appear to be substantially modified during development. Unlike newborns, who mostly appear to be indifferent or averse to the taste of salt, 4-month-old infants begin to prefer saline solutions and 6-month- to 2-year-old infants have developed a clear preference for salted solutions (0.1–0.2 M) and foods (Beauchamp et al., 1986, 1994). This could be related to maturation of salt receptor function as reported in other mammals (for review, see Hill and Mistretta, 1990) (see also Chapter 36) or central nervous system developmental factors that have not yet been elucidated. Human infants may be born “hard-wired” to discriminate tastes with survival value (e.g., potentially harmful bitter and sour-tasting stimuli from potentially nutritious sweet and umami stimuli) (see Scott, 1992) but “soft-wired” so that a particular culinary environment can influence taste (and flavor) preferences essential for culture-specific survival (see, e.g., Beauchamp and Mennella, 1998; Gerber, 1991; Hudson and Distel, 1999; Mennella, 1997a). Considering taste alone, Beauchamp and Moran (1982) reported that infants fed sweetened water any time during the first 6 months maintained their high sweet preference established at birth, while those not so fed diminished their sweet preference during this time interval. This effect was still apparent 1.5 years later (Beauchamp and Moran, 1984). Likewise, salt intake may be affected by early experience. Salt preference is sometimes unexpectedly high at around 16–17 weeks for infants exclusively breastfed until testing (Harris and Booth, 1987). However, this study did not take into account possible fetal exposure to electrolyte imbalances and accompanying dehydration. Such events may up regulate later tendencies to prefer higher salt concentrations. For example, 16-week-old infants of mothers who had experienced morning sickness, or vomiting during pregnancy, were more willing to consume 0.1 and 0.2 M NaCl (i.e., showed higher preference ratios compared to water) and to ingest larger volumes of 0.2 M NaCl, as compared to control infants whose mothers did not express this symptom (Crystal and Bernstein, 1998). Furthermore, infants of the sick mothers were less likely to express aversive facial reactivity patterns, and more likely to exhibit hedonically positive responses, to the salty solutions, especially to 0.2 M NaCl. These data might also support the proposition that preterm infants taste NaCl but under most normal testing conditions are indifferent to it. This altered salt preference apparently carries through to adulthood, as reflected by (1) greater self-reported use of salt, (2) greater intake of salt in the laboratory, and (3) preferences for salty snack food compared to those whose mothers had mild or no morning sickness symptoms (Crystal and Bernstein, 1995). The mechanism is not

Ontogeny of Human Flavor Perception

understood, but maternal renin-angiotensin involvement and consequential elevated salt intake are no doubt important elements (e.g., Argüelles et al., 2000; Crystal and Bernstein, 1998). 3.

Threshold

Almost no research has been directed towards establishing taste thresholds in infants. Osepian (1958) used classical conditioning techniques to establish discrimination thresholds to sweet, salt, and sour in infants 2–9.5 months of age. The resulting values were within the normal range of detection and recognition thresholds reported for young adults (Cowart, 1981), although the study’s methodology has been questioned (Beauchamp and Moran, 1986). 4.

Palatability and Pain Measures

In comparison to perinatal ages, “sucrose analgesia,” measured by reduced crying tendencies, was modest for 2-and 4-month-old infants receiving diptheria-tetanuspertussis immunizations (Barr et al., 1995). A slight improvement (~40% crying reduction) occurred with an increased concentration (from 50% to 75% sucrose), an increased volume (from 0.75 to 2 ml), and the addition of cuddling and pacifier availability (Lewindon et al., 1998). Neither study attempted to stratify results by age. Critical stimulus parameters need to be better defined for this age group and older. E.

Early Childhood

1.

Intake and Preference

No age effects were found when solutions of urea (0–0.48 M) and citric acid (0–0.012 M) were tested: 2-year-olds decrease intake with increasing concentrations, as expected (Vasquez et al., 1982). Similarly, 2- to 3-year-old children increase the amount of sucrose solution they consume linearly with increasing concentration (0–0.4 M); however, unlike younger children, they decrease NaCl consumption (0–0.2 M) relative to water (Beauchamp et al., 1986; Vasquez et al., 1982). From 2.5 to 5 years of age, rejection of NaCl dissolved in water is quite pronounced (Beauchamp et al., 1986). On the other hand, when food (soup or cereal) is the vehicle for salt, 2-year-old children prefer salted to unsalted foods (e.g., Beauchamp and Moran, 1984). Experience and context may play a role here, since salty water would be a rather unusual beverage for 2-year-olds (see Beauchamp and Engleman, 1991). The important role experience and context play in determining salt and sweet preference is also demonstrated

831

in an experiment wherein 4- to 5-year-olds were exposed to either salty or sweet or plain tofu tasted many times over several weeks (Sullivan and Birch, 1990). When tested later, they preferred the tofu with the taste they were accustomed to, but the taste preference did not generalize to other foods of similar color/texture (e.g., ricotta) that were made sweet or salty. 2.

Thresholds and Intensity Scaling

Individual food aversions could be, in part, determined by variability in the perception of bitterness by different children. Five- to 7-year-old children have been tested for their sensitivity to the bitter substance 6-n-propylthiouracil (PROP). Unlike most other bitter substances (e.g., quinine), the ability to perceive PROP is a genetically determined trait, with the general population recessive for nontasting. “Tasters” among the children had lower thresholds and provided higher intensity ratings across the four PROP concentrations tested. However, there were proportionally fewer “nontasters” than predicted by adult data, suggesting that PROP thresholds rise with age and may partially account for greater food finickiness often exhibited by younger children (Anliker et al., 1991). 3.

Palatability and Pain

In contrast to 2-week-old infants, it appears that experimental conditions become increasingly important in obtaining the analgesic effect as maturation progresses. Toddlers (to 18 months) only showed sucrose (2 ml 12%) analgesia if they were administered one rather than 2 injections (Allen et al., 1996). This reinforces findings in 2- and 4- months-old infants (see above). F.

Late Childhood/Adolescence

1.

Thresholds

Taste thresholds bear little relation to measures of dietary preference (e.g., Adams and Butterfield, 1979; Nilsson and Holm, 1983) and, thus, may provide insight into development of the gustatory system per se. Procedural difficulties have precluded extensive research on this topic. Some studies support a general tendency for lower thresholds in 6- to 15-year-olds relative to 16- to 25-year-olds (Adams and Butterfield, 1979; Catalanotto et al., 1979; Nilsson and Holm, 1983). However, other studies have reported the opposite effect (see Cowart, 1981, for review). Attention span difficulties in younger children and environmental or ethnicity factors including within-group variability in taste receptor endowment were thought to contribute to these

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Ganchrow and Mennella

discrepancies (e.g., Okoro et al., 2000). However, a recent well-controlled study of 8- to 9-year-olds and young adults revealed the importance of sex and age: James et al. (1997) noted that the gustatory system of the prepubertal male subgroup had not reached maturity. This was expressed by significantly higher detection thresholds for sucrose and sodium chloride than those observed in men and women as well as prepubertal girls. Interestingly, the citric acid thresholds of the boys were seven times higher, on average, than that of the adults, although caffeine thresholds were similar across subgroups (see Table 1). A longitudinal study following up on these remarkable results is needed. 2. Intensity and Discrimination Scaling Early studies of taste intensity perception met with mixed results, some suggesting that children are more sensitive and some that they are less sensitive to taste intensity changes (e.g., Enns et al., 1979; Shapera et al., 1986). Age is a factor in obtaining reliable results of intensity differences, as those from children 2–3 years of age are practically random, unlike those of children 6 years and older (Kimmel et al., 1994). Recently, successful magnitude scaling results have been obtained from children 8 years of age. Direct magnitude scaling (James et al., 1999) of sucrose sweet intensity produced similar functions for 8to 9-year-olds and adults if the diluent was water. When the diluent was orange juice, the children’s intensity slopes flattened compared to adults, making context an important issue. Using a 5-point category scale to assess sweet intensity, comparisons between older children, adolescents, and young adults yielded borderline (p
0.08) significant differences for the age factor. When orangeade was the diluent, no significant age effect was observed, but the ageconcentration interaction was significant, with younger children providing lower intensity estimates (DeGraaf and Zandstra, 1999). The impression is that sweet taste intensity perception in children is quite stabilized, but testing parameters may affect how they it is expressed. Other taste stimuli need to be examined.

3.

Preference

Preference for salty foods established by the second year is maintained throughout early childhood and early adolescence before stabilizing at adult levels (Beauchamp and Cowart, 1987). Thus, a comparison of NaCl preferences among 3- to 6-year-old children, 7- to 10-year-old children, and 18- to -26-year-old young adults (0.0–1.0 M concentrations dissolved in vegetable soup) found the adults to have diminished preference for the highest concentration (65, 78, and 13% of these respective groups maximally preferred the highest concentration), with children preferring on average around 0.4 M NaCl, compared to adults at around 0.2 M (Beauchamp and Cowart, 1987, 1990). Similar results have also been obtained with chicken soup as the diluent (Nijjar et al., 1991). Hispanic preschoolers expressed higher sodium preference than their North American non-Hispanic white counterparts, again suggesting possible cultural/local environmental influences. The most comprehensive pioneering study examined taste preferences for sucrose (0.075–0.6 M), lactose (0.1–0.4 M), and sodium chloride (0.05–0.40 M) in 618 9-to 15-year-olds and 140 adults. This study found that, relative to adults, a greater percentage of children preferred the higher concentrations of each stimulus (Desor et al., 1975a). Within the younger group there were also sex and race differences, namely, males and African Americans tended, on average, to prefer higher concentrations. The age-related decrease in most-preferred sucrose concentration is a consistent finding and has been recently suggested to be related to the higher energy requirements of younger children (De Graaf and Zandstra, 1999), providing them with a sensory signal for energy content. A follow-up longitudinal study of a subsample of the younger group tested at 19–25 years of age in the Desor et al. (1975a) study confirmed that preference declines within individuals during maturation and that the results are not simply due to a generation-related change in preference behaviors (Desor and Beauchamp, 1987).

Table 1 Taste Detection Thresholds of Adults and Children Adult Tastant Sucrose Sodium chloride Citric acid Caffeine aExpressed

Female  0.0023  0.0005 0.0002  0.0001 0.0008  0.0002

0.0062a

as molar concentration. error. Source: From James et al., 1997. bStandard

0.0017b

Children Male 0.0068 0.0034 0.0002 0.0018

   

Female 0.0010 0.0005 0.0003 0.0004

0.0072 0.0027 0.0005 0.0011

   

0.0026 0.0005 0.0001 0.0003

Male 0.0170 0.0061 0.0014 0.0020

   

0.0033 0.0008 0.0005 0.0008

Ontogeny of Human Flavor Perception

Developmental differences in taste preferences may be colored by several influences, including (1) experience, (2) hormonal effects, (3) differences in physiological need within an environment, (4) expression of genetic programs, or some combination of these factors. For example, hormonal influences may contribute to the wide variations in salt preferences expressed across individuals over the life span such that, for instance, pregnant teenagers express increased avidity for salty snack foods during, but not after, pregnancy (Skinner et al., 1998). Sugar content did not differentially affect preference during those conditiions. Long-term effects of physiological challenges experienced early in development have been reported. Prenatal mineral or fluid loss resulting from maternal vomiting, and/or electrolyte imbalances associated with excessive infantile diarrhea and vomiting, increased adolescent avidity for salt, but not for sugar, corroborating and extending similar previous findings (Crystal and Bernstein, 1995, 1998; Leshem, 1998; see also Leshem et al., 1998). Furthermore, adolescents who were exposed during infancy to a chloride-deficient feeding formula favored highly salted foods and expressed a relative dislike for foods classified as being lower in saltiness as compared to their nonexposed siblings. Sugar preferences were similar in the two groups (Stein et al., 1996). Still, preterm infants undergoing neonatal furosemide (diuretic, natriuretic, and chloriuretic) therapy did not express unusual salt preferences in a laboratory setting, although increased dietary salt intake was implied by higher sodium excretion in a subsample (Leshem et al., 1998). It has been pointed out that a combination of sensory measures is probably required to properly assess salt preference, since different individuals may have different ways of expressing this preference (Stein et al., 1996), not to mention other environmental factors that may influence an individual’s salt habits. Still, taken together, these findings suggest that perinatal experience may have long-lasting effects on salt appetite. It is not known how much taste receptor level alterations or organizational central nervous changes during this period of plastic maturational events may have contributed to these outcomes.

4. Palatability and Pain Measures Sweet stimuli may continue to suppress pain in prepubertal children (Miller, 1994), as previously described for infants. Children in the 8- to 11-year-old range expressed prolongation of tolerance threshold times (cold pressor test) while holding 24% sucrose in their mouths as compared to water.

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IV. ONTOGENY OF OLFACTORY BEHAVIORAL RESPONSES AS RELATED TO FEEDING A.

Fetus

Taste notwithstanding, the human fetus can respond to a wide variety of other sensory stimuli that occur naturally in its environment. For example, the fetus responds to extrauterine auditory stimuli, as indicated by changes in heart rate (Fifer and Moon, 1988), and will blink when a bright light source is applied to the mother’s lower abdomen (Birnholtz, 1988). Furthermore, prenatal sensory experiences can subsequently affect the behavior of the newborn. This is best illustrated by the finding that human newborns exhibit a preference for a specific voice, melody, or passage experienced prenatally (De Casper and Spence, 1986; Hepper, 1998a) and the fact that they can discriminate between human voices (De Casper and Fifer, 1980). Such findings can be extended to the sense of smell—newborns can discriminate and will prefer their own amniotic fluid for at least 2 days after birth (Marlier et al., 1998; Schaal et al., 1995a, 1998). This preference for amniotic fluid odors shifts to olfactory cues emanating from mother’s milk shortly thereafter (Marlier et al., 1998). Learning about the dietary choices of the mother may also be occurring prenatally since the environment in which the fetus lives, the amnion, can indeed be odorous. Its odor not only can indicate certain disease states (Mace et al., 1976), but can reflect the flavors of foods eaten by the pregnant mother (Hauser et al., 1985; Mennella et al., 1995). This has been demonstrated experimentally in a study in which amniotic fluid samples were obtained from 10 pregnant women undergoing routine amniocentesis who ingested either garlic or placebo capsules approximately 45 minutes before the procedure (Mennella et al., 1995). The odor of the amniotic fluid obtained from the women who ingested the garlic, as determined by adult human evaluators, was judged to be stronger or more like garlic than amniotic fluid from the control women who did not consume garlic. Animal studies have revealed that flavor experiences in utero can result in preferences postnatally (for reviews, see Bilkó et al., 1994; Schaal and Orgeur, 1992; Smotherman and Robinson, 1988) (see also Chapter 15). For example, rat pups born by Caesarean section preferred their mother’s fluid to that of an unrelated rat when tested immediately after birth, indicating a prenatal acquisition of this odor preference (Hepper, 1987). Moreover, the fetus can acquire information about the dietary choices of the mother. For example, offspring of mother rabbits who had eaten garlic or juniper during pregnancy exhibited a

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preference for these flavors when compared to offspring of mothers who were not so exposed (Hepper, 1988b; Bilkó et al., 1994). Such findings have recently been extended to human infants (Mennella et al., 2001). Weanling-aged infants, who had several weeks of exposure to the flavor of carrots in either amniotic fluid or mothers’ milk, consumed significantly more carrot-flavored cereal and were perceived by their mothers as enjoying the carrot-flavored cereal more when compared to plain cereal. In contrast, control infants, whose mothers drank water during pregnancy and lactation, exhibited no such preference. These findings are the first experimental demonstration that prenatal and early postnatal exposure to a flavor enhances the acceptance and enjoyment of that flavor during weaning. As will be discussed in the next section, these very early flavor experiences may provide the foundation for cultural and ethnic differences in cuisine.

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B.

Infancy

Shortly after birth, human infants can detect a wide variety of volatile chemosensory stimuli, and they appear to be as sensitive to odors as are adults, if not more so. Newborns can detect and discriminate among qualitatively distinct odorants as evidenced by changes in their facial responses (see Fig. 3), body movements, and heart and respiratory rates (for review see Engen, 1982; Mennella and Beauchamp, 1993a, 1997a, 1999; Rovee, 1972; Soussignan et al., 1997; Steiner, 1977, 1979a,b). Perhaps the most salient odors for the newborn are those emanating from the mother. Within hours after birth, mothers and infants can recognize each other through the sense of smell alone (Cernoch and Porter, 1985; Macfarlane, 1975; Schaal, 1986, 1988) (for further discussion, see Chapter 15). Such recognition, with little postpartum contact, may be

Figure 3 Examples of facial reactions to food-related odors (BA., banana; VA., vanilla; FI., fish; BU., butter; R. E., rotten egg) as compared to the odorless control (C.) expressed in term-born infants in their first postnatal hours. (From Steiner, 1977.)

Ontogeny of Human Flavor Perception

due, in part, to mothers learning about the olfactory identity of their infants via changes in their own body odor during pregnancy, a change that reflects the odor type of the child (Beauchamp et al., 1995) (see also Chapter 17). Maternal odors have both a calming and a feeding preparatory effect on the infant. This is demonstrated by the findings that newborns spend more time orienting toward a breast pad previously worn by their lactating mothers than toward one worn by an unfamiliar lactating woman (Schaal, 1986, 1988), and they move their head and arms less, mouth more, and cry less during exposure to their mothers’ body odors (Schaal, 1986; Sullivan and Toubas, 1998). Moreover, shortly after birth infants prefer their mothers’ breast unwashed as compared to when it has been thoroughly washed and is therefore less odorous (Varendi et al., 1994). Thus, like other mammalian young, the recognition of and preference for maternal odors may play a role in guiding the infant to the nipple area and facilitating early nipple attachment and breast-feeding. 1.

Retronasal Perception of Flavors

a. Milk Feeding. Before the development of infant formulas, postpartum flavor experiences until the time of weaning were largely restricted to those obtained through mothers’ milk or the milk of another woman. Considerable research indicates that the milk of mammals, including humans, is rich in flavors that directly reflect the foods and spices eaten by the mother (see Mennella, 1995, for review). The retronasal route of olfaction may also be particularly salient for infants, allowing them to experience the many odors present in their mother’s milk. The following section focuses on human milk as a medium for early sensory experiences for the infant and how these sensory experiences impact on later preferences. In a study of the flavor properties of human milk (Barker, 1980; McDaniel, 1980), milk samples were collected from 24 lactating women during the morning hours on 3 consecutive days. Within 3 hours of expression, a trained sensory panel evaluated the milk samples for taste quality and for textural properties such as viscosity and mouth-coating. Each of these sensory attributes varied from mother to mother, with the primary taste quality of the milk being its sweetness. The sensory panelists also reported that off-flavors were present in some of the samples. Of particular interest was the finding that panelists used the verbal descriptors hot, spicy, and peppery to describe the milk of one woman who had consumed a “spicy” meal during the test period. Thus, the types and intensity of flavors experienced by each infant in their mother’s milk may be unique.

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More recent studies have systematically explored whether flavors from the woman’s diet are transmitted to her milk and what effects, if any, this has on the behavior of her breast-fed infant. Two approaches have been used to determine whether the odor, and consequently the flavor, of human milk is distinctively altered following the ingestion of different foods and beverages. First, it was determined whether a sensory panel of adults could detect a change in the odor of human milk as a function of maternal ingestion of flavors. Milk samples obtained from each woman at fixed intervals before and after the ingestion of the flavor or placebo were evaluated by a sensory panel of adults within hours of expression (Mennella and Beauchamp, 1991a,b, 1993b, 1996b, 1998a, 1999). Second, mothers “tasted” their own expressed milk samples to determine whether they could detect a difference in the flavor of their milk following ingestion of the flavor (Mennella, 1999b; Mennella and Beauchamp, 1999). In each case mothers were asked to maintain a bland diet and avoid eating foods that were flavored with the flavor of interest during the 3 days preceding each milk collection session to ensure that the milk was devoid of these volatiles. These methods revealed that human milk is rich in flavors and directly reflect the foods and spices eaten (e.g., garlic, mint, alcohol, carrot, vanilla) or substances inhaled (e.g., cigarette) by the mother (Mennella and Beauchamp, 1991a, 1993b, 1996, 1998, 1999). For example, maternal alcohol consumption significantly and consistently increased the perceived intensity of the milk odor; this increase in odor intensity peaked in strength 0.5–1 hour after ingestion and decreased thereafter, paralleling the changing concentrations of ethanol in the milk (see Fig. 4). There was no perceived change in the odor of the milk on the days the mothers ingested the control beverage. As seen in Table 2, infants can detect these flavors in milk as evidenced by changes in their patterning and duration of feeding and suckling (for review see Mennella and Beauchamp, 1997a). For the majority of flavors, they will respond by suckling more at the breast or bottle, especially if they have not experienced that flavor in the recent past. One exception to this rule is the breast-feeding response following maternal ingestion of alcohol (Mennella and Beauchamp, 1991b, 1993b). Infants breast-fed less during the initial hours when the milk was flavored with alcohol. However, further studies revealed that this diminished intake is due, in part, to a direct effect of alcohol on milk production by the mother, indicating that the infants are not rejecting the flavor of alcohol in their mothers’ milk (Mennella, 1998). When the infants were observed drinking their mothers’ milk from a bottle, no such decrease was

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Figure 4 Ethanol content of milk samples obtained at baseline and at 30 minutes and 1, 2, and 3 hours after the ingestion of an alcoholic or nonalcoholic beverage (open circles) and percentage of time panelists chose milk samples as smelling “more like alcohol” or “stronger” (closed circles). According to a forced-choice paradigm, panelists were presented individually with pairs of milk samples and asked to indicate which of the pair smelled “more like alcohol” or “stronger.” A value of 50% would be expected if there were no difference in the odor of the samples, and hence the panelists responded at random. (Data represent mean  SEM). (From Mennella and Beauchamp, 1991b.)

observed (Mennella, 1997b). Rather, the infants consumed significantly more and sucked more frequently when drinking the alcohol-flavored milk as compared with the unaltered milk. That experience with the flavor of alcohol in mother’s milk modified the infants’ responses to alcohol flavor is suggested by the relationship between the reported frequency of mothers’ drinking during lactation and the infants’ rhythm and frequency of sucking when feeding the alcohol-flavored milk. Consistent with that

observed for other flavors, more experience with alcohol, as assessed by maternal frequency of drinking, was associated with a diminished suckling response to alcoholflavored milk when feeding from a bottle. These findings indicate that infants can readily detect the flavor of alcohol in mother’s milk and experience will modify how they respond to it in subsequent feedings. That experience with a flavor in mothers’ milk will modify the infants’ response to that particular flavor has also been demonstrated (Mennella and Beauchamp, 1993c). For example, the infants of mothers who had repeatedly consumed garlic capsules during the week before testing, breast-fed for similar periods of time during the 4-hour test session in which their mothers’ consumed garlic, as compared with the session in which their mothers ingested the placebo capsules. In contrast, the infants who had no or minimal exposure to garlic volatiles in their mother’s milk during the previous week spent more time breast-feeding when their mothers ingested garlic compared to when their mothers ingested the placebo (Mennella and Beauchamp, 1991a). In other words, the infants’ suckling response to a particular flavor in mothers’ milk or formula depends not only on the flavor but the recency of exposure (Mennella and Beauchamp, 1993c, 1996b). Perhaps the garlic flavor became monotonous to infants who were repeatedly exposed to it in mother’s milk. Over the short term, children (Birch and Deysher, 1986) and adults (Rolls et al., 1982) report that the palatability of a food, and the amount of it consumed, declines following repeated consumption of that food, whereas less recently consumed foods are considered more palatable and stimulate food intake. Moreover, the garlic-flavored milk may have aroused the infants who were exposed to a diet of mother’s milk relatively low in flavor, garlic-like compounds, or both. When studying how a change in the flavor of mother’s milk affects the behavior of the infant at the breast, it is dif-

Table 2 Infants’ Suckling and Feeding Response to Flavored Breast Milk or Formula as a Function of Previous Sensory Experiencesa Flavor

Medium

Condition prior to testing

Length of feeding/ suckling response

Milk intake

Garlic

Human milk

Bland diet

Increase

Increase?

Garlic diet

No effect

No effect

Human milk Human milk

Bland diet Bland diet

Increase Increase

Increase Increase

Formula Formula

Bland diet Vanilla formula

Increase No effect

No effect No effect

Alcohol Vanilla

aSee

text for further details.

Ref. Mennella and Beauchamp, 1991a Mennella and Beauchamp, 1993c Mennella, 1997b Mennella and Beauchamp, 1996b

Ontogeny of Human Flavor Perception

ficult to separate the direct effects on the infant from other possible influences the consumed flavors could have on the mother (e.g., changes in the odor of the mother’s breath or sweat) (see Mennella and Beauchamp, 1991a, for further discussion). Consequently, one cannot unequivocally conclude that the flavor change in the mother’s milk was responsible for the alteration of the infants’ suckling behavior. To examine the effects of flavoring directly, vanilla was added to the infants’ formula. Consistent with what was found for the breast-fed infant, the bottle-fed infants’ response to the vanilla-flavored formula was altered relative to their response to the unflavored formula. In the first, short-term preference test, the infants sucked more vigorously when feeding the vanilla-flavored formula, and in the second test, which encompassed an entire feeding, they spent more time feeding on the first bottle when it contained the vanilla flavor (Mennella and Beauchamp, 1996). Following 2 days of exposure to this flavor, however, their suckling response to the flavor diminished. Why do infants respond to flavored milk by enhanced suckling? As mentioned previously, in the studies demonstrating enhanced suckling response to a flavor in breastfed infants (e.g., vanilla, garlic), the nursing mothers were asked to eat bland diets devoid of these flavors during the 3 days preceding each testing day (Mennella and Beauchamp, 1993c, 1996b). In the study with formula-fed infants, their mothers clearly were providing a very monotonous diet since formula is virtually unchanging (Mennella and Beauchamp, 1996b). Perhaps the novelty was sufficient to induce increased suckling because these flavors are inherently positive or arousing either as a result of prior exposure (an unlikely explanation for the formulafed infant, although in utero experience is possible) or, like sweet taste, a hard-wired (innate) response. When aroused, mammalian infants will suck more (Bridger, 1962) and exhibit a variety of other oral behaviors (Korner et al., 1968; Terry and Johanson, 1987). These data, along with those reported previously (Mennella and Beauchamp, 1991a, b, 1993b, c, 1996b), demonstrate that infants can detect flavors either added to formula or transmitted to human milk from the mother’s diet. The experience with a flavor in either medium altered how they responded to that flavor in subsequent feedings. The retronasal perception of odors in mothers’ milk may provide the infant with the potential for a rich source of varying chemosensory experiences and a possible route for the development of preference for a diet similar to that of the mother’s. This is due in part to the fact that the context in which the flavor is experienced, with the mother and during feeding, consists of a variety of elements (e.g., tactile stimulation, warmth, milk, mother’s voice), which

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have been shown to be reinforcers for early learning. Moreover, a substantial body of animal research indicates that weanling animals actively seek and prefer the flavor of the diet eaten by the mother during nursing (for review, see Mennella, 1995). In summary, exposure to flavors in mother’s milk may be one of several ways in which the mother teaches her young what foods are “safe” (Rozin, 1976). Consequently, young animals may tend to choose a diet similar to that of the mother’s when faced with their first solid meal. b. Solid Foods. For centuries the custom of many cultures has been to introduce grain products, such as precooked cereal, as the infant’s first solid food (Hervada and Newman, 1992; Raphael, 1982). The cereal was sometimes prepared with mother’s milk because many believed that it was essential that the food be simple, smooth in texture, and consistent in flavor (Fildes, 1986; Wachenheim, 1915). In present times, baby food manufacturers and child care manuals advise parents to prepare the cereal with water or either mother’s milk or formula, depending on the feeding regimen of the infant. Still, little was known about the infants’ acceptance of differently flavored cereals. Thus, a study was designed to determine (1) whether breast-fed infants’ acceptance of their first solid food— cereal—is enhanced when it is flavored with mothers’ milk and (2) whether the infants’ willingness to accept such flavored cereal is correlated with their mothers’ reported willingness to try novel foods and flavors (Mennella and Beauchamp, 1997b). The study revealed that breast-fed infants who had been fed cereal for approximately 2 weeks but had only experienced cereal prepared with water readily accepted and preferred the cereal when it was prepared with mothers’ milk (Mennella and Beauchamp, 1997). They not only consumed more of the cereal flavored with mothers’ milk, they displayed a series of behaviors signaling their preferences. In addition, the more varied the mother’s diet, the greater the acceptance of the flavored cereal. Because an infant’s first flavor experience may occur even before birth in amniotic fluid, breast milk may bridge the experiences of flavors in utero to those in solid foods. It is also possible that experience with a variety of flavors (e.g., in amniotic fluid, the mother’s milk) may predispose the infant to be more willing to accept novel flavors during the weaning process. As discussed earlier, animal studies support this hypothesis, and an additional study found that the more varied the mother’s diet, the more likely the infant would accept a novel-flavored formula (Mennella and Beauchamp, 1996c). Because human milk is rich in flavors that directly reflect the dietary choices of the mother, the transition

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from a diet exclusively of human milk to a mixed diet may be facilitated by providing the infant with bridges of familiarity such that the infant experiences a commonality of flavors in the two feeding situations. In this case, human milk would be familiar and would facilitate the introduction of a novel food, cereal. Animal model studies have documented this phenomenon (Bilkó et al. 1994; Galef and Clark, 1972), and, in fact, it has been exploited in the development of weaning foods for pigs (Campbell, 1976). Because breast-fed infants’ diet is more rich and varied than that of infants fed a monotony of flavors in standard formulas, Mennella and Beauchamp (1991a) hypothesized that breast-fed infants should be more accepting of novel foods. This was supported by a recent study of 4- to 6month-old infants’ acceptance of novel food (e.g., green beans, peas) both before and after 10 daily feedings of the food (Sullivan and Birch, 1994). The infants’ reaction to and intake of the food increased significantly after the exposure period; similar findings have been reported for preschool children (Sullivan and Birch, 1990). Of particular interest, however, was the finding that the breast-fed infants’ acceptance of the vegetable was greater after the exposure period when compared to those who were formula fed. The breast-fed infants’ enhanced acceptance of the vegetables could be due to the breast-fed infants’ familiarity with specific flavors associated with the vegetables in mothers’ milk, because they had much more experience with flavor variety than did the formula-fed infants, or both. As discussed earlier, amniotic fluid is a potential flavor carrier. Because amniotic fluid and mother’s milk are under the common priming of maternal diets, their aromatic profiles may overlap and serve as a “thread of chemical continuity between the pre- and postnatal niches” (Schaal and Orgeur, 1992). Indeed, recent findings clearly show that experience with the flavor of carrots, either in amniotic fluid or mothers’ milk, results in preference for the flavor of carrots during the time of weaning (Mennella et al., 2001). Whether the apparent redundancy in exposure to flavors in amniotic fluid and mother’s milk ensures that the young animal can acquire preferences for a variety of foods eaten by the mother during different stages of development is clearly an area in which further research is needed in humans (see Bilkó et al., 1994; Capretta et al., 1975). These questions have ramifications not only for understanding the development of food habits, but also for understanding such nutritional concerns as excessive salt and fat consumption, which may lead to hypertension and obesity, respectively, and the effects of early exposure to drugs such as alcohol and tobacco on later preferences.

Ganchrow and Mennella

C.

Young Children

Food preference predispositions are susceptible to conditioning influences throughout childhood (for review, see Birch, 1999). For example, conditioned flavor aversions have been demonstrated in 73% of children ranging in age from 2 to 15 years undergoing chemotherapy (Bernstein, 1978), and comparable shifts in hedonic assessment of nausea-associated flavors have been observed throughout adulthood (e.g., Schwartz et al., 1996). Human taste conditioning alone has not been investigated in the developmental context, although research suggests that both taste and smell may independently contribute to flavor aversion (e.g., Kiefer et al., 1982; see Bernstein, 1999, for review). The taste-smell combination is sufficient to support conditioned aversions even when pairing with an aversive stimulus occurs in utero in rats who are then tested more than 2 weeks postnatally (Stickrod et al., 1982). The few published studies that have focused on olfactory preferences in the verbal child indicate that they indeed have likes and dislikes relating to a range of odors and flavors, but that their hedonic experience may be different from that of adults. For example, children less that 5 years of age appear to respond positively to some odors, such as the odor of synthetic sweat and feces. This led investigators to conclude that young children do not have aversions to certain odors that adults and older children find offensive and are generally more tolerant of odors than are adults (e.g., Engen, 1982). In contrast to those studies, androstenone, a component of sweat, is rated as a “bad” odor by 92% of children (Schmidt and Beauachamp, 1988) and 59% of adults. Although androstenone is not a flavor, it is of special interest because it can be perceived as smelling urinous, sweaty, musky, like sandalwood, or having no smell; twin studies revealed that genetic differences between individuals contribute to these differences in the perception of androstenone (Wysocki and Beauchamp, 1984). Approximately half of the adult population has a specific anosmia to it. (Specific anosmia is defined as the inability of an individual, who otherwise has normal olfactory functioning, to smell a specific molecule or related class of molecules.) The fact that such a high percentage of children rated this odor as bad suggested that most or perhaps all 3-year-old children can detect it. A developmental shift in the perception of androstenone occurs at or near adolescence, when approximately 50% of individuals lose the ability to smell this odor. The mechanisms underlying this change in perception remain unknown. Recent evidence suggests that children’s thresholds are comparable to

Ontogeny of Human Flavor Perception

young adults for a number of other odorants, although children aged 4–11 were less accurate at odor identification, and their semantic memory for odors was less well developed (Lehrner et al., 1999). Some of the discrepancies as to when olfactory preferences and aversions arise can be traced to methodological and technical difficulties in testing young children. For example, children younger than 6 years of age tend to answer a positively phrased question in the affirmative, and they have a shorter attention span than older children (Engen, 1974; see also Richman et al., 1995). Thus, their responses may be biased and not reflect their actual reaction to the odor. By using methodologies sensitive to these behavioral limitations and embedding the task in the context of a game, research has demonstrated that olfactory preferences and aversions are evidenced in children as young as 3 years (Schmidt and Beauchamp, 1988). Like adults, children preferred the odor of C-16 aldehyde (strawberry), phenylethyl-methylethyl carbinol (floral), L-carvone (spearmint) and methyl salicylate (wintergreen), but disliked the odor of butyric acid (strong cheese/vomit) and pyridine (spoiled milk).

V.

CONCLUSIONS

The sensory world of a fetus or infant is, no doubt, different from that of a child or an adult. Moreover, the sensory world of the newborn infant is different than that of a 6-month-old infant. In adults, input from taste, retronasal olfaction, and oral irritation appears to fuse into one sensory gestalt—that of flavor. It is not known whether the same is true for the infant. Certainly the balance of these three systems may be very different with relative insensitivity to some taste components (e.g., salt, some bitter compounds) combined with adult-like or perhaps even heightened sensitivity to others (e.g., odors, perhaps irritants). Early gustatory and olfactory receptor development prepares even premature infants to respond appropriately to elementary chemosensory properties associated with nutrients essential for survival. Some spontaneous behaviors and preferences associated with taste or smell drop out during development, while others endure. For example, the pain-protective function of palatable (e.g., sweet) tastes, apparent in early infancy, endures into adulthood under certain conditions (Mercer and Holder, 1997; Zmarzty et al., 1997), while some reflex-like motor patterns diminish or vanish. Sweet may be a uniquely potent stimulus in its general durability as a preferred tastant unless repeatedly associated with a negative consequence of intake,

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such as nausea. Indeed, initial attempts to reverse sweet preference in rats following taste deprivation during the suckling period met with failure (Bernstein et al., 1986). This procedure is known to profoundly alter the normal presynaptic development in the gustatory nucleus of the solitary tract in this same species (Lasiter and Diaz, 1992). Further research is required to sort out the critically important central nervous system factors acting to control flavor perception and behavior during development. What qualifies as palatable is susceptible to change with experience. For example, it has been demonstrated that salt preference in infants may change with age and that it can be influenced by prenatal maternal fluid/salt imbalances. By adulthood, the impact of early experience and childhood food experiences, as well as current physiological state, may impact on salt preferences. This is even more apparent when flavor is considered. Although much research is still needed to fully understand the impact of early flavor experiences of the human infant, it is clear that they are not passive recipients of flavored foods. Rather, based on genetic predisposition and experience, they will avidly accept some flavors while decidedly rejecting others. It is intriguing to begin to unravel how exposure to flavors, most often experienced in amniotic fluid, mothers’ milk, or formula, might affect later preferences, the development of food habits, and the willingness to accept new foods at weaning or thereafter. It appears that early sensory experiences may be particularly important in human development and the advent of formula feeding may not only deprive infants of important immunological and perhaps psychological benefits (see Goldman et al., 1987, for review), but may also limit their exposure to an important source of information and education about the flavor world of their mother, family, and culture (Mennella, 1995; Mennella and Beauchamp, 1991, 1993a). Many questions remain to be answered. For example, are there sensitive periods during early development when experiences with flavors produce particularly enduring effects? How do these intersect with possible influences of the mothers’ physiological state? The scientific study of sensitive periods during the development of behavior, made popular by the ethologist Konrad Lorenz (1965), implies that there is a period during early development when the organism is primed to receive and perhaps permanently encode important environmental information. An early knowledge of what are safe, appropriate, and nutritious foods would intuitively be important information for the fetus, infant, and young child. This is not to say that later learning is not important, but it highlights the possible significance of these very early experiences.

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844 Mennella, J. A., and Beauchamp, G. K. (1997a). The ontogeny of human flavor perception. In Handbook of Perception and Cognition: Tasting and Smelling, G. K. Beauchamp and L. Bartoshuk (Eds.). Academic Press, San Diego, pp. 199–221. Mennella, J. A., and Beauchamp, G. K. (1997b). Mother’s milk enhances the acceptance of cereal during weaning. Pediatr. Res. 41:188–192. Mennella, J. A., and Beauchamp, G. K. (1998). Smoking and the flavor of breast milk—letter. N. Engl. J. Med. 339:1559–1560. Mennella, J. A., and Beauchamp, G. K. (1999). Experience with a flavor in mother’s milk modifies the infant’s acceptance of flavored cereal. Dev. Psychobiol. 35:197–203. Mennella, J. A., Jagnow, C. P., Beauchamp, G. K. (2001). Prenatal and postnatal flavor learning by human infants. Pediatrics 107:E88. Mennella, J. A., Johnson, A., and Beauchamp, G. K. (1995). Garlic ingestion by pregnant women alters the odor of amniotic fluid. Chem. Senses 20:207–209. Mercer, M. E., and Holder, M. D. (1997). Antinociceptive effects of palatable sweet ingesta on human responsivity to pressure pain. Physiol. Behav. 61:311–318. Miller, A., Barr, R. G., and Young, S. N. (1994). The cold pressor test in children: Methodological aspects and the analgesic effect of intraoral sucrose. Pain 56:175–183. Miller, I. J. (1988). Human taste bud density across adult age groups. J. Gerontol. 43:B26–B30. Mistretta, C. M. (1972). Topographical and histological study of the developing rat tongue, palate and taste buds. In Third Symposium on Oral Sensation and Perception. The Mouth of the Infant, J. F. Bosma (Ed.). Charles Thomas, Springfield, IL. pp. 163–186. Mistretta, C. M., and Bradley, R. M. (1975). Taste and swallowing in utero. Br. Med. Bull. 31:80–84. Neumann, H. (1896). Bemerkung über die Geschmacksempfindung bei kleinen Kindern. Jahrb. Kinderheilk. 41: 155–159. Nijjar, R., Straits, K., Sniffen, J., Gilmore, M., and Murphy, C. (1991). Salt preference in anglo and hispanic preschoolers. Chem. Senses 16:563. Nilsson, B., and Holm, A. K. (1983). Taste thresholds, taste preferences and dental caries in 15-year-olds. J. Dent. Res. 62:1069–1072. Nisbett, R. E., and Gurwitz, S. B. (1970). Weight, sex and the eating behavior of human newborns. J. Comp. Physiol. Psych. 73:245–253. Nowlis, G. H., and Kessen, W. (1977). From reflex to representation: Taste-elicited tongue movements in the human newborn. In Taste and Development: The Genesis of Sweet Preference, J. M. Weiffenbach (Ed.). U.S. Dept. H.E.W. Publications, Bethesda, MD, pp. 190–202. Okoro, E. O., Brisibe, F., Jolayemi, E. T., and Hadizath Taimagari, G. (2000). Taste sensitivity to sodium chloride and sucrose in a group of adolescent children in northern Nigeria. Ethn. Dis. 10:53–59. Osepian, V. A. (1958). Development of the function of the taste analyzer in the first year of life. Pavlov. J. Higher Nerv. Activ. 8:766–772.

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40 Genetics of Human Taste Perception Adam Drewnowski University of Washington, Seattle, Washington, U.S.A.

I.

and other poisons generally have an unpleasant bitter taste (Rouseff, 1990). Abnormal or extreme bitterness usually denotes toxicity and is the main reason for food rejection (Drewnowski and Gomez, 2000). Being able to identify and reject bitter poisons offers a major evolutionary advantage. Bitter compounds, including extremely toxic ones, are detected by humans in micromolar amounts. While there seems to be no linear relation between bitter taste thresholds and compound toxicity (Hladik and Simmen, 1996), quinine is detected at 25 mol/L concentrations and bitter plant toxins at levels far below that. In contrast, taste detection thresholds for sucrose are in the order of 10,000 mol/L. Some researchers have suggested that PROP responsiveness among women is hormone-driven, being enhanced during pregnancy and diminished following menopause (Bartoshuk, 2000), although empirical data on this point are sparse. Nonetheless, infants, young children, and pregnant women are most likely to avoid bitter taste and to reject bitter-tasting foods (Drewnowski and Gomez, 2000). This taste-mediated rejection of bitter compounds is present at birth (Drewnowski, 1997). Although humans accept a low level of bitterness in some foods, intensely bitter tastes are aversive to most people (Drewnowski, 2001; Drewnowski and Gomez, 2000). Bitter taste sensation tends to be more prolonged as compared to sweet, salty, or sour (McBurney, 1978). As a result, food industry has taken steps to modulate the amount of bitterness present in the food supply (Drewnowski, 2001; Drewnowski and Gomez, 2000). Furthermore, individual sensitivity to bitter taste can vary

INTRODUCTION

The study of human taste genetics is largely the study of bitter taste. Research on the genetics of human taste perception began in 1931 with the accidental finding that crystals of phenylthiocarbamide (PTC) tasted bitter to some people but were tasteless to others (Blakeslee, 1931; Fox, 1932). One in three people showed a heritable “taste blindness” to PTC (Blakeslee, 1931; Snyder, 1931) and to a related compound, 6-n-propylthiouracil (PROP). Since that time, the phenotypic taste responses to PTC/PROP have been well described in the literature (Guo and Reed, 2001). However, the genes responsible for this trait have not been identified, and their exact location is still unknown (Reed, 2000). The ability to taste PTC/PROP is a unique example of genetic taste polymorphism in humans. Though initially viewed as an all-or-none phenomenon, the ability to taste PTC/PROP is a continuously distributed variable. Sensitivity to PTC/PROP is influenced, moreover, by race/ethnicity, sex, and age. How researchers characterize taste responses to PROP among individuals or groups, and what that tells us about genetic influences on human taste preferences, is the main focus of this review. II.

THE ROLE OF BITTER TASTE

While sweet taste is associated with dietary energy, bitterness tends to be equated with dietary danger (Hladik and Simmen, 1996; Rouseff, 1990) (see Chapter 35). Hydrolyzed proteins, rancid fats, plant-derived alkaloids, 847

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widely. There is also a strong and inverse relationship between perceived bitterness and acceptance ratings. While there is no consistent relationship between sweetness intensity perception and liking for sweet taste, increased bitterness always predicts a greater dislike of bitter compounds and bitter-tasting foods.

Drewnowski

allele models (Morton et al., 1981; Olson et al., 1989). Recent studies in humans have linked the ability to taste PROP to at least two genetic loci—at chromosome 5p15 and at chromosome 7 (Guo and Reed 2001). Such studies critically depend on an accurate classification of respondents by PROP taster status. However, the definition of what constitutes the ability to taste PROP has also evolved with time.

III. BITTER TASTE GENETICS As noted above, biology of bitter taste perception is poorly understood. Among bitter-tasting compounds are amino acids and peptides, esters and lactones, phenols and polyphenols, flavonoids and terpenes, methylxanthines (caffeine), sulfimides (saccharin), ureas and thioureas (PTC, PROP), and organic and inorganic salts (Rousseff, 1990). These structurally unrelated compounds all give rise to a uniform sensation of bitter taste. That would suggest that bitterness is perceived through a variety of receptors and multiple transduction mechanisms. At least three different types of bitter taste receptors were once thought to exist, sensitive to quinine, urea, and to PTC/PROP, respectively (McBurney, 1978). Since that time, the estimated number of bitter taste receptors has been revised upward. Studies have placed the number of gustducin-linked bitter taste receptors in humans at between 40 and 80 (Adler et al., 2000). According to recent studies (Adler et al., 2000), these candidate taste receptors (T2Rs) are organized in clusters and are genetically linked to loci that govern the perception of bitter taste. Candidate taste receptors were expressed in all taste buds of circumvallate, foliate, and palate papillae (Adler et al., 2000). A follow-up study linked different receptors to specific compounds. Human bitter taste receptor (hT2R-4) responded only to denatonium and PROP, while a mouse receptor MT2R-5 responded only to cycloheximide (Chandrashekar et al., 2000). The recognition of multiple bitter tastants may occur at the level of the individual cell. These studies are consistent with the notion that humans are capable of recognizing bitter substances, but not always of distinguishing among them. PTC tasting in humans was initially thought to follow a simple Mendelian model, with taste deficiency inherited through a single genetic locus as an autosomal recessive trait (Blakeslee 1931; Snyder, 1931). PTC tasters were thought to be homozygous (TT) or heterozygous (Tt or tT) for the dominant allele (T), while nontasters were thought to carry two recessive alleles (tt) (Fischer, 1967; Kalmus, 1958, 1971). However, linkage studies later showed that the traditional single locus two-allele model of PTC sensitivity did not provide the best fit to the data, and there have been suggestions of multilocus and multi-

IV.

TASTE DETECTION THRESHOLDS

In early studies, taste responses to PTC crystals or PTCimpregnated filter papers served to establish genetic taste “blindness” (Fox, 1932; Snyder, 1931). PTC filter papers were distributed by the American Genetic Association as early as 1931. Later studies used PTC solutions or solutions of 6-n-propylthiouracil or PROP (Blakeslee and Salmon, 1935; Fischer and Griffin, 1964; Harris and Kalmus, 1949). PROP lacks the faint sulfur smell of concentrated PTC solutions and is reportedly safer and easier to use (Kalmus, 1971; Lawless, 1980). Traditionally, tasters and nontasters of PTC or PROP were identified using threshold detection procedures (Harris and Kalmus, 1949). In contrast to most other substances, detection thresholds for PTC and PROP show a bimodal distribution, allowing for an easy segregation by taster groups (Fischer, 1967; Fischer and Griffin, 1964; Kalmus, 1971). The classic studies used a series of 15 PROP solutions that ranged in concentration from 0.001 to 3.2 mmol/L PROP, increasing in quarter-log steps (Bartoshuk, 1979; Fischer, 1984; Harris and Kalmus, 1949). The most concentrated solution (number 15 in the PROP series) contained 0.54 g/L PROP, the next solution contained 0.30 g/L, and so on (Fischer, 1967; Kalmus, 1971). Each subject was first presented with the least concentrated solution of PROP (solution 1) and then with increasingly higher solutions until he or she reported detecting a taste distinct from that of water. The subject was then presented with two identical cups, one containing the detected concentration of PROP and the other containing deionized water. The water was at the same temperature and was stored in the same location as the PROP solution. The subject was asked to judge which of the two samples had the bitter taste (Bartoshuk et al., 1988; Drewnowski et al., 1997a,b,c; Fischer and Griffin, 1964). Wrong answers led to the presentation of the more concentrated PROP solution, while correct answers led to a second presentation of the same solution. Two consecutive correct answers at the same concentration led to the presentation of a less concentrated PROP solution. Reversal points were defined as the concentration at which

Genetics of Human Taste Perception

a series of correct responses turned to an incorrect response or vice versa (Drewnowski et al., 1997a,b,c). Generally, thresholds were based on a mean of at least five reversals. Participants rinsed thoroughly with deionized water after tasting each PROP stimulus. PROP thresholds for a total of 541 women, ranging in age from 18 to 80 years, are shown in Figure 1. Consistent with past studies, the data show an antimode around 0.1 mmol/L PROP. PROP tasters are commonly defined as having thresholds below 0.1 mmol/L (solution 9), while nontasters are defined as having thresholds in excess of 0.2 mmol/L (equivalent to solution 10). Bartoshuk (1979, 1993). Respondents with PROP threshold between solutions 9 and 10 are often rejected as unclassifiable, since the taster and nontaster distributions are said to overlap in that range (Bartoshuk et al., 1994). Since 1949 (Harris and Kalmus, 1949), taste detection thresholds have come to dominate laboratory research on PTC/PROP. Some studies explored links between PROP taster status and taste perception of other bitter or sweet compounds (Bartoshuk, 1980). Other studies explored how the proportion of tasters and nontasters varied with sex, age, ethnicity, maturation, or disease status (Bhalla, 1972; Fischer and Griffin, 1964; Parr, 1934; Whissell-Buechy and Wills, 1989). In virtually all cases, the distinction between tasters and nontasters was treated as a dichotomous variable. This led to questions whether the distribution of PTC/PROP thresholds was truly bimodal or whether the data could support more than two subgroups. The broad range of PTC thresholds within the taster group suggested that the ability to taste PTC was continuously distributed.

Figure 1 Distribution of PROP detection thresholds for 541 women.

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(Kalmus, 1958). Furthermore, tasters who had taster siblings had lower PTC thresholds than tasters with at least one nontaster sibling, suggesting that PTC tasting was an incomplete dominant (Kalmus, 1958). A two-component model would support a single locus dominant model of inheritance, whereas a three-component model would support a single locus additive model of inheritance. Reed et al. (1995) tested these hypotheses by analyzing PROP taste thresholds collected from 1015 unrelated subjects, using maximum likelihood estimation and likelihood ratio tests. The results were inconclusive. Assuming equal variances, a three-component model was more likely, but the two models were equally likely if the variances could be unequal. However, unequal variances lead to statistical problems, since maximum likelihood is unbound and the likelihood ratio test is no longer appropriate. Applying a Bayesian Finite Mixture Linear Model to PROP thresholds for a sample of 359 adult women, including 180 breast cancer cases, we found that threshold distribution, adjusted for age and cancer status, was consistent only with the two-component model. No three-component model fit the data. In other words, thresholds can distinguish between tasters and nontasters, but do not support a multicomponent model. In particular, PROP thresholds cannot be used to determine whether individuals are homozygous or heterozygous for the dominant allele (Bartoshuk et al., 1994).

V.

THE FILTER PAPER METHOD

For the most part, threshold-based studies are limited to small groups of subjects tested in the laboratory. Studies conducted in the field, or with larger groups, have made use of the filter paper method. Those studies presented subjects with a piece of filter paper, which had been impregnated with a saturated solution of PTC or PROP (Parr, 1934). In early anthropological studies, responses to PTC or PROP filter paper were used as a measure of genetic inheritance. Those studies focused on the proportion of tasters and nontasters in different populations and ethnic groups (Das, 1958; Parr, 1934). Filter papers are prepared by soaking laboratory filter paper in a supersaturated solution of PROP, heated to close to boiling point (Bartoshuk, 1993, Kaminski et al., 2000). Some studies systematically varied the concentration of PTC solution to produce papers of different degree of bitterness. The paper was placed on the tongue and the subject was asked about its taste. In some cases, subjects were simply asked if the paper was bitter or not, in others they were asked to estimate the intensity of bitter taste.

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Blakeslee and Salmon (1931) were the first to note that women were more sensitive than men to the bitter taste of PTC filter paper, an effect later confirmed by Bartoshuk et al. (1994). The filter papers provide a crude but rapid way of assigning taster status. However, different recipes for producing PTC/PROP papers could lead to different classifications for the same individual. The time of contact with the tongue and the achieved degree of hydration could also make a difference. In most studies, subjects were simply instructed to place the paper flat on the tongue. In other studies, subjects who tasted nothing were asked to chew the filter paper, a practice that might lead to false-positive responses (Lawless, 1980). Pooled group data collected from attendees in the course of different lectures and presentations have also found their way into the literature (Bartoshuk et al., 1994). These practices have produced a diversity of results. The filter paper method has been criticized both for producing false negatives among tasters and for producing false positives among nontasters (Lawless, 1980). Its agreement with detection threshold data was reported as being only moderate (Lawless, 1980). Even so, the filter paper method has been the screening procedure of choice in many field studies. Under optimal conditions, the testretest reliability of PROP filter papers from the same batch, tasted following uniform instructions can be very high. In our studies, subjects were instructed that the paper should remain in the mouth for a minimum of 3 seconds (Ly and Drewnowski, 2000). Using three consecutive tests some days apart, we obtained reliability coefficients of 0.90 (Ly and Drewnowski, 2000).

Drewnowski

The correlation between PROP detection thresholds and bitterness intensity ratings was high, r
0.84. Intensity scaling of PROP solutions is a much simpler procedure that the traditional detection threshold method. However, threshold methods have come to dominate the field to such an extent that Lawless’s approach was not followed up until recently. In our studies, subjects tasted and rated 5 PROP solutions at concentrations of 0.032, 0.1, 0.32, 1.0, and 3.2 mmol/L PROP. Following Lawless’s (1980) approach, the solutions were arranged around the antimode. We used standard 9-point category scales, where 1
“not at all bitter” and 9
“extremely bitter.” Respondents also ranked each stimulus along a 9-point hedonic preference scale, initially developed by the U.S. Army Quartermaster Corps (Peryam and Pilgrim, 1957). That scale ranges from 1
“dislike extremely” to 9
“like extremely,” with a neutral point at 5 (“neither like nor dislike”). Allowing subjects to taste and rate several solutions across a perceptual range provides further information about their taste response profile. Figure 2 shows the distribution of summed intensity ratings for the five PROP solutions obtained with 541 women. The distribution was a mirror image of the distribution of thresholds, with a suggestion of bimodality. So far there are no studies as to whether the distribution of bitterness intensities supports a two- or a three-component model, since all of the existing genetic linkage studies have been conducted using threshold data (Olson et al., 1989; Reed et al., 1995; Whissell-Buechy and Wills, 1989). The distribution if hedonic ratings was found less useful in this study. Since nobody likes PROP solutions much, hedonic responses tended to occupy the bottom part of the scale, from 1 through 5. However, as shown in Figure 3,

VI. BITTERNESS INTENSITY SCALING OF SOLUTIONS Detection thresholds do not always predict taste experience at above-threshold levels. Sensory acuity for very dilute tastants provides a limited picture of everyday taste function, especially in relation to eating habits (Bartoshuk, 2000; Drewnowski, 2001). Looking for a middle ground between threshold procedures and the filter paper method, some researchers have used a single solution of PTC or PROP to determine taster status (Fischer and Griffin, 1961; Lawless, 1979). A near-antimode concentration was presented to subjects in the expectation that nontasters would find it tasteless, whereas tasters would find it very bitter. Lawless (1980) tested samples of 0.1 mmol/L PTC and 0.56 mmol/L PROP using an 8-point category scale that ranged from 0 (no taste) to 7 (very strong taste). There was good agreement among threshold data and category-scale ratings of single stimuli.

Figure 2 Distribution of PROP bitterness ratings, summed over 5 solutions, for 541 women.

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Figure 4 Bitterness intensity ratings as a function of PROP concentrations. The data are for 541 women, classified into quintiles by PROP detection thresholds. Highest threshold quintile equals lowest responsiveness. Figure 3 Distribution of PROP hedonic ratings, summed over 5 solutions, for 541 women.

some respondents gave neutral ratings to PROP solutions. Those respondents were typically nontasters. As might be inferred from the two figures, greater perceived bitterness was linked to a progressively greater dislike of PROP at an individual level. Summed bitterness and summed hedonic ratings for the 5 PROP solutions were strongly and inversely linked (r
0.80; p  0.01). This relationship held for both tasters and nontasters. Assessing the dislike for PROP, as opposed to bitterness intensity scaling, may be a viable way of determining PROP taster status among children (Drewnowski et al., 1997). VII. INTENSITY SCALING AND PROP TASTER STATUS The use of bitterness intensity scaling of multiple PROP solutions, instead of the traditional detection threshold procedure, is a relatively novel approach to the determination of PROP taster status (Ly and Drewnowski, 2000). The data of the study under discussion are based on 541 women for whom both threshold data and intensity ratings were available. Initially, we rank-ordered the threshold data into 5 equal-size groups (quintiles). Mean bitterness intensity profiles were then established for each group as a function of PROP concentration, as shown in Figure 4. PROPinsensitive women or nontasters (top 20% of thresholds) showed a profile that was different from the other four groups. For PROP tasters, thresholds were a poor predictor of bitterness intensity, consistent with past observations of Bartoshuk et al. (1994). Threshold data allowed for a clear segregation of tasters and nontasters but did not

distinguish well between medium and high responders to PROP. The same respondents were then sorted by quintiles of summed bitterness ratings for the 5 solutions of PROP. Mean bitterness intensity functions for each group as a function of PROP concentration are shown in Figure 5. Whereas a better separation between the curves was only to be expected, the shape of the response profiles was very different. Respondents in the bottom quintile by bitterness ratings showed a flat curve that only left the floor at 1 mmol/L PROP (solution 13). That is consistent with the notion that respondents in that group were nontasters, with a detection threshold above 0.2 mmol/L PROP. In contrast, respondents in the top quintile perceived even the 0.03 mmol/L PROP solutions as intensely bitter, consistent with the notion that they had lower thresholds and elevated

Figure 5 Bitterness intensity ratings as a function of PROP concentrations. The data are for 541 women, classified into quintiles by PROP bitterness ratings, summed over 5 solutions. Highest intensity quintile equals highest responsiveness. Mean thresholds for each group are also indicated.

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sensitivity to PROP. Those respondents also reached the ceiling of the scale much sooner than the nontaster group. Technically, the elbow of the curve should correspond roughly to the mean detection threshold for that subject group. Figure 5 also shows the relationship between bitterness response profiles and the mean detection thresholds for each group. Whereas mean threshold for the bottom quintile (nontasters) was above solution 10 (0.2 mmol/L), consistent with the traditional definition of nontasters, the mean threshold for the top quintile was around solution 5 (0.01 mmol/L PROP), in the middle of the taster range. Evidence was present for both floor and ceiling effects, as shown in Figure 5. Bartoshuk (2000) has argued that rating single stimuli along a 9-point scale does not allow for a good separation among different categories of tasters. Indeed, in our study bitterness ratings of the most concentrated 3.2 mmol/L solution of PROP clustered toward the top of the scale and did not distinguish between medium and high responders. Conversely, ratings of the most dilute 0.03 mmol/L solution did not discriminate between low and medium responders. On the other hand, there were no ceiling effects when subjects used the same 9-point scales to rate PROP solutions closer to the antimode, 0.1–0.3 mmol/L PROP. That is, of course, the reason why previous researchers used concentrations around the antimode to distinguish between tasters and nontasters of PROP.

Drewnowski

intensity. Respondents were therefore ranked according to the ratio of bitterness intensity ratings for two PROP solutions (1.0 and 3.2 mmol/L) relative to the perceived saltiness of two solutions of sodium chloride (0.32 and 1 mol/L). Respondents in the top 25% of the values were identified as supertasters. In other words, the cutpoint was wholly arbitrary and was simply devised to produce 25% supertasters. Respondents whose data did not fit that particular model were eliminated as unclassifiable. Figure 6 (Bartoshuk et al., 1994) shows a scatterplot of log PROP threshold versus PROP/NaCl ratio for 269 subjects. First, respondents with thresholds between 0.1 and 0.2 mmol/L PROP were eliminated since the taster and nontaster distributions overlap in this range. Second, respondents with high PROP/NaCl ratios and low thresholds were eliminated, as were respondents with low thresholds and high ratios. Third, the arbitrary cutoff point was used to separate medium tasters from supertasters. In reducing the total number of classifiable subjects to 220, these procedures yielded 16% nontasters, 56% medium tasters, and 28% supertasters. Other studies on PROP supertasters adopted the procedures of Bartoshuk et al. (1994) with some modifications. Rather than using magnitude estimation and the two highest concentrations of PROP and NaCl, we used category scales and all five solutions of PROP and NaCl. PROP solutions had concentrations as described above. Sodium chloride solutions had concentrations of 0.01,

VIII. TASTERS AND “SUPERTASTERS” OF PROP Being able to separate medium tasters from intense tasters of PROP has some important implications. The wide variability of PTC detection thresholds among PTC tasters suggested to Kalmus (1958) and later to Bartoshuk (1993) that tasters could be subdivided into two groups. Bartoshuk et al. (1994) speculated that nontasters had two recessive alleles (tt); medium tasters were heterozygotes with one dominant allele (Tt); while the most sensitive “supertasters” had two dominant alleles (TT). The proportion of “supertasters” expected on the basis of this singlelocus, two-allele model was 25%. Since supertasters could not be identified using threshold procedures, Bartoshuk used the ratio of perceived bitterness of PROP to the perceived saltiness of NaCl to distinguish between the two groups. Studies had shown that 3.2 mmol/L PROP was much more bitter to intense PROP tasters than 1.0 mol/L NaCl was salty. Medium tasters found the two stimuli to be of comparable

Figure 6 A plot of mean PROP/NaCl ratios against PROP detection thresholds. The figure illustrates the classification of nontasters, tasters, and supertasters of PROP using a combination of detection threshold and intensity scaling procedures. (Reprinted from Physiol. Behav. 56:1165–1171, 1994 with permission from Elsevier Science.)

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hedonic response profiles were mirror images of each other. The question is whether a similar distribution can be obtained using bitterness intensity ratings for 3 solutions, as opposed to 5. Using the three middle solutions only (0.01, 0.3, and 1.0 mmol/L), we obtained essentially the same classification by PROP taster status. Mean thresholds associated with each distribution are shown in Table 1. It would appear that bitterness intensity scaling of three PROP solutions around the antimode can provide an acceptable screening tool for PROP taster status. Figure 7 Bitterness intensity (left panel) and hedonic ratings (right panel) as a function of PROP concentration for 541 women. Respondents are classed by summed bitterness intensity ratings for 5 solutions of PROP: bottom 25% (nontasters), 25–75% (tasters), and top 25% (supertasters).

0.032, 0.1, 0.32, and 1.0 mol/L (Drewnowski et al., 1997c, 1998). The PROP/NaCl ratio was based on the mean of five solutions of PROP and NaCl to salt. In one study with young women, such procedures yielded 33% nontasters, 41% medium tasters, and 26% supertasters, although the exact percentages varied somewhat from one study to another. However, there may be no need to use NaCl solutions at all. Mean PROP/NaCl ratios and summed PROP intensity ratings were highly correlated (r
0.72). Using summed bitterness intensity ratings, we have assigned respondents to three taster groups by using a distribution based on four equal groups or quartiles. Respondents in the bottom 25% were classified as nontasters, respondents in the 25–75% group were medium tasters, and respondents in the top 25% were supertasters. Bitterness intensity and hedonic response profiles obtained using this classification are shown in Figure 7. As expected, bitterness intensity and Table 1 Percentage Distribution of Respondents (n
541) by Summed Bitterness Intensity Ratings of 5 Versus 3 PROP Solutions Summed bitterness intensity for 3 solutions Summed bitterness intensity for 5 solutions Bottom 25% 25–75% Top 25% Mean threshold (solution #)

Bottom 25%

25–75%

94 5

6 89 10 7.1

9.7

Top 25%

5 90 5.5

Mean threshold (solution #) 9.9 7.1 5.6

IX.

SCALING METHODOLOGIES

There has been some disagreement as to which method for intensity scaling best captures different taste responses to PROP. Among the more popular choices are those between 9-point category scales, magnitude estimation, and labeled magnitude estimation scales (Drewnowski et al. 1997, 1998; Green et al., 1993, 1996; Marks et al., 1991) (see also Chapters 10 and 37). Some researchers have argued that category scale anchors, such as “very bitter,” may mean different things to PROP tasters than to nontasters, given that the experience of bitterness can be so different between the two groups (Bartoshuk, 2000; Prutkin et al., 2000). The anchored 9-point category scale remains the conventional approach in laboratory studies, sensory evaluation tests, and in much of consumer research (Drewnowski, 1997a,b,c; Meiselman et al., 1974). Such scales are anchored at either end with adjective ratings, such as “not at all bitter” and “very” or “extremely bitter.” In early studies (Kalmus 1958), subjects who indicated that a PTC filter paper was “very” or “extremely” bitter were classified as tasters, whereas those who rated the paper as “not at all” or “slightly” bitter were classified as nontasters. Later on, respondents were divided into three taster groups: those who rated bitterness intensity as 1 or 2, those who rated it between 3 and 7, and those who rated it 8 or 9 (Bartoshuk et al., 1994; Drewnowski, 2001). That classification corresponds almost exactly to the 25:50:25 percentile split described above. The second approach, magnitude estimation, relies on ratio scaling. At the beginning of the session, each subject tastes a standard stimulus and is asked to give it a specific number, say 15. Perceived intensities of subsequent stimuli are then given numbers such that a stimulus that is five times as intense is given a number that is five times as large. In contrast, a stimulus that is half as intense is given a number that is one-half the standard, and so on. Since one subject’s responses cannot be directly compared to another’s, a normalization procedure serves to bring all

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responses into a common range. Magnitude estimation procedures were developed at Harvard and have been tested with college populations (Stevens, 1969) (see Chapter 10). However, the general public does not have the mental arithmetic skills of college students, and asking people to divide by two or multiply by five can be a problem. Neither classical magnitude estimation nor its refinement, cross modality-matching (Marks et al., 1988; Stevens and Marks, 1965), is in common use today, least of all in community-based studies. In cross-modality matching, respondents were asked to match sensory intensity of different tastants with the brightness of lights or the loudness of tones (Marks et al., 1988). The most recent alternative, the labeled magnitude estimation (LME) scale (Green et al., 1993, 1996) combines features of adjective based and ratio scales. Adjective labels are spaced such that the scale has ratio properties. Instead of being labeled “very” or “extremely” bitter, the top of the scale is labeled “strongest bitter sensation imaginable.” Effectively, the LME or the “Green” scale allows for more response options at the top of the range. However, whereas Green et al. (1993, 1996) initially used the words “strongest imaginable” to refer to taste sensations only, other researchers have modified the instructions to refer to the strongest sensation of any kind (Prutkin et al., 2000). In those studies, moreover, the LME scale was applied to pleasure or hedonic ratings, with mixed results. One cannot help but wonder about those study participants who rated ordinary grapefruit juice as providing them with “the strongest imaginable pleasant sensation of any kind” (Prutkin et al., 2000). Category scales may suffer from ceiling effects when used to scale intensely bitter PROP solutions. In some studies, the LME scale was said to allow for a better segregation of respondents rating a single concentrated solution of a given compound. However, studies pitting the LME scale against 9-point category scales always used intense solutions of 3.2 mmol/L PROP, 1.0 mol/L sucrose, or 1.0 mol/L sodium chloride (Bartoshuk, 2000; Prutkin et al., 2000). Such strong stimuli are rated in the upper part of the conventional 9-point scale such that ceiling effects may indeed obscure potential differences in taste perception among respondents. As shown in Figure 6, bitterness intensity ratings of solution 15 (3.2 mmol/L PROP) did not discriminate between medium and high tasters of PROP. Bartoshuk has taken the position that new psychometric scales need to be developed to deal with the perceptions of intense solutions (Bartoshuk, 2000). An alternative approach might be to keep the existing scales and dilute the solutions by a factor of 10. While the LME scale provides room at the top of the perceptual range, our experience suggests that the response options at the bottom of

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the range are more limited. The LME scale is most useful in helping to reduce ceiling effects when rating strong solutions; however, that advantage is traded in for floor effects when the solutions are weak. As documented above, weak solutions show no ceiling effects, even with 9-point category scales. It has also been argued that the use of category scales might even lead to paradoxical effects, whereby PROP tasters adjust their ratings downward and rate PROP solutions as less bitter than do nontasters (Bartoshuk, 2000; Prutkin et al., 2000). However, there is real danger in arguing that psychophysical events are observable only with certain variants of perceptual scales. If a phenomenon is sufficiently robust, it ought to be observed with magnitude estimation, labeled magnitude estimation, and category scales. Indeed, there is extensive literature showing that the three scales provide equivalent results.

X.

PROP TASTING AND TONGUE ANATOMY

PROP taster status appears to be associated with differential innervation of the tongue. There are anatomical differences in tongue papillae between tasters and nontasters of PROP (Miller and Reedy, 1990; Reedy et al., 1993). Blue food dye can be used to stain taste pores and the surface of the tongue, leaving fungiform papillae to appear as pink dots against a blue background. Counts of fungiform papillae generally reveal that tasters have more papillae, on average, than do nontasters; supertasters tend to have on average, the most, papillae of all. Furthermore, women tend to have more taste pores than do men. Nonetheless, not every supertaster has more papillae than every medium taster or nontaster, and not every woman has more papillae than every man (Bartoshuk et al., 1994). Given that fungiform papillae are innervated both by the chorda tympani and the trigeminal nerve, Bartoshuk (1993) made the connection between bitter taste sensitivity and pain sensation. Trigeminal neurons surround each taste bud and terminate in the fungiform papillae. Their location together with the presence of substance P suggests that many may mediate pain sensations (Whitehead et al. 1994). Studies soon showed that supertasters perceived greater irritation from ethanol and a greater oral burn with piperine (black pepper) and capsaicin (chili peppers) (Karrer et al., 1995). Further studies have explored whether the number of fungiform papillae influence the intensity of all taste sensations. If so, then persons with most papillae would be expected to give the highest ratings not only for PROP, but also for other bitter tastes, as well as for sour, salty, and sweet tastes. However, as will be shown below, the ability

Genetics of Human Taste Perception

to taste PROP has translated into a heightened ability to taste some, but not all, other compounds. Whether this is only a scaling issue or a broader issue in human taste genetics is a good question.

XI. PROP TASTER STATUS BY ETHNICITY, SEX, AND AGE PTC taste blindness served initially as a marker of genetic inheritance. A number of studies explored the frequency of the nontaster gene in different populations and different cultures (Allison and Blumberg, 1959; Bhalla, 1972). Generally, Europeans and North Americans were found to have the highest proportions of PTC nontasters (26–30%), while the nontasting gene was found to be less common among Asians (6–10%), Africans, and Native Americans. Data from early studies based on PTC/PROP filter papers have been confirmed recently in a number of studies (Drewnowski, 2001; Guo et al., 1998). It would appear that the highest proportion of nontasters is found among Caucasians. There are some interesting suggestions that PROP sensitivity may be somehow related to endocrine factors, including associations with adolescent growth and development. According to one report (Whissell-Buechy and Wills, 1989), PTC-taster girls reached maturation some 3.8 months earlier than did nontasters, suggesting that the PTC polymorphism may involve the hypothalamic-pituitarygonadal axis and the reproductive endocrine pathways. There are also anecdotal observations that PTC/PROP

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thresholds were reduced during the first trimester of pregnancy (Fischer, 1967). Another set of studies reported an unexpectedly high proportion of PTC tasters among women with hormone-dependent cancers, including cancers of the breast and uterus (Milunicova, 1969). There are some suggestions that the heightened perception of bitterness, modulated by hormonal events, may serve to protect the mother and child from bitter toxins. PROP sensitivity appears to decline with age (Fischer, 1984). The observed decline seems to be moderate for PROP detection thresholds (Whissell-Buechy and Wills, 1989), but more marked for bitterness ratings of PROP filter papers. A recent study (Drewnowski, 2001) found (Fig. 8) that the percentage of low responders increased as a function of age, whereas the percentage of high responders declined. Similar effects were observed for both women and men. Age-associated decline in taste responsiveness is often modality specific, and other studies have found that bitter taste responsiveness is affected the most. Whether the decline in taste response to PROP is gradual or whether it drops sharply following menopause (in women) is an unresolved question. One persistent notion has been that PTC/PROP tasters eat less food and are therefore thinner than nontasters (Bartoshuk, 2000; Prutkin et al., 2000). PROP tasting has even been said to protect against obesity. Unfortunately, the evidence for this notion is weak, given that studies reporting this phenomenon have employed only a few subjects and are published only in abstract form (e.g. Duffy et al., 1999; Tepper and Ullrich, 1999). It should be

Figure 8 Distribution of low, medium, and high PROP responders within each age category. The data are for 364 men (left panel) and 378 women (right panel). (From Drewnowski et al., 2001.)

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remembered that the body mass index (BMI) is a transform of body weight that is moderately correlated with body fat (r
0.70), and that BMI values vary with age, sex, and ethnicity, as well as with a wide range of sociodemographic variables, including education and income. Proper epidemiological procedures require that any associations with BMI be properly controlled for known covariates, preferably using multiple regression analysis. Two published studies, one conducted with college-age women and another with a clinical sample of female adults, controlled for such confounds and did not find any meaningful relationship between PROP tasting and BMI (Drewnowski et al., 1998, 1999). In those studies, taste responsiveness to PROP was treated as a continuous or a categorical variable (taster status) and the analyses controlled for known covariates. Another study, conducted with 364 adult men and 378 adult women, found no relationship between BMI and PROP taster status, determined using the filter paper procedure (see Fig. 9). Again, regression methods were used to control for known covariates (Drewnowski et al., 2001).

XII.

PROP AND OTHER BITTER TASTES

How PROP tasting is related to the perception of other bitter tastes remains a matter of debate. As noted earlier, genetic studies based on PTC taste data for 1152 individuals from 120 families obtained the best fit with a two-locus model, in

Figure 9 Absence of a relationship between PROP responsiveness (a composite measure of intensity and hedonic ratings) and body mass index values (BMI
kg/m2). (From Drewnowski et al., 2001.)

which one locus controlled PTC taste perception and the other locus controlled a more general taste acuity (Olson et al., 1989). Indeed, some taste studies showed that PTC nontasters may also be divisible into two groups (Frank and Korchmar, 1985). One group was characterized by a specific PTC taste deficit, while the other showed a low sensitivity to a wide range of taste stimuli (Frank and Korchmar, 1985). Inherited taste blindness was initially thought to involve only those bitter compounds that carried the -N-C
S group (Kalmus, 1971). However, later studies showed that other chemical structures could also be involved, including compounds found in everyday foods (Blakeslee and Salmon, 1935; Fischer et al. 1963). It had been noted early on that PTC/PROP tasters rated caffeine and quinine solutions, though not urea, as more bitter (Fischer, 1971; Hall et al., 1975). The level of caffeine found in a cup of brewed coffee (0.004 M) was below threshold for most nontasters but above threshold for most tasters (Hall et al, 1975). A time-intensity study (Neely and Borg, 1999) confirmed that PROP tasters gave higher aftertaste intensity ratings to a low concentration of caffeine (0.018 mol/L) than did nontasters. Most recently, Ly and Drewnowski (2000) showed that PROP tasters rated caffeine as more intensely bitter than did nontasters. Tasters of PTC/PROP have also been shown to perceive sodium benzoate and potassium chloride as more bitter than do nontasters (Bartoshuk et al., 1988). Sodium benzoate is a widely used preservative, while potassium chloride is the most frequently used salt substitute for people consuming low-sodium diets. At levels close to those used in soft drinks (0.0015 mol/L), saccharine has also been found to taste more bitter to tasters than to nontasters (Bartoshuk, 1979). One potential implication of such findings is that PTC/PROP tasters should avoid coffee and saccharin-sweetened soft drinks (Bartoshuk, 1993). Later reports that PROP tasting was associated with enhanced oral burn of capsaicin, the active ingredient of hot peppers, led to the suggestion that PROP tasters might also avoid hot and spicy foods (Karrer and Bartoshuk, 1995). PROP tasters were also said to be more sensitive to the trigeminal irritation by ethanol, and reportedly avoided alcoholic beverages as well (diCarlo and Powers, 1998; Pelchat, 1992). In a series of studies, we examined taster responses to bitter phytochemicals as a function of PROP taster status (Akella et al., 1998; Drewnowski et al., 1997, 1998; Kaininsky et al., 1999). All studies were conducted with women. One study examined perceptions and preferences for solutions of naringin, a bioactive flavonoid that is the principal bitter component of grapefruit juice (Drewnowski et al., 1997b). Naringin does not occur in orange juice. Though tasters and supertasters of PROP

Genetics of Human Taste Perception

rated 5 naringin solutions in 4% sucrose as more bitter than did nontasters, this effect failed to reach significance. However, an analysis of hedonic ratings showed that PROP supertasters disliked naringin more than did medium tasters and nontasters. In another study (Akella et al., 1998), the ability to taste PROP was linked to lower acceptance of bitter Japanese green tea. Green tea contains bitter polyphenols catechin and epicatechin (Drewnowski and Gomez-Carneros, 2000). In addition, PROP tasters were more likely to dislike soy foods, including miso and tofu (Akella et al., 1998). Soy products, especially fermented ones, contain bitter isoflavones, genistein, and dadzein (Rouseff, 1990). Although PROP tasters appear to be more sensitive to some bitter phytochemicals found in vegetables and fruit, the effect was by no means universal and the observed effects on eating habits were small. Some past studies have linked PTC tasting with a series of food aversions (Fischer et al., 1961; Glanville and Kaplan, 1965). Data showing that PTC tasters found caffeine more bitter were taken as evidence that PTC tasters would avoid coffee and caffeinated beverages. However, perceived bitterness is effectively reduced by the addition of fat, sugar, or salt (Breslin and Beauchamp, 1995; Ly and Drewnowski, 2000). A recent study confirmed that PROP tasters did indeed rate caffeine solutions as more bitter than did nontasters. However, the differences between the two groups were sharply reduced when caffeine solutions were sweetened with neohesperidin dihydrochalcone, an intense sweetener. There was no longer a differences in hedonic ratings for caffeine between the two groups (Ly and Drewnowski, 2000). Rather than avoid bitter foods altogether, PROP tasters may seek to reduce bitter taste by the addition of fat, sugar, or salt.

XIII.

BITTER, SWEET, AND FAT

The question whether PROP tasters are also more sensitive to sweetness and fat is another controversial issue. Early studies (Gent and Bartoshuk, 1983) showed that low concentrations of sucrose, saccharin, and neohesperidin dihydrochalcone (an intense sweetener) tasted more intensely sweet to PROP tasters than to nontasters. However, the effects were minuscule. PROP tasters were also reported to dislike sweet sucrose solutions (Looy and Weingarten, 1995) and the oral sensation of dairy fat. These data were taken to mean that PROP tasters would therefore avoid sweet and fat-rich foods. However, the observations linking PROP to enhanced perception of sucrose, saccharin, and neohesperidin DC were not replicated (Drewnowski et al., 1997, 1998; Ly

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and Drewnowski, 2000). There was no effect of PROP taster status on intensity or preference ratings for sucrose solutions (Drewnowski et al., 1997). There was no effect of PROP taster status on sensory ratings or preferences for sweetened dairy products with different concentrations of sugar (Drewnowski et al., 1998). Furthermore, there was no effect of PROP taster status on self-reported preferences for sweet foods (Drewnowski et al., 1998). PROP tasting and sweet taste preferences were unrelated even if response to PROP was treated as a continuous as opposed to a categorical variable. There have also been suggestions that supertasters perceive food textures differently. Duffy et al. (1996, 1999) reported that PROP supertasters are highly sensitive not only to all four tastes and to trigeminal sensation, but also to the mouthfeel and texture of fats. One study reported that supertasters gave higher “fatness” ratings to a single stimulus, a high-fat salad dressing (Tepper and Nurse, 1997). An abstract, based on ratings for 5 stimuli, reported that supertasters gave higher ratings to 2 milk products with the highest fat content (Duffy et al., 1996). Using 15 different taste stimuli of varying sugar and fat content, we found no differences in sweetness, creaminess, or overall acceptability ratings by PROP taster status in a sample of 118 young women (Drewnowski et al. 1998). Hence, there is no convincing evidence at this point that PROP tasters dislike high-fat foods.

XIV. GENETIC TASTE MARKERS AND FOOD CHOICES While many factors, including taste, contribute to food acceptance, unpleasant taste is the chief reason for food avoidance (Drewnowski and Gomez-Carneros, 2000). Humans, conditioned through evolution to be wary of bitter plant-derived alkaloids and other toxins, find excessive bitterness objectionable. Bitterness is the most commonly cited reason for the dislike for a particular food and often leads to food rejection (Drewnowski et al., 1997a,b; Rouseff, 1990). The very existence of PTC-tasting genes in humans may be linked to the genetic advantage of being able to detect and avoid bitter poisons (Boyd, 1950). The reported avoidance by PTC tasters of bitter antithyroid compounds found in raw cruciferous vegetables, broccoli, cabbage, and Brussels sprouts is the most often cited example in this area (Boyd, 1950; Niewind et al., 1990). Goitrin (1,5-vinyl-2-thiooxazolidone) and isothiocyanates are bitter PTC-related compounds caused by hydrolysis of glucosinates naturally present in raw cabbage (Fenwick et al., 1983). However, their antithyroid activity is no longer the main research focus. Some of

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these compounds have been shown to have antioxidant and anticarcinogenic effects and a wide spectrum of tumor-blocking properties (Steinmetz and Potter, 1995). Their effectiveness in cancer prevention is the focus of intense study (Steinmetz and Potter, 1995). Antioxidant phytochemicals in vegetables and fruit also include phenols and polyphenols, flavonoids, isoflavones, and glucosinolates. Phenolic compounds are directly responsible for the bitterness and astringency of many foods and beverages, from vegetables, fruits and legumes to tea, cocoa, coffee, and wine (Rouseff, 1990). Most of the bioactive phytochemicals are bitter, acrid or astringent, and unacceptable to the consumer (Drewnowski and Gomez, 2000). PROP tasters may be affected the most. In early studies, based on food preference checklists, PTC/PROP tasters tended to dislike cruciferous and green vegetables, rhubarb, sauerkraut, beer, coffee, and various sharp cheeses (Boyd, 1950; Fischer and Griffin, 1964; Fischer et al., 1961; Forrai and Bankovi, 1984; Glanville and Kaplan, 1965). However, other studies have failed to link PTC or PROP sensitivity with a consistent pattern of food dislikes (Anliker et al., 1991; Mattes and Labov, 1989). Our studies (Drewnowski et al., 1999, 2000) have confirmed earlier reports that genetically mediated sensitivity to the bitter taste of PROP was associated with lowered acceptability of Brussels sprouts, cabbage, spinach, and coffee (Drewnowski et al. 2000). In turn, self-reported food preferences were associated with dietary outcome variables. Reduced acceptability of vegetables and fruit was associated with lower intakes of carbohydrate, fiber, and -carotene, as estimated from 3-day food records. These data suggest that genetic responsiveness to PROP may alter food choices and affect eating habits. Women who are tasters of PROP may be less likely to comply with dietary strategies that emphasize increased consumption of bitter-tasting cruciferous vegetables and salad greens. Alternatively, PROP-sensitive women may seek to mask bitter taste by the addition of fat, sugar, or salt (Drewnowski et al, 2000). Genetic taste factors may influence food preferences and may have an impact on the selection of healthful diets. ACKNOWLEDGMENT Supported by National Cancer Institute grant CA 61680. REFERENCES Adler, E., Hoon, M. A., Mueller, K. L., Chandrashekar, J., Ryba, N. J. P., and Zuker, C. S. (2000). A novel family of mammalian taste receptors. Cell 100:693–702.

Drewnowski Akella, G. D., Henderson, S. A., and Drewnowski A. (1998). Sensory acceptance of Japanese green tea and soy products is linked to genetic sensitivity to 6-n-propylthiouracil. Nutr. Cancer 29:146–151. Allison, A. C., and Blumberg, B. S. (1959). Ability to taste phenylthiocarbamide among American Eskimos and other populations. Hum. Biol. 31:352–359. Anliker, J. A., Bartoshuk, L., Ferris, A. M., and Hooks, L. D. (1991). Children’s food preferences and genetic sensitivity to the bitter taste of 6-n-propylthiouracil (PROP). Am. J. Clin. Nutr. 54:316–320. Bartoshuk, L. M. (1979). Bitter taste of saccharin related to the genetic ability to taste the bitter substance 6-n-propylthiouracil. Science 205:934–935. Bartoshuk, L. M. (1980). Separate worlds of taste. Psychol. Today 14:48–63. Bartoshuk, L. M. (1993). The biological basis of food perception and acceptance. Food Qual. Pref. 4:21–32. Bartoshuk, L. M. (2000). Comparing sensory experiences across individuals: recent psychophysical advances illuminate genetic variation in taste perception. Chem. Senses 25:447–460. Bartoshuk, L. M., Rifkin, B., Marks, L. E., et al. (1988). Bitterness of KCl and benzoate: related to genetic status for sensitivity to PTC/PROP. Chem. Senses 13:517–528. Bartoshuk, L. M., Duffy, V. B., and Miller, I. J. (1994) PTC/PROP tasting: Anatomy, psychophysics, and sex effects. Physiol. Behav. 56:1165–1171. Bhalla, V. (1972). Variations in taste threshold for PTC in populations of Tibet and Ladakh. Hum. Hered. 22:453–458. Blakeslee, A. F. (1931). Genetics of sensory thresholds: taste for phenylthiocarbamide. Proc. Natl. Acad. Sci. 18:120–130. Blakeslee, A. F., and Salmon, M. R. (1935). Genetics of sensory thresholds: individual taste reactions for different substances. Proc. Natl. Acad. Sci. 21:84–90. Boyd, W. C. (1950). Taste reactions to antithyroid substances. Science 112:153. Breslin, P. A., and Beauchamp, G. K. (1995). Suppression of bitterness by sodium: variation among bitter taste stimuli. Chem. Senses 20:609–23. Chandrashekar, J., Mueller, K. L., Hoon, M. A., Adle,r E., Feng, L., Guo, W., Zuker, C. S., Ryba, N. J. (2000). T2Rs function as bitter taste receptors. Cell 100:703–711. Das, S. R. (1958). Inheritance of the PTC taste character in man: An analysis of 126 Rarhi Brahmin families of West Bengal. Ann. Hum. Genet. 22:200–212. DiCarlo, S. T., Powers, A. S. (1998). Propylthiouracil tasting as a possible genetic association marker for two types of alcoholism. Physiol. Behav. 64:147–152. Drewnowski, A. (1997). Taste preferences and food intake. Ann. Rev. Nutr. 17:237–253. Drewnowski, A. (2001). The science and complexity of bitter taste. Nutr. Rev. 59:163–169. Drewnowski, A., and Gomez-Carneros, C. (2000). Bitter taste, phytonutrients and the consumer: A review. Am. J. Clin. Nutr. 72:1424–1435. Drewnowski, A., Henderson, S. A., and Shore, A. B. (1997a). Genetic sensitivity to 6-n-propylthiouracil (prop): and hedo-

Genetics of Human Taste Perception nic responses to bitter and sweet tastes. Chem. Senses 22:27–37. Drewnowski, A., Henderson, S. A., and Shore, A. B. (1997b). Taste responses to naringin, a flavonoid, and the acceptance of grapefruit juice are related to genetic sensitivity to 6-n-propylthiouracil (PROP). Am. J. Clin. Nutr. 66:391–397. Drewnowski, A., Henderson, S. A., Shore A. B., and BarrattFornell A. (1997c). Nontasters, tasters, and supertasters of 6n-propylthiouracil (PROP) and hedonic response to sweet. Physiol. Behav. 62:649–655. Drewnowski, A., Henderson, S. A., and Barratt-Fornell, A. (1998). Genetic sensitivity to 6-n-propylthiouracil and sensory responses to sugar and fat mixtures. Physiol. Behav. 63:771–777. Drewnowski, A., Henderson, S. A., Hann, C. S., Berg, W. A., and Ruffin M. T. (2000). Genetic taste markers and preferences for vegetables and frui of female breast care patients. Am. J. Dietet. Assoc. 100:191–197. Drewnowski, A., Kristal, A., and Cohen, J. (2001). Genetic taste responses to 6-n-propylthioracil among adults: a screening tool for epidemiological studies. Chem. Senses 26:41–47. Duffy, V. B., Bartoshuk, L. M., Lucchina, L. A., Snyder D. J., and Tym A. (1996). Supertasters of PROP (6-n-propylthiouracil) rate the highest creaminess to high-fat milk products (abstr). Chem. Senses 21:598. Duffy, V. B., Fast K., Cohen, Z., Chodos, E., and Bartoshuk, L. M. (1999). Genmetic taste status associates with fat food acceptance and body mass index in adults (abstr). Chem. Senses 24:545–546. Fenwick, G. R., Heaney, R. K., and Mulling, W. J. (1983). Glucosinolates and their breakdown products in food and food plants. CRC Crit. Rev. Food Sci. Nutr. 18:123–201. Fischer, R. (1967). Genetics and gustatory chemoreception in man and other primates, In The Chemical Senses and Nutrition, Kare, M., and Maller, O. (Eds.). Johns Hopkins University Press, Baltimore, pp. 61–81. Fischer, R., and Griffin, F. (1961). ‘Taste-blindness’ and variations in taste threshold in relation to thyroid metabolism. J. Neuropsychol. 3:98–104. Fischer, R., and Griffin, F. (1964) Pharmacogenetic aspects of gustation. Drug Res. 14:673–686. Fischer, R., Griffin F., England, S., and Garn, S. M. (1961). Taste thresholds and food dislikes. Nature 191:1328. Fischer, R., Griffin F., and Kaplan, A. R. (1984) Taste thresholds, cigarette smoking and food dislikes. Med. Exp. 9:151–167. Forrai, G., and Bankovi, G. (1984). Taste perception for phenylthiocarbamide and food choice—a Hungarian twin study. Acta Physiol. Hungar. 64:33–40. Fox, A. F. (1932). The relationship between chemical constitution and taste. Proc. Natl. Acad. Sci. USA 18:115–120. Frank, R. A., and Korchmar, D. L. (1985). Gustatory processing differences in PTC tasters and non-tasters: A reaction time analysis. Physiol. Behav. 35:239–242. Gent, J. F., and Bartoshuk, L. M. (1983). Sweetness of sucrose, neohesperidin dihydrochalcone, and saccharin is related to the genetic ability to taste the bitter substance 6-n-propylthiouracil. Chem. Senses 7:265–272.

859 Glanville, E. V., and Kaplan, A. R. (1965). Food preference and sensitivity of taste for bitter compounds. Nature 205:851–853. Green, B. G., Shaffer, G. S., and Gilmore, M. M. (1993). A semantically-labeled magnitude scale of oral sensation with apparent ratio properties. Chem. Senses 18:683–702. Green, B. G., Dalton, P., Cowart, B., Shaffer, G., Rankim, K., and Higgins, J. (1996). Evaluating the ‘Labeled magnitude Scale’ for measuring sensations of taste and smell. Chem. Senses 21:323–34. Guo, S. W., and Reed, D. R. (2001). The genetics of phenylthiocarbamide perception. Ann. Hum. Biol. 28:111–142. Guo, S. W., Shen F. M., Zheng, C. J., and Wang, Y. (1998). Threshold distributions of phenylthiocarbamide (PTC) in the Chinese population. Ann. N. Y. Acad. Sci. 855:810–812. Hall, M. J., Bartoshuk, L. M., Cain, W. S., and Stevens, J. C. (1975). PTC taste blindness and the taste of caffeine. Nature 253:442–443. Harris, H., and Kalmus, H. (1949). The measurement of taste sensitivity to phenylthiourea (PTC). Ann. Eugen. (Lond.) 15:24–31. Hladik, C. M., and Simmen, B. (1996). Taste perceptions and feeding behavior in nonhuman primates and human populations. Evol. Anthropol. 5:58–71. Kalmus, H. (1958). Improvements in the classification of the taster genotypes. Ann. Hum. Gen. 22:222–230. Kalmus, H. (1971). Genetics of taste, In Handbook of Sensory Physiology, Beidler, L. M. (Ed.). Springer-Verlag, Berlin, pp. 165–179. Kaminski, L. C., Henderson, S. A., and Drewnowski, A. (2000). Young women’s food preferences and taste responsiveness to 6-n-propylthiouracil. Physiol. Behav. 68:691–697. Karrer, T. and Bartoshuk, L. (1995). Effects of capsaicin desensitization on taste in humans. Physiol. Behav. 57:421–429. Lawless, H. T. (1979). Evidence for neural inhibition in bittersweet taste mixtures. J. Comp. Physiol. Psychol. 93: 538–547. Lawless, H. T. (1980). A comparison of different methods used to assess sensitivity to the taste of phenylthiocarbamide (PTC). Chem. Senses 5:247–256. Looy, H., and Weingarten, H. P. (1992). Facial expressions and genetic sensitivity to 6-n-propylthiouracil predict hedonic response to sweet. Physiol. Behav. 52:75–82. Ly, A., and Drewnowski, A. (2001). PROP (6-n-propylthiouracil) tasting and sensory responses to caffeine, sucrose, neohesperidin dihydrochalcone, and chocolate. Chem. Senses 26:41–47. Marks, L. E. (1991). Reliability of magnitude matching. Percept. Psychophysics 49:31–37. Mattes, R., and Labov, J. (1989). Bitter taste responses to phenylthiocarbamide are not related to dietary goitrogen intake in human beings. J. Am. Dietet. Assoc. 89:692–694. McBurney, D. H. (1978). Psychological dimensions and perceptual analyses of taste. In Handbook of Perception: tasting and smelling, Vol. VIA, E. C. Carterette, and M. P. Friedman (Eds.). Academic Press, New York, pp. 125–155. Meiselman, H. L., Waterman, D., and Symington, L. E. (1974). Armed Forces Food Preferences. Technical Report 75-63FSL. U.S. Army Natick Development Center, Natick, M. A.

860 Miller, I. J., and Reedy, F.E. (1990). Variations in human taste bud intensity and taste intensity perception. Physiol. Behav. 47:1213–1219. Milunicova, A., Jandova, A., and Skoda, A. (1969). Phenylthiocarbamide tasting ability and malignant tumors. Hum. Heredity 19:398–401. Morton, C. C., Cantor, R. M., Corey, L. A., and Nance, W. E. (1981). A genetic analysis of taste threshold for phenylthiocarbamide. Acta Gen. Med. Gemellol. (Roma) 30:51–57. Neely, G., and Borg, G. (1999). The perceived intensity of caffeine aftertaste: Tasters versus nontasters. Chem. Senses 24: 19–21. Niewind, A., Krondl, M., and Shrott, M. (1988). Genetic influences on the selection of Brassica vegetables by elderly individuals. Nutr. Res. 8:13–20. Olson, J. M., Boehnke, M, Neiswanger, K., et al. (1989). Alternative genetic models for the inheritance of the Phenylthiocarbamide (PTC) taste deficiency. Gen. Epidemiol. 6:423–434. Parr, L. W. (1934). Taste blindness and race. J. Hered. 25:187–190. Pelchat, M. L., and Danowski, S. (1992). A possible genetic association between PROP-tasting and alcoholism. Physiol. Behav. 51:1261–1266. Peryam, D. R., and Pilgrim, P. J. (1957). Hedonic scale method for measuring food preferences. Food Technol. 11:9–14. Prutkin, J., Duffy, V. B., Etter L., Fast K., Gardner E., Lucchina L. A., Snyder, D. J., Tie, K., Weiffenbach, J., and Bartoshuk, L. M. (2000). Genetic variation and inferences about perceived taste intensity in mice and men. Physiol. Behav. 69: 161–173. Reed, D. R. (2000). Gene mapping for taste related phenotypes in humans and mice. Appetite 35:189–190.

Drewnowski Reed, D. R., Bartoshuk, L. M., Miller, I. J., Duffy, V. B., Marino, S., and Price R. A. (1995). Propylthiouracil tasting: Determination of underlying threshold distributions using maximum likelihood. Chem. Senses 20:529–533. Reed, D. R., Nanthakumar, E., North, M., Bell, C., Bartoshuk, L. M., and Price R. A. (1999). Localization of a gene for bittertaste perception to human chromosome 5p15. Am. J. Human Genet. 64:1478–1480. Reedy, F. E., Bartoshuk, L. M., Miller, I. J., Duffy, V. B., and Yanagisawa K. (1993). Relationship among papillae, taste pores and 6-n-propylthiouracil (PROP) suprathreshold taste sensitivity. Chem. Senses 18:618–619. Rouseff, R. L. (1990). Bitterness in Foods and Beverages, Elsevier, Amsterdam. Snyder, L. H. (1931). Inherited taste deficiency. Science 74:151–152. Steinmentz, K. A., and Potter, J. D. (1996). Vegetables, fruit, and cancer prevention: a review. J. Am. Dietet. Assoc. 96:1027–1039. Stevens, S. S., and Galanter, E. H. (1957). Ratio scales and category scales for a dozen perceptual continua. J. Exp. Psychol. 54:377–411. Tepper, B. J., and Nurse, R. J. (1997). Fat perception is related to PROP taster status. Physiol. Behav. 61:949–954. Tepper, B. J., and Ullrich, N. (1999). Dietary restraint influences the relationship between PROP taster status and body weight in women (abstr). Appetite 33:234–235. Whissell-Buechy, D., and Wills, C. (1989). Male and female correlations for taster (PTC) phenotypes and rate of adolescent development. Ann. Hum. Biol. 16:131–146. Whitehead, MC, Beeman, CS, and Kinsella, BA (1985). Distribution of taste and general sensory nerve endings in fungiform papillae of the hamster. Am. J. Anat. 173:185–201.

41 Psychophysical Evaluation of Taste Function in Nonhuman Mammals Alan C. Spector University of Florida, Gainesville, Florida, U.S.A.

I.

laboratory (see Chapters 18 and 19 for parallel issues in assessing olfactory function). Taste function can be experimentally classified into at least three domains: identification, ingestive motivation, and digestive preparation (see Spector, 2000). Identification involves the innate or learned association of the sensory/discriminative features of a taste stimulus with internal or external events. Ingestive motivation refers to the ability of taste stimuli to promote or discourage feeding and drinking; it can be considered the hedonic aspect of taste. Digestive preparation deals with the reflexive physiological reactions (e.g., salivation) that are triggered by some taste stimuli serving to facilitate digestion and assimilation of ingested foods and fluids; such processes are generally referred to as cephalic phase responses. Animal psychophysical procedures primarily deal with the measurement of either sensory/discriminative or hedonic taste function. In choosing a particular method, a researcher must consider which aspect of taste function is of relevance to the experimental question at hand.

INTRODUCTION

Broadly defined, animal taste psychophysics is the scientific determination of the relationship between the physical properties of gustatory stimuli and the behavioral actions of nonhuman animals. Because perceptual experience cannot be measured directly, it must be inferred from behavior, a fact no less true for humans. The application of animal psychophysical procedures to the study of taste began in earnest in the 1950s concomitant with the surge of research on operant and classical conditioning theory and practice. Before this time, intake measures were primarily used to gauge responsiveness of nonhuman animals to taste compounds (e.g., Blum et al., 1941; Patton et al., 1944; Richter, 1936; Richter and Campbell, 1940). Although the intake measure remains the staple of behavioral taste testing, a variety of more sophisticated procedures have been developed and used over the last few decades to assess taste function in animals. These procedures have successfully been applied to measure taste processes primarily in the rat, but there is every reason to believe that they could be adapted for use with a diversity of mammals (e.g., Aspen et al., 1999; Reilly et al., 1994). These test paradigms all possess specific attributes and limitations—some practical, some conceptual. The goal of this chapter is to describe the basic types of procedures available and discuss methodological and interpretive issues associated with their application in the

II. ONE- AND TWO-BOTTLE INTAKE TESTS A.

Description of Method

The two-bottle intake test remains the most popular test employed to measure taste function in animals. One bottle contains a chemical solution and the other contains water. 861

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In some designs, both bottles contain different chemical compounds. These bottles are usually placed on the animal’s home cage for a prescribed period of time, most commonly 24 hours. To guard against the possibility of bottle position preferences, researchers generally repeat the test with the positions of the solutions on the cage reversed. A single-bottle variation is sometimes used to promote stimulus sampling and eliminate side bias. The so-called preference-aversion function is derived by increasing the concentration of a taste compound across pairs of sessions and measuring the amount ingested relative to water (e.g., Pfaffmann, 1957). A preference ratio can be calculated by dividing the intake of the taste stimulus by the total intake (water  taste). A ratio of 1.0 represents complete preference, a ratio of 0 represents complete avoidance, and a ratio of 0.5 represents indifference. In the case of the single-bottle test, the amount consumed is the measure. Order of presentation can be a critical factor in the nature of such curves. For example, ascending series of concentrations can produce different types of functions from descending and randomized series (Flynn and Grill, 1988a; Fregly and Rowland, 1992). B.

M NaCl in a 1-hour test by simultaneously infusing various concentrations of the salt directly into the stomach (Fig. 1). These examples demonstrate the potential for certain taste stimuli to exert effects on postoral body compartments, ultimately influencing the ingestive behavior. In two-bottle preference tests, measurement floors and ceilings can be problematic. For example, preference ratios for sucrose (vs. water) approach an asymptotic value of 1.0 at rather low concentrations. Although intake can be measured at this point, providing some further information, postingestive factors may limit interpretation. For aversive compounds such as quinine, an asymptotic minimum is reached beyond which further increases in concentration provide no further information regardless of whether preference or intake is measured—an animal cannot drink less than nothing. Of course, many behavioral measures have floors and ceilings, and one should consider them carefully in interpreting the outcome of experimental manipulations. Perhaps the most important caveat concerning intake tests is that they assess taste function in the hedonic domain. In other words, it is the response-reinforcing properties of the taste solution that drives the ingestive

Attributes and Limitations

The one- and two-bottle intake tests are favored by many researchers for good reasons. Foremost among these are their simplicity and ease of use. There is no need for sophisticated equipment or extensive training (of animals or laboratory personnel). In many cases there is no need for food or water restriction schedules. Indeed, this test is excellent for a first approximation of the animal’s responsiveness to various taste compounds, but the features of the procedure impose unavoidable restrictions on interpretation. First, many factors can influence intake in addition to taste (see Grill et al., 1987; Stellar, 1967). Notably, postingestive stimulation can play a large role in guiding intake (see Davis and Levine, 1977). For example, in short-term tests (1 hr) with rats, an inverted-U shape characterizes the intake of sugar solutions as a function of concentration, but in sham drinking preparations in which the ingested solution drains out of a surgically implanted gastric or esophageal cannula, the function becomes monotonic (McCleary, 1953; Nissenbaum and Sclafani, 1987b; Shuford, Jr., 1959; Weingarten and Watson, 1982). The difference between the shapes of the respective curves is thought to reflect the caloric density of the higher concentrations inhibiting intake in the normal drinking condition (i.e., without an open gastric cannula). Rabe and Corbit (1973) were able to reconstruct an inverted-U concentration-intake function for animals drinking 0.01

Figure 1 Mean (  SD) intake (mL) of five 22-hour waterdeprived rats drinking a NaCl solution during a 1-hour singlebottle test. The animals had intragastric feeding tubes chronically implanted such that solutions could be infused into the stomach concomitant with drinking. In the normal drinking condition, the orally ingested and intragastrically infused concentrations of NaCl were the same. In the taste-held-constant condition, the rats drank a 0.01 M NaCl solution and the concentration of NaCl infused into the stomach was adjusted such that the final intragastric concentration equaled the values listed on the horizontal axis. Note that the characteristic inverted U–shaped intake function is obtained even when the oral concentration of NaCl is held constant. (Data replotted from Rabe and Corbit, 1973.)

Taste Function in Nonhuman Mammals

behavior. There are many taste compounds that are equally preferred or avoided but are nevertheless discriminable. Consequently, these tests do not provide information on taste quality, per se. Moreover, these tests are not particularly effective for measuring sensitivity to taste compounds in the low concentration range. This is because animals may be able to detect the presence of the chemical stimulus, but may treat it indifferently to water with respect to its relative hedonic value. Likewise, these tests are not useful with clearly discriminable taste compounds that are neither preferred nor avoided by animals relative to water.

III. GENERAL PROCEDURAL REQUIREMENTS IN THE BEHAVIORAL ASSESSMENT OF TASTE FUNCTION IN ANIMALS A. Small Stimulus Volumes and Immediate Responses To circumvent the influence of postingestive factors in the behavioral assessment of taste function, researchers can

A

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incorporate two important methodological features into their protocols. First, small volumes of taste solutions can be used. Second, immediate responses to the application of the chemical stimulus can be measured. These two features increase the confidence that the observed behavior is controlled by oral sensory events. B.

Stimulus Delivery Apparatus

The application of these methodological features requires the use of a stimulus-delivery system and a lickometer (with the exception of the taste reactivity procedure discussed below). Such apparatuses are commercially available or can be custom-built (e.g., Aspen et al., 1999; Brosvic and Slotnick, 1986; Reilly et al., 1994; Shaber et al., 1970; Spector and Grill, 1988; Spector et al., 1990a; Thaw and Smith, 1992). These devices are sometimes referred to as gustometers (Fig. 2). An apparatus of this type that has been successfully used to assess taste-guided behavior in rats in my laboratory includes 12 fluid-filled reservoirs that are slightly pressurized (~5 psi) (Spector et al., 1990a). These

B

Figure 2 Two gustometers used to assess taste function in rodents. (Panel A reproduced from Spector et al., 1990a, with permission from Elsevier Science. Panel B reproduced from Thaw and Smith, 1992, with permission from Oxford University Press.)

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reservoirs are attached through TeflonTM tubing to micropore filters, which in turn are connected to miniature solenoid valves. The valves are connected to 18 G stainless steel tubes circularly arranged in the cap of a funnel-shaped drinking spout. This drinking spout is machined from either Teflon or Kel-F TM (harder material) and has a small stainless steel ring placed around the orifice where the animal licks. A wire connects the metal ring to the lick circuit, which passes less than 50 nA of current through the animal, a value that is below the reported thresholds for electrical current detection and electric taste effects (Bujas et al., 1979; Herness, 1985; Weijnen, 1977). A taste trial begins with the shaft of the drinking spout being filled. Because the inside of the spout is under partial negative pressure, a small drop appears at the end and stays in place. When the animal licks, a ~5 L drop (the approximate lick volume for the rat) is deposited into the shaft. Between trials a stepping motor rotates the drinking spout to a drainage funnel over which the spout is thoroughly rinsed with distilled water and the remaining fluid is evacuated with a burst of pressurized air. In the design of such apparatuses, it is important to minimize and test for cross contamination of test stimuli and to verify that the lick circuit is accurately representing contact. In the case of the former, some researchers eliminate the potential for cross contamination by using a gustometer that rotates separate drinking spouts, each connected to a different stimulus, into position for the animal to lick. However, if this turntable design is used in conditioned signal discrimination experiments, it is important to insure that the drinking spouts are uniform and do not have somatosensory (e.g., nicks, gouges) or visual (e.g., size of the orifice) signatures that can identify them. In the case of rodents, and perhaps other mammals, lick circuit performance can be evaluated by the close inspection of the interlick interval (ILI) distribution, which is largely unimodal with a mode at about 150 msec (e.g., Davis and Smith, 1992; Halpern, 1977; Smith et al., 1980; Spector et al., 1998). If there are substantial peaks at integer multiples of the fundamental interval, then this could be indicative of missed licks (Weijnen, 1977, 1998). In some cases, this may be because the animal’s tongue did not contact the spout, but in others it could be because the lick detection circuit was not sufficiently sensitive, or, if photobeams are used, it could mean that the LED and phototransistors are out of alignment. Although the design, construction, maintenance, and operation of these devices can be laborious, such gustometers are very versatile procedurally and provide the type of experimental control that builds interpretive strength.

Spector

C. Potential Confounding Due to Stimulation of Olfactory and Trigeminal Receptor Systems Although the incorporation of the methodological features above raises the confidence that the behavior is based on oral sensory events, it does not necessarily guarantee that responsiveness is under the control of gustatory input. The possibility that either olfactory or trigeminal stimulation is contributing to the behavior is a vexing issue in taste research. With the exception of organic acids (e.g., acetic acid), prototypical taste compounds are not considered especially effective odor stimuli because of their low vapor pressure. Nonetheless, there is evidence that rats can detect midrange or even in some cases low concentrations of certain taste stimuli (e.g., NaCl, sucrose) apparently using olfactory cues (Miller and Erickson, 1966; Rhinehart-Doty, et al., 1994; Van Buskirk, 1981). The potential effectiveness of olfactory cues can be somewhat diminished through apparatus design. For example, in my laboratory the rat must lick a dry drinking spout before a stimulus is deposited. This forces the animal to at least lick the stimulus. Also, because of the vertical topography of the drinking spout, the rat’s snout is positioned upward during licking. These features, coupled with the short stimulus access period, appear to minimize olfactory cues, as confirmed by the fact that gustatory nerve transections severely compromise performance on a variety of tasks involving the gustometer (e.g., Spector et al., 1996b, St. John and Spector, 1998; St. John et al., 1994). Shaber et al. (1970) found that detection thresholds for saccharin, quinine hydrochloride, NaCl, and HCl were similar between bulbectomized (i.e., olfactory bulbs ablated) and shamoperated rats. Trigeminal stimulation is likely not a problem at low stimulus concentrations, but at higher values it cannot be easily dismissed. Carbohydrate solutions increase in viscosity as the concentration is raised. At high concentrations, salt and acid stimuli have been reported to be irritating by human subjects (Gilmore and Green, 1993). There are no easy solutions to these problems. In some cases, the contribution of olfactory and trigeminal factors can be refuted experimentally, but in all cases the potential for nongustatory cues to influence performance must be considered in the interpretation of the results. IV. PROCEDURES FOR ASSESSING AFFECTIVE (HEDONIC) RESPONSIVENESS TO TASTE STIMULI IN ANIMALS There are a variety of procedures that can effectively evaluate an animal’s hedonic responsiveness to a taste stimulus.

Taste Function in Nonhuman Mammals

A.

Brief-Access Taste Test

The brief-access taste test is a very capable alternative to the two-bottle preference test. The procedure involves the presentation of very small samples of taste stimuli for very brief duration trials (e.g., 10 sec) and the animal’s unconditioned licking responses are measured (e.g., Breslin et al., 1993a,b; Contreras et al., 1995; Davis, 1973; Glendinning et al., 2002; Markison et al., 1995, 2000; O’Keefe et al., 1994; Smith et al., 1992; Spector, 1995; Spector et al, 1993a,b, 1996b; St. John et al., 1994; Young and Trafton, 1964). Several concentrations can be presented in a random order and a concentration-response function can be derived in a single session. In other cases, different chemical compounds can be presented and an across-chemical profile of responsiveness can be established (Breslin et al., 1993b; Markison et al., 1995, 2000). For example, sodiumdepleted rats will selectively lick NaCl relative to water and other nonsodium salts (Breslin et al., 1993b; Markison et al., 1995). For taste compounds that are naturally aversive, water deprivation can be used to motivate stimulus sampling and provide a high baseline of licking from which concentration-dependent decreases can be quantified (e.g., Spector, 1995; St. John et al., 1994). For naturally preferred stimuli, animals can be tested in a nondeprived state or under the conditions of a food restriction schedule (e.g., Davis, 1973; Spector et al., 1996b) (Fig. 3). One of the principal benefits of the brief-access taste test is that relative degrees of responsiveness to

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various chemical stimuli can be assessed as in a twobottle preference test, but this can be done with more stimuli in a single session. Moreover, stimulus sampling is promoted by presenting one stimulus at a time on a given trial. In practice, it is not uncommon for researchers to conduct several test sessions to increase the number of trials for each stimulus, thus improving the estimate of responsiveness. When water deprivation is used, individual differences in lick rate or physiological state between animals can be adjusted by using water licks as the denominator in ratio to taste stimulus licks. In general, the concentration-licking functions that are derived by these procedures are monotonic and can be fit by sigmoidal functions; the parameters of such functions can then be analyzed (see Spector et al., 1996b; St. John et al., 1994). It is important to recognize that affective responses to taste fall into two categories: appetitive and consummatory. Appetitive responses refer to the behavior that serves to bring the animal into contact with the stimulus (e.g., approach behavior). Consummatory responses refer to reflex-like behavior that is elicited by stimulus contact with sensory receptors (Craig, 1918). There is growing evidence that these two domains of affective responsiveness can be neurally dissociated (Berridge, 1996; Flynn and Grill, 1988b; Grill and Norgren, 1978b; Parker, 1995). The brief-access taste test involves appetitive and consummatory responses because spout licking has features of both (Hulse, 1967).

Figure 3 (A) Mean ( SE) licks during 10-second trials as a function of sucrose concentration before and after sham gustatory nerve surgery (n
7). Water licks were subtracted out from the scores. Note the prototypical sigmoidal shape that distinguishes it from inverted U–shaped functions characterizing intake in one- and two-bottle tests with sucrose. (Replotted from Spector et al., 1996.) (B) Mean ( SE) licks during 10-second trials as a function of the concentration of quinine hydrochloride before and after sham gustatory nerve surgery (n
9). Licks to quinine were divided by licks to water to form a ratio. (Replotted from St. John et al., 1994.)

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B.

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Oromotor Responses to Intraorally Delivered Taste Stimuli

The measurement of appetitive and consummatory responses depends on the mode of stimulus delivery. Purely consummatory responses can be measured by delivering taste solutions through a surgically implanted intraoral cannula (Berridge, 1996; Grill and Norgren, 1978a; Grill et al., 1987; Spector et al., 1988). Thus, the pattern, rate, and duration of stimulus delivery is entirely under experimenter control. Oromotor responses can then be videotaped for later analysis. These responses can be classified into two general hedonic domains: ingestive and aversive. Ingestive behaviors include tongue protrutions, lateral tongue protrusions, paw licking, and mouth movements and are associated with the ingestion of normally preferred taste compounds such as sucrose. Aversive behaviors include gapes, chin rubs, forelimb flails, and head shakes and are associated with the rejection of normally avoided compounds such as quinine. These behaviors are concentration-dependent and have been most extensively studied in the rat. However, such behaviors have been observed in other rodent species as well (Brining et al., 1991). Even human neonates express similar oromotor responses to prototypical taste compounds (Steiner, 1977). Aside from its theoretical significance, one of the primary merits of this procedure is that it can be used with severely neurally compromised animal preparations. Indeed, it was developed for use in supracollicular decerebrate rats (Grill et al., 1978a,b). These rats, which have the forebrain neurally disconnected from the rest of the central nervous system, do not eat and drink voluntarily and do not regulate body temperature. Nevertheless, they do display appropriate concentrationdependent increases and decreases in both ingestive and aversive taste reactivity responses to taste compounds in a similar fashion to intact control rats. One of the drawbacks to this procedure is that the behavior is sometimes “noisy” across animals and test sessions. This variability requires that a sufficient number of animals be used in experimental designs and that the effect size is adequate. Given the labor-intensive energy that is placed in objectively analyzing the videorecord of the behavior, there should be a strong theoretical or procedural rationale supporting the choice of this method. C. Conditioned Operant Responses Involving Taste Reinforcers

response in order to receive a taste reinforcer (the use of taste compounds as reinforcers should not be confused with their use as signals in operant tasks). Accordingly, the measured responses are performed when the stimulus is not in explicit contact with the receptors. Close to 40 years ago, Gutman (1954) was able to train food-deprived rats to press a lever to receive a small amount of a sugar solution (i.e., the reinforcer). Reinforcement was delivered on a variable interval schedule such that, on average, the sugar reinforcer was delivered every 60 seconds. Under these contingencies, rate of lever pressing increased monotonically with the concentration of the sugar solution (a single concentration was presented in a given session) and was greater for sucrose compared with glucose (i.e., the glucose curve was shifted to the right on the concentration axis). An alternative method developed several decades ago by Hodos (1961) for measuring purely appetitive responses is the progressive ratio procedure. In this protocol, the animal is required to perform a progressively greater number of responses (e.g., lever pressing) before the reinforcer (e.g., sucrose solution) is delivered. The animal completes each successively greater ratio until the requirement becomes too great relative to the value of the reinforcer, at which point, referred to as the breakpoint, the animal ceases to respond. Breakpoints vary as a function of the properties of the reinforcer (Hodos, 1961; Reilly, 1999). Consequently, it is an excellent way to quantify the appetitively based affective properties of taste solutions (Reilly, 1999; Reilly and Trifunovic, 1999). Moreover, because of the nature of the reinforcement schedule, the amount of taste solution delivered during a session is low, minimizing the contribution of postingestive factors to the measured responses. If animals are placed on a restricted water-access schedule, the progressive ratio procedure can then also be used to measure responsiveness to aversive taste compounds such as quinine (Reilly, 1999). The breakpoint should be highest for water and should progressively decrease with rising concentration of the aversive compound being tested. As is the case with the brief-access taste test, one must keep in mind the physiological state of the animal during testing and consider that in the interpretation of the results. V. GENERAL REQUIREMENTS FOR ASSESSING TASTE SIGNAL DETECTION AND DISCRIMINATION IN ANIMALS A.

The measurement of purely appetitive responses can be conducted with the use of operant conditioning procedures in which the animal is trained to perform some type of

Hedonics Versus Signal Processing

The procedures described in the prior sections depend on the hedonic characteristics of the stimuli to drive

Taste Function in Nonhuman Mammals

behavior. There are a variety of procedures that have been designed to assess taste sensitivity and discrimination independent of the motivational properties of the taste stimuli themselves. These techniques exploit the principles of classical and operant conditioning procedures. In the former, taste compounds serve as conditioned stimuli. In the latter, taste compounds can serve as discriminative stimuli and provide the context in which specific operant responses are reinforced or punished. In both learning paradigms, a taste compound serves as a signal for the occurrence of other events regardless of its affective valence. In these procedures, the motivation to respond is usually provided by the experimenter imposing a schedule of restricted access to food or water. This promotes stimulus sampling and, in the case of operant conditioning, can motivate the animal to respond correctly so as to obtain a food or water reinforcer.

B.

Stimulus Control

Regardless of the specific conditioning procedure employed, it is essential to demonstrate that the animal is under taste stimulus control. In other words, it is important to provide evidence that the animal is behaving in response to the sensory features of the taste compound as opposed to some other extraneous cue. For example, there are often extraneous events associated with stimulus delivery (clicks of solenoids, temperature, fluid flow rate, etc.) that can potentially be differentiated by the animal and might serve as reliable cues. This is every psychophysicist’s nightmare. Of course, these issues should be considered in the design of the testing apparatus, but there is no guarantee that a researcher’s vigilant efforts in this regard will be necessarily successful. Therefore, the best way to demonstrate that the animal is under stimulus control is through behavioral means. It is important to show that changes in the animal’s behavior match changes in the stimulus. For example, if an animal is trained to discriminate between water and, say, NaCl, and the animal’s performance begins to approach chance levels once the concentration reaches a certain weak value, then this would constitute reasonable evidence that the animal’s discrimination behavior is concentration dependent and thus under stimulus control. Such a conclusion, however, should be confirmed at the end of an experiment by removing the chemical cues from the solutions (i.e., presenting only water) while leaving the contingencies in place and seeing if the animals can perform competently. If they cannot, then that would provide testimony that the animals need the chemical in the solution to reliably perform the discrimination.

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C.

Perception Inferred from Behavior

Perception is inferred from behavior. Researchers are limited to asking nonverbal animals two basic questions: Are these stimuli different, and if different, how different (or similar) are they? In terms of animal learning principles, the first question pertains to discrimination, and the second question pertains to generalization. In discrimination tasks, the animal is trained to respond one way to a given taste stimulus (e.g., press righthand lever when NaCl is delivered) and another way to a different taste stimulus (e.g., press lefthand lever when sucrose is delivered). In generalization tasks, the animal is trained to respond in a specific way to a taste stimulus and then other chemical compounds are delivered as test stimuli and the animal’s response noted. The quantitative degree to which the response to the test stimulus matches that to the training stimulus is taken as an index of similarity. Although these two psychophysical questions are conceptually simple, they can provide very powerful and objective insight into the taste world of animals. Some of the specific methodological and interpretive issues related to discrimination and generalization procedures are discussed in the following sections. VI.

ASSESSMENT OF PERCEIVED INTENSITY

A.

Detection Threshold

1.

Procedures

One of the most fundamental issues in psychophysical research deals with the assessment of an observer’s sensitivity. Over the last 5 decades a variety of procedures have been employed to do this in animal subjects, mostly rats (e.g., Brosvic and Hoey, 1990; Brosvic et al., 1986, 1992; Carr, 1952; Geran and Spector, 2000; Geran et al., 1999; Harriman and MacLeod, 1953; Koh and Teitelbaum, 1961; Morrison and Norrison, 1966; Shaber et al., 1970; Slotnick, 1982; Slotnick et al., 1991; Spector et al., 1990b, 1995; St. John and Spector, 1996; Thaw, 1996; Thaw et al., 1992; Willner et al., 1990). Some investigators have used a two-alternative, forced-choice procedure in which two bottles, one containing a taste solution and the other containing water, were presented, and if the water-deprived animal licked from the “incorrect” solution (based on various criteria across experiments), it received an electric shock (Carr, 1952; Harriman and MacLeod, 1953). Koh and Teiltelbaum (1961) used a similar procedure in which nondeprived rats were trained to lick the “correct” stimulus 10 times in succession before 8 licks were recorded on the other fluid. Correct responses prevented shock delivery. These investigators also included an additional group in

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which food-deprived animals were rewarded with a food pellet when a correct choice was made. Using a yes/no procedure in which a single stimulus is presented on a trial and the animal indicates whether the solution contains the relevant chemical compound, Morrison and Norrison (1966) trained food-deprived rats to press one lever if the stimulus was a taste compound and another lever if the stimulus was water; correct responses were rewarded with a food-pellet. Geran et al. (Geran and Spector, 2000; Geran et al., 1999) applied a similar strategy but used water reinforcement in water deprived rats (Fig. 4). Spector et al. (1990b, 1995) used a conditioned avoidance procedure to train thirsty rats to lick a dry drinking spout to receive a 5-second fluid pre-

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sentation. If the fluid was a taste stimulus, then the animal was required to suppress licking within 2 seconds to avoid shock. If the fluid was water, then the animal was allowed to lick freely during the entire trial and was required to lick during the latter 3 seconds or else it received a timeout, further delaying the opportunity to receive another fluid presentation. Slotnick (1982) trained water-deprived rats to lick a sample spout at a high rate if the stimulus was a taste. A correct response was rewarded with water delivered from a seperate reinforcer delivery tube. If the animal licked the sample spout at a high rate when the stimulus was water, then it received a time-out. Brosvic and Slotnick (1986) modified the latter procedure such that when the taste stim-

Figure 4 Flow chart depicting the sequence of events and contingencies that characterize a stimulus trial in the psychophysical procedure used by Geran et al. (1999) to measure detection thresholds to KCl. This procedure was modified from that used by St. John et al. (1997) to train rats in a two-taste discrimination task. The latter was, in turn, modified from a procedure developed by Morrison and Norrison (1966).

Taste Function in Nonhuman Mammals

ulus was delivered the animal had to stop licking the sample spout within 9 licks and switch to the reinforcement spout to receive a water reinforcer. When water was the stimulus, the animal had to maintain licking the sample spout (9 licks) or else it received a time-out. In all of the procedures described briefly above, after the animal was trained, the concentration of the chemical solution was systematically varied and performance measured. The comprehensive assessment of an animal’s sensitivity to a taste compound is, in general, at odds with the conditioning principles underlying the procedures used to accomplish the goal. That is, there are trials in which the concentration of the taste stimulus is undetectable by the animal and will likely lead to the occurrence of punished responses, even though, from the animal’s perspective, the response was correct. Of course, from a conditioning perspective this can be viewed as an extinction trial; in anthropomorphic terms, on such trials the animal could question whether the rules have changed. Once again, this is an issue of stimulus control. Animal psychophysicists in general have grappled with this issue for many years and different investigators have devised different strategies to deal with it (see Berkley and Stebbins, 1990; Blough and Blough, 1977; Smith, 1970). In my own laboratory, we train the animal with a range of clearly detectable concentrations, and then, across sessions, we progressively extend the lower limit of the range until it includes undetectable stimulus values. One benefit of this strategy is that the number of perithreshold concentrations offered in a single session can be limited and embedded in a random fashion (referred to as the method of constant stimuli) among trials with semi-detectable and clearly detectable stimulus values which serves to both maintain, and importantly, measure stimulus control (i.e., assess performance at the detectable concentrations). Some investigators have used a tracking (or staircase) procedure in which the stimulus value presented on a given trial depends on the animal’s performance on the previous trial (Koh and Teitelbaum, 1961). Other researchers have used a modified descending method of limits in which a block of trials with a single clearly detectable concentration is presented and then the concentration is lowered for the next series provided a criterion level of performance was met (Brosvic and Slotnick, 1986; Harriman and MacLeod, 1953; Slotnick, 1982; Slotnick et al., 1991). 2.

The Comparison of Detection Thresholds Across Studies

There is a strong tendency for researchers to compare detection thresholds for a given taste compound across studies (e.g., Table 1). This practice, however, is conceptu-

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ally dangerous and can lead to misleading conclusions. Not only do the behavioral procedures differ on potentially important variables (water rinses, water deprivation, food deprivation, one vs. two stimuli per trial, etc.), but, as Morrison eloquently argued close to 30 years ago, the definitions of threshold differ as well. Some researchers have defined the concentration at the performance midpoint on the psychometric function as the threshold (e.g., Geran and Spector, 2000; Geran et al., 1999; Slotnick, 1982; Slotnick et al., 1991). Others have defined it as the concentration at which a specified number of trial blocks have more than a criterion number of errors (e.g., Harriman and MacLeod, 1953). When a tracking procedure is used, threshold is generally calculated as the average of the concentrations at which reversals occur (Koh and Teitelbaum, 1961). Morrison and Norrison (1966) defined absolute detectability as the concentration at which the false alarm rate (the proportion of water trials with an error) equaled the hit rate (the proportion of taste stimulus trials that are correct) (i.e., d’
0; see below). There is nothing inherently wrong with comparing thresholds measured in different procedures as long as it is done cautiously. Indeed, not only might it provide a general sense of the animal’s sensitivity to a given taste compound measured under a variety of contexts, but it may also reveal interesting factors that interact with taste sensitivity. The fact of the matter is that it is truly debatable whether an absolute detection threshold actually exists. If it does exist, then it likely varies under a variety of conditions. Indeed, early psychophysicists believed in the principle of a threshold in which stimulus intensities above that value would be detected 100% of the time and intensities below that value would be detected 0% of the time (Gescheider, 1997). However, to explain the gradual non–step-like slope of the psychometric function, they reasoned that the threshold varied from moment to moment generating a normal distribution of threshold values. Thus, the psychometric function was merely the respective cumulative probability distribution. The concentration at the midpoint of the function represented the mean of the normal distribution of varying threshold values. More modern psychophysicists challenged the notion of an absolute threshold and incorporated the principle of observer response bias in their assessment of sensitivity leading to the application of signal detection theory (Green and Swets, 1966). In this view, the sensory system is “noisy”, resulting in a normal distribution of neural activity in the absence of an adequate stimulus and the presentation of the latter shifts the distribution laterally to higher values. An observer must determine what value of neural activity will be reported as arising from the signal  noise distribution (i.e., reporting that a stimulus is pre-

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Table 1 Detection Thresholds for Prototypical Taste Compounds in Rats Across Experiments Taste compound Sucrose

NaCl

Study

Procedure

Mode of stimulus presentation

Food or water restriction schedules

Reported (mM) threshold

Koh and Teitelbaum, 1961

2-Alternative forced choice —shock avoidance

Tracking

No restriction

13.6a

Koh and Teitelbaum, 1961

2-Alternative forced choice —food reward

Tracking

Food restriction

6.25a

Willner et al., 1990

Yes/No— sucrose reward

Modified method of limits

No restriction

0.0351

Morrison and Norrison, 1966

Yes/No— food reward

Modified method of limits

Food restriction

0.0924b

Brosvic and Slotnick, 1986

Yes/No— water reward

Modified method of limits

Water restriction

1.02a

Slotnick et al., 1991

Yes/No— water reward

Modified method of limits

Water restriction

7.40a

Thaw and Smith, 1992

Yes/No—conditioned suppression

Modified method of constant stimuli

Food restriction

2.26a

Spector et al., 1995

Yes/No— shock avoidance

Modified method of constant stimuli

Water restriction

10.7cd 15.1ce

Carr, 1952

2-Alternative forced choice— shock avoidance

Modified method of limits

Water restriction

1.53a

Harriman and MacLeod, 1953

2-Alternative forced choice— shock avoidance

Modified method of limits

Water restriction

0.0678a

Koh and Teitelbaum, 1961

2-Alternative forced choice— shock avoidance

Tracking

No restriction

0.780a

Koh and Teitelbaum, 1961

2-Alternative forced choice— food reward

Tracking

Food restriction

0.705a

Morrison and Norrison, 1966

Yes/No— food reward

Modified method of limits

Food restriction

0.170b

Shaber et al., 1971

Yes/No—conditioned suppression

Modified method of limits

Water restriction

3.59a

Slotnick, 1982

Yes/No— water reward

Modified method of limits

Water restriction

0.650a

Brosvic and Slotnick, 1986

Yes/No— water reward

Modified method of limits

Water restriction

0.839a

Spector et al., 1990

Yes/No— shock avoidance

Modified method of constant stimuli

Water restriction

0.41cf

Slotnick et al., 1991

Yes/No— water reward

Modified method of limits

Water restriction

0.80a

Thaw and Smith, 1992

Yes/No—conditioned suppression

Modified method of constant stimuli

Food restriction

0.630a

Spector et al., 1995

Yes/No— shock avoidance

Modified method of constant stimuli

Water restriction

1.66cd 1.66ce

Geran and Spector, 2000

Yes/No— water reward

Modified method of constant stimuli

Water restriction

4.27g

Taste Function in Nonhuman Mammals

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Table 1 Continued Taste compound

Study

Procedure

Mode of stimulus presentation

Food or water restriction schedules

Reported (mM) threshold

Modified method of constant stimuli

Water restriction

33.0g

2-Alternative forced choice— shock avoidance

Tracking

No restriction

0.0145a

Koh and Teitelbaum, 1961

2-Alternative forced choice— food reward

Tracking

Food restriction

0.0101a

Shaber et al., 1971

Yes/No—conditioned suppression

Modified method of limits

Water restriction

0.00313a

Thaw and Smith, 1992

Yes/No—conditioned suppression

Modified method of constant stimuli

Food restriction

0.005a

St. John and Spector, 1996

Yes/No— shock avoidance

Modified method of constant stimuli

Water restriction

0.0105cg 0.0184ch

Quinine sulfate Morrison and Norrison, 1966

Yes/No— food reward

Modified method of limits

Food restriction

0.00277b

HCl

Koh and Teitelbaum, 1961

2-Alternative forced choice— shock avoidance

Tracking

No restriction

0.352a

Koh and Teitelbaum, 1961

2-Alternative forced choice— food reward

Tracking

Food restriction

0.608a

Shaber et al., 1971

Yes/No—conditioned suppression

Modified method of limits

Water restriction

0.342a

Thaw and Smith, 1992

Yes/No—conditioned suppression

Modified method of constant stimuli

Food restriction

0.085a

KCl

Geran, Guagliardo, and Spector, 1999

Quinine-HCl

Koh and Teitelbaum, 1961

Citric acid

Yes/No— water reward

aArithmetic

averages. reported concentration for which d’ equaled 0. cGeometric means. dFrom Experiment 1. eFrom Experiment 2. fPresurgical thresholds collapsed across both experiments. gPresurgical threshold for all rats in Experiment 1. hSecond presurgical threshold for all rats in Experiment 2. bNearest

sent) as opposed to arising from the noise distribution alone (i.e., reporting that a stimulus is not present). Different subjects place this criterion at different values and this is reflected in the type of response biases expressed (e.g., the tendency to report that the stimulus is present when it is not). The use of this particular psychophysical model carries with it certain assumptions about the underlying distributions. These assumptions can be confirmed by establishing what is known as receiveroperating characteristic (ROC) curves in which response biases are experimentally manipulated by either altering

the payoff matrix (relative values of rewards and punishments for correct and incorrect responses) or by varying the probability of stimulus presentation and then plotting the probability of false alarms (reporting that a stimulus is present when it is not) against the probability of hits (reporting that a stimulus is present when it is). Signal detection theory predicts that such plots will have a distinctive curvilinear shape. This is a very brief account of signal detection theory, and more comprehensive treatments of the concept can be found elsewhere (see Gescheider, 1997; Green et al., 1966)

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(see Chapter 10). As a matter of theory these psychophysical models are different in significant ways, but in practice the difference may, in some cases, be irrelevant in terms of discerning the nature of effects. For example, Figure 5 presents replotted data from an experiment (Kopka and Spector, 2001) in which thirsty rats were trained to press one lever when water was presented and to press another lever when NaCl was the stimulus; correct responses were rewarded with extra access to water and incorrect responses were punished with a time-out (see Fig. 4). The performance of these animals can be evaluated in at least three ways. The first involves the traditional correction procedure for adjusting the hit rate relative to the false alarm rate, as shown by the equation below: P(Hit)c
[P(Hit)  P(FA)]/[1.0  P(FA)] where P(Hit)c is the corrected hit rate, P(Hit) is the proportion of taste stimulus presentations with a correct response, and P(FA) is the proportion water presentations with an error (i.e., false alarm). The second involves the calculation of d’ based on signal detection theory. This represents the relative separation of the noise distribution from the signal  noise distribu-

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tion. When d’
0, the probability of a hit is equal to the probability of a false alarm, reflecting complete overlap of the two distributions. The value of d’ is calculated as: d’
ZP(Hit)  ZP(FA) where ZP(Hit) is the Z-score corresponding to the area under the normal curve defined by P(Hit) and ZP(FA) is the Z-score corresponding to the area under the normal curve defined by P(FA). The third involves a nonparametric analog of d’ based on signal detection theory as suggested by Frey and Colliver (1973): SI
[P(Hit)  P(FA)]/[2[P(Hit)  P(FA)]  [P(Hit)  P(FA)]2] where SI is the sensitivity index and P(Hit) and P(FA) are defined as above. The SI ranges from 1.0 reflecting complete stimulus detection to 0 reflecting no stimulus detection (see Brosvic et al., 1990, 1992). Despite the differences between these models, the similarities in the logistic functions fit to the data are more notable than the disparities, especially with respect to locus of rise (i.e., concentration at half maximal perfor-

Figure 5 Psychometric functions from rats (n
11) trained to discriminate water from NaCl in the two-lever task depicted in Figure 4 (Kopka and Spector, 2001). Three different scales were used to derive the functions. Means SE are shown. See text for more details.

Taste Function in Nonhuman Mammals

mance) and slope. Moreover, when the NaCl was mixed in the epithelial sodium channel blocker amiloride, which suppresses one of the sodium taste transduction pathways, the curves shifted laterally and increased slope to relatively similar degrees. It is true that d’ is noticeably different, and this could be of theoretical significance, but as a practical matter all three measures indicate relatively similar degrees of effect. B.

Suprathreshold Intensity

Up to this point attention has been placed on the measurement of stimulus detection performance. As important as it may be, this only provides a measure of the starting point of stimulus sensibility along the dimension of concentration. Once the stimulus is detected, the growth of its perceived intensity can potentially vary among taste compounds. Magnitude estimates of stimulus intensity grow as a power function of concentration in human subjects (Stevens, 1961, 1969). The exponents of these functions vary for different compounds. Despite some creative attempts (Herrnstein and Sommers, 1962), magnitude estimation procedures are not amenable for use in animal subjects. There are, however, several ways to assess the processing of intensity in the suprathreshold concentration range. One way to assess suprathreshold processing of intensity is through the use of stimulus generalization gradients. This strategy involves training the animal to respond to a given concentration of a taste compound and then testing the animal’s response to lower concentrations. In classical and operant conditioning procedures, as the test stimulus intensity is lowered, departing progressively from the intensity of the training stimulus, the strength of the response decreases. Intensity generalization gradients are commonly monotonic such that increases in test stimulus strength past the training stimulus level result in equivalent or even stronger responses (Mackintosh, 1974). Taste aversion conditioning is the most common procedure used to establish intrachemical (i.e., intensity) generalization functions (Bealer, 1978; Harder et al., 1984; Nowlis, 1974; Richter and Campbell, 1940; Scott and Giza, 1987; Shaw, 1983; Spector et al., 1988; Tapper and Halpern, 1968). A given concentration of a taste compound is used as a conditioned stimulus (CS) and its ingestion by thirsty animals during a limited exposure period (e.g., 15 min) is followed immediately by the administration of a viscerally aversive stimulus (e.g., an emetic) referred to as the unconditioned stimulus (US). Upon subsequent presentation of the CS, animals will avoid ingesting it (e.g., Garcia et al., 1955). The degree of avoidance is related to the strength of the aversion as well

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as the concentration of the test stimulus (Nowlis, 1974). Concentrations weaker than the CS lead to less avoidance (Harder et al., 1984; Nowlis, 1974; Richter et al., 1940; Scott and Giza, 1987; Shaw, 1983; Spector et al., 1988; Tapper and Halpern, 1968). The factors controlling the steepness of such functions have not been systematically delineated. So, for example, the concentration of the CS, the number of conditioning trials, and the potency of the US all likely contribute, but the exact parametric manner in which they do has yet to be fully described (but see Nowlis, 1974). Also, because animals are tested in extinction (i.e., without the presentation of the US), it is optimal to measure responses to the full range of test stimuli within a single session using the brief-access procedure. Accordingly, the test stimuli should be presented in randomized blocks. Otherwise, responses to successive stimulus presentations may reflect the extinction of the conditioned response rather than the relative intensity of the CS. Finally, failure to see a difference between the responses elicited by a test and a training stimulus does not necessarily mean that the perceived intensities generated by the different concentrations are equivalent to the animal. It simply means that they are sufficiently similar for generalization of the conditioned response to take place. Nevertheless, with this caveat in mind, the conditioned taste aversion generalization procedure is an effective way to assess the effect of various manipulations (e.g., taste inhibitors, brain lesions, nerve transections, etc.) on the processing of intensity in the suprathreshold domain. Morrison (1969) used an operant conditioning procedure to train rats to press one lever when the sampled stimulus was a single high concentration of a taste compound (e.g., sucrose) and to press another lever when the stimulus was water. He then introduced test trials with lower concentrations of the taste stimulus and measured which bar the animal pressed. As the concentration departed from the training stimulus, the proportion of trials in which the “water” lever was chosen increased. It is important to differentiate the generalization gradient generated by Morrison’s method from similar procedures used to derive psychometric sensitivity functions described earlier (Morrison and Norrison, 1966). The primary difference is that the test trials in the generalization procedure were not differentially reinforced. In other words, regardless of which lever was chosen on a given test trial, reward was always delivered. Under those conditions, responses to the test stimuli are free to vary because they are not explicitly related to a reinforcement contingency and therefore the animal’s behavior appears to be under the control of the relative similarity of the test stimulus to the two training stimuli.

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Another technique that has yet to be widely adopted to assess suprathreshold intensity processing in animals is the measurement of difference thresholds (see Brosvic and Slotnick, 1986). This can be accomplished with any of the procedures used to assess detection thresholds except that instead of the animal being trained to discriminate water from a range of concentrations, it is trained to perform one response to a standard concentration of the stimulus and another response to all other concentrations (comparison stimuli). As the concentrations of the comparison stimuli approach the standard, the ability to discriminate between the two should become more difficult, and this should be reflected as a decline in performance accuracy. A psychometric function can be derived and a threshold can be defined as the midpoint concentration of the curve. Such difference thresholds (expressed as the absolute value of the change from the standard concentration; I) can be plotted as a function of the standard concentration (I), and, if linear starting from the origin, the slope would represent the Weber Fraction for that taste stimulus.

VII.

ASSESSMENT OF PERCEIVED QUALITY

Researchers can operationally define the taste quality of chemical compounds by testing how well animals can discriminate among them, as well as by constructing profiles of similarity across these stimuli through the use of generalization gradients. These data can then be used to rigorously classify compounds into perceptual groups. The same operant and classical conditioning procedures described above can be applied to achieve this goal. There are, however, some methodological and interpretive issues that are worthy of consideration. A.

Discrimination of Taste Compounds

If an animal can be trained to discriminate between two chemical compounds, then clearly the neural signals generated by those stimuli must be distinguishable. The question then becomes, is the discrimination on the basis of taste quality? For example, it is likely that rats would be able to discriminate between a weak and a moderate concentration of sucrose on the basis of intensity even though the quality of the sensation is probably constant. Thus, the choice of concentrations to be employed in a discrimination task is critical. The strategy that my colleagues and I have adopted to deal with this concern in a two-taste discrimination procedure is to use a range of concentrations of both chemical stimuli during training and testing (e.g., Spector and Grill, 1992; Spector et al., 1996a, 1997; St. John et al., 1995, 1997a, 1998).

Accordingly, intensity is rendered an irrelevant cue. The assumptions associated with this strategy are that (1) the range of concentrations chosen have overlapping intensities and (2) the respective taste quality of each compound tested does indeed remain constant across the relevant concentration range. Knowledge of how the animals respond in tasks designed to assess intensity processing can be helpful in addressing the first assumption. The second assumption is a bit more vexing. For example, saccharin is a complex taste stimulus that is reported by human subjects as strongly “sweet” tasting and only weakly “bitter” tasting at low concentrations, but the “bitter” component grows significantly as the concentration is raised. The use of stimulus generalization procedures, discussed further below, can help provide support for the second assumption. Finally, it is worth recalling that the discrimination, while solidly linked to the chemical nature of the respective compounds, could be based in part on olfactory or trigeminal input, and this possibility must be experimentally dismissed, as noted earlier in this chapter. On a more theoretical level, it is possible that animals might be able to discriminate taste compounds because of the relative distribution of receptors in the oral cavity despite the fact that the perceptual experience generated by the two compounds in question are identical. For example, if compound A stimulates receptor A and compound B stimulates receptor B and receptor A is exclusively found in the taste buds of the anterior tongue and receptor B is exclusively found on the back of the tongue, then potentially the animals could use locus of stimulation as a cue even if the resulting sensations were qualitatively the same. Humans can apparently identify the locus of taste stimulation on the tongue (Shikata et al., 2000). In a given experiment, if animals are using locus of stimulation as the primary cue guiding the discrimination, then there should be molecular or electrophysiological evidence that the receptors for the relevant compounds are indeed differentially distributed in the oral cavity. Moreover, if the respective receptors were distributed in taste bud fields innervated by different nerves, then differential neurotomies should yield differential effects on the detectability of the two compounds. Another possibility is that animals might be able to use temporal cues (e.g., rise and decay of the taste sensation relative to somatosensory stimuluation) to distinguish between taste compounds that may nonetheless lead to the same perceptual quality. To the extent that such cues vary as a function of stimulus intensity, the use of a range of concentrations in training and testing might help to minimize them. Of course, if animals can indeed use temporal cues to distinguish between chemical compounds, then this raises

Taste Function in Nonhuman Mammals

the complex issue of defining what is meant by the term “taste quality,” a matter beyond the scope of this article. If an animal cannot be trained to discriminate between two chemical compounds, then this at least implies that the neural signals generated by those stimuli are very similar—a conclusion that could be confirmed through the use of generalization procedures. Of course, this conclusion assumes that general learning and performance deficits can be ruled out. It is difficult, if not impossible, to conclude with absolute certainty that the two signals are indistinguishable, because there is always the possibility that if training had continued or a different procedure had been used, some degree of competent discrimination performance would have become evident. If, however, the procedure used in the failed discrimination training can be shown to be effective with other pairs of chemical compounds, then one could at least speak to the relative discriminability of the respective taste compounds (see Spector and Kopka, 2002). B.

Generalization Across Taste Compounds

In the assessment of taste quality, the complement to stimulus discrimination is stimulus generalization. Although an animal may be able to discriminate between two chemical compounds, it may nonetheless find them very similar relative to other taste stimuli. Stimulus generalization tests can help address this issue. Because stimulus generalization can occur in both the intensity and quality domains, it is important for researchers to try to dissociate the two. For example, after the conditioning of a taste aversion to 0.1 M sucrose in rats, a midrange concentration of maltose (0.3 M) is avoided about as much as a low concentration of sucrose (0.03 M); low maltose concentrations are not avoided at all (Spector et al., 1988). This could be interpreted in at least two ways. It may be that maltose tastes identical to sucrose, but that it is less effective at stimulating “sucrose-binding” taste receptors and so at isomolar concentrations, is treated as a less intense stimulus. Alternatively, it may be that sucrose and maltose do not generate qualitatively identical perceptions, but that as the concentration is raised, the two sugars become qualitatively more similar. To distinguish between these two interpretations, Harvey Grill and I conditioned an aversion in a separate group of rats using 0.1 M maltose as the CS, and then examined the generalization profiles across the same test concentrations of the two sugars that we used when 0.1 M sucrose was the CS (Spector et al., 1988). If the two sugars are qualitatively identical but differ in their intensity at isomolar concentrations, then every test concentration of sucrose should have been avoided at least as much or more

875

than all of the concentrations of maltose. In addition, the order of avoidance should have been consistent with the generalization gradients established when 0.1 M sucrose was the CS. However, maltose, on average, was avoided more than the sucrose, suggesting that, although similar, the two sugars are not qualitatively identical. This conclusion confirmed by the fact that rats can be trained to discriminate between the two compounds across a range of concentrations in an operant conditioning procedure (Spector et al., 1997). The conditioned taste aversion generalization procedure has been a very popular and effective tool for assessing qualitative similarity among taste compounds (Dugas du Villard et al., 1981; Jakinovich, Jr., 1982; Nachman, 1963; Ninomiya et al., 1984; Nissenbaum and Sclafani, 1987a; Nowlis et al., 1980; Spector and Grill, 1988; St. John et al., 1997b; Tapper and Halpern, 1968). Incorporation of the procedural recommendations mentioned above should help minimize potential complications and strengthen interpretation of the results. As with any generalization procedure, it is important to discern along which stimulus dimension generalization is taking place. The best way to address the intensity issue in interpreting the generalization of a conditioned taste aversion is to design experiments in which each stimulus also serves as a CS. A failure for a taste aversion to a CS to generalize to a given test stimulus should also be observed when the roles of the taste stimuli are reversed in a separate group. If not, then the possibility that the two stimuli differ primarily in intensity, as opposed to quality, cannot be dismissed. Morrison (1967) used an operant conditioning procedure to determine qualitative generalization profiles. He trained three groups of rats to discriminate NaCl from either sucrose, HCl, or quinine sulfate by reinforcing presses on the correct lever of the two available. He then introduced test stimuli on 20% of the trials and on these presentations responses on either lever were reinforced. The test stimuli represented a variety of sodium and nonsodium salts. The relative proportions of responses on the lever associated with the non-NaCl stimulus (either quinine, sucrose, or HCl) for each of the groups were measured, and these profiles were compared across salt stimuli. This was a very creative use of an operant conditioning task to derive generalization patterns, but the interpretation of the results from this experiment was not as straightforward as it may first appear. First, concentration of the standard stimuli was not varied. Therefore, even if we accept the likelihood that the animals could distinguish the taste quality of NaCl from that of sucrose, quinine, and HCl, it is still possible, even likely, that the sensations of the two standard stimuli were not equal in intensity. This means that some portion of the generalized responding may have

876

Spector

been under the control of intensity especially if the quality of the test stimulus happened to be equally dissimilar from the two standard stimuli. Second, the meaning of responses to a test stimulus that were equally divided between the two levers is unclear. If a test stimulus shared qualitative features with both of the standard stimuli (e.g., similar to a mixture), then such an outcome would be expected. However, a similar outcome would be expected if the test stimulus had no qualitative feature in common with either of the standard stimuli. Thus, two markedly different inferences about the qualitative nature of the perception can be derived from a single outcome. Finally, as it turned out, when water was a test stimulus, the generalization pattern was similar to that generated by most of the nonsodium salt test stimuli. This means that it was difficult to distinguish patterns that arose because the test compounds were extremely weak from those that reflected something about the qualitative nature of a clearly detectable taste stimulus. Nevertheless, one could at least conclude from these patterns that the nonsodium salts were more similar to water than to sodium salts at the concentrations chosen. Some of the interpretive limitations just discussed could perhaps be relieved by varying the concentrations of the standard stimuli in training and varying the concentrations of the test stimuli. VIII.

FINAL REMARKS

Given the rapid developments in molecular biology and the technological advances in neuroscience research in general, the demand for rigorous methodology to assess taste function in animal models is likely to increase. What is the best psychophysical procedure to use? The answer depends on what aspect of taste processing one is interested in assessing. Practical constraints and the relevant theoretical framework should be considered in the choice. Because of the complex nature of the analysis, however, the use of a variety of taste-related behavioral tasks is preferable, because it synergetically strengthens the interpretation of results from each type of procedure, leading to a more comprehensive understanding of gustatory function in nonhuman animal models. ACKNOWLEDGMENTS I would like to thank Shachar Eylam, Laurie Geran, and Stacy Kopka for providing feedback on an earlier draft of the manuscript. I would also like to acknowledge the support of the National Institute on Deafness and Other Communication Disorders (K04-DC00104, R01-DC01628, R01-DC04574, P01-DC02641).

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42 Nutritional Implications of Taste and Smell Richard D. Mattes Purdue University, West Lafayette, Indiana, U.S.A.

pressures for more palatable products would have appeared, thus initiating the first purposeful genetic modifications of the food supply. As the diversity and abundance of foods increased, the freedom to express preferences also grew, thereby spawning a wide range of cuisines. The flavor principles defining these cuisines functioned, in part, to signal the safety of foods. However, the protection this offered was often subverted by unscrupulous vendors who discovered methods to manipulate the sensory properties of foods and beverages so as to mask product defects or limitations. Indeed, the problem became so widespread that it prompted passage of the Food Drug and Cosmetic Act of 1938, which made such practices illegal. A sharp expansion of both basic and applied research on the sensory properties of foods and ingestive behavior occurred during the mid-twentieth century. This was sparked by the growth of consumer concern over the loss of flavor in processed foods, as well as recognition by the government that wholly new food systems were required to support the nation’s poorly nourished military personnel. Only a few decades later, problems of overconsumption, rather than underconsumption, dominated the public health agenda. The evolving epidemic of overweight and diet-related chronic diseases (e.g., heart disease, diabetes) stimulated a new wave of research aimed at developing products that maintained their sensory characteristics with less of various problematic ingredients (e.g., sugar, salt, fat). The nutritional implications of modifying the food supply in this manner were (and are) unknown, as this was the first time in human evolution that the sensory properties of foods were purposively disassociated, on a

I. INTRODUCTION The chemical senses have been inextricably linked to food selection, nutritional status, and health throughout human evolution. Procurement of adequate energy and nutrients was central to survival, and human chemosensory systems played a key role in locating, ingesting, and metabolizing foods and the nutrients they supply. Olfaction, the distance sense, is often viewed as instrumental in locating foods in the environment. Taste, on the other hand, is commonly regarded as the final gatekeeper for ingestive decisions, with the different qualities conveying information about the nutrient content and/or safety of foods. Sweetness is generally associated with carbohydrates, saltiness with electrolytes, umami with protein, sourness with acids, and bitterness with toxic or harmful agents. Although current views hold that fat is largely identified by its textural properties, accumulating evidence suggests there may be a taste component and this could aid in identification of this energy-dense food constituent (Gilbertson, 1998; Mattes, 1996). While such teleological arguments are compelling, they are clearly overly simplistic, since the associations are not absolute. For example, proteins and electrolytes can impart sweetness to foods, and bitter items are commonly ingested. Such inconsistencies can be explained, in part, by the flexibility afforded by learning. Cautious experimentation would have conveyed an advantage to our inquisitive ancestors by expanding the food supply (Glendinning, 1994; Mattes and Beauchamp, 2000). Further, as crops were cultivated and livestock domesticated, selective 881

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large scale, from their metabolic consequences. Today, the complexity of this interaction is widely recognized. Provision of adequate nutrition without oronasal chemosensory stimulation (e.g., total parenteral nutrition) commonly does not result in complete satiety (McCutcheon and Tennissen, 1989; Padilla and Grant, 1985), and use of high-intensity sweeteners and fat replacers have not led to reduced consumption of sugar, fat, or total energy (Mattes, 1998). The present chapter begins with a consideration of the interaction between hunger and chemosensory responsiveness. Current knowledge on the inherent associations between sensory function and food selection is then presented, along with evidence relating to whether these associations may be modified, intentionally (e.g., therapeutically) or otherwise. This is followed by a review of sensory influences on food digestion, nutrient absorption, and metabolism. Finally, current understanding of the effects of taste and smell abnormalities on diet and nutritional status is considered, and guidelines for the nutritional management of patients with chemosensory disorders are presented. A review of the importance of individual nutrients on chemosensory function has been published elsewhere (Mattes, 1999).

II.

HUNGER AND SENSORY FUNCTION

Hunger and taste sensations are components of multiple interrelated mechanisms that regulate energy and nutrient balance. Hunger promotes the initiation of feeding and sensory factors influence food choice. Often is it assumed that a common factor (e.g., blood glucose) (Bedard and Weingarten, 1989) influences both and that they work in tandem, an assumption that receives some support from electrophysiological studies in primates (Rolls, 1997; Scott, 1992). Although several early studies reported a reliable direct relationship between appetite and both olfactory and taste thresholds (Goetzl et al., 1947, 1950), other work has failed to note associations with sensitivity measures of either sensory system (Janowitz and Grossman, 1949; Pangborn, 1959). A recent study observed a direct association between a food’s satiety value (inhibition of feeding) and ability to reduce threshold sensitivity to glucose (Crovetti et al., 1997), but this work did not control for cognitive factors (e.g., expected satiety value of the experimental food and impact on taste) and reporting biases (Moore et al., 1965). Intensity scaling of glucose, sodium chloride, citric acid, and quinine sulfate also appear to be invariant with hunger status (Laeng et al., 1993; Looy and Weingarten, 1991; Moskowitz et al., 1976; Rolls et al., 1983; Scherr and King, 1982).

A strong and more consistent relationship is reported between hunger and food liking or preferences (e.g., Cabanac, 1971; Lozano, et al., 1999; Rolls et al., 1983; Scherr and King, 1982). The most pronounced effects have been observed when hedonic ratings were obtained for a sweetener before and after consumption of a satiating load of glucose (Esses and Herman, 1984; Moskowitz et al., 1976). However, interpretation of these data may require qualification based on an individual’s general hedonic reaction to strong sweet stimuli. It has been proposed that hunger status exerts an effect on hedonic ratings to sucrose only in those who are not “sweet likers” (Laeng et al., 1993; Looy and Weingarten, 1991). Interestingly, greater effects have been noted in women than in men (Laeng et al., 1993). Hunger can be altered by both metabolic and sensory factors. The latter is evidenced by the common practice of the desire to indulge in a palatable dessert after having eaten a satiating meal. Evidence that sweeteners stimulate hunger comes from the observation that preloads of high-intensity sweeteners in water produce a more rapid rebound in hunger sensations than does water alone (Blundell et al., 1988). A heightened appetite has also been reported after chewing gum sweetened with aspartame, although the effect was not dose related and varied according to sex and time postexposure (Tordoff and Alleva, 1990). In contrast to these observations are studies finding no effect on hunger from high-intensity sweeteners added to beverages (Birch et al., 1988; Black et al., 1991; Canty and Chan, 1991; Lavin et al., 1997), breakfast cereal (Mattes 1990a), cheese (Drewnowski et al., 1994), gelatin (Rolls et al., 1986), or a milkshake (Brala and Hagen, 1983). Furthermore, with rare exception (e.g., Blundell et al., 1988; Brala and Hagen, 1983), chronic use of highintensity sweeteners does not appear to promote increased food intake (Stellman and Garfinkel, 1986). Thus, while sweet stimuli may enhance appetite under conditions where foods or beverages contain no energy value, the effect is ameliorated when sweetness is accompanied by ingestion of an energy load. Interestingly, nonsweet oral stimuli may also influence hunger. For example, exposure to salt in energy-free soup broth increases hunger sensations relative to ratings following ingestion of hot water or broth containing an energy source (Mattes 1994). It may be that sensory exposure elicits cephalic phase responses (described below) that initiate digestive processes that, in the absence of a metabolic challenge, heighten awareness of the need for energy (Tordoff, 1988). Palatability has also been reported to enhance hunger (Hill et al., 1984; Rogers and Schutz, 1992), possibly through the same mechanisms (Rogers and Schutz, 1992).

Nutritional Implications of Taste and Smell

III. SENSORY INFLUENCES ON MACRONUTRIENT DETECTION AND SELECTION Surveys of consumers and health care professionals in academia, medicine, industry, and government consistently support a primary role for sensory factors in food selection (Giese, 1994; National Dairy Council, 1995). Despite growing concerns about the health implications of dietary constituents and food safety, these factors are less important than sensory properties in influencing food selection. Sensory factors govern food choice by several mechanisms. First, the sensory variety provided by different foods is rewarding (Young, 1959). Monotonous diets have long been promoted for weight loss. They are effective because intake declines due to decreased acceptability. However, weight loss diets based on monotony are typically unsuccessful in the long term because the motivation for more varied sensory input eventually overwhelms the desire to lose weight. By promoting ingestion of items with varied sensory characteristics, diet diversity and the likelihood of adequate and balanced nutrient consumption is enhanced (Rolls, 1986). Second, certain forms of sensory stimulation are inherently appealing (e.g., sweetness, saltiness) or aversive (bitterness). Where food is abundant, convenient and inexpensive, these qualities can promote food ingestion or lead to its rejection. Third, chemical cues provide an early warning about potential food spoilage and toxicants. The high incidence of food poisonings, 76 million illnesses and 5 million deaths per year (Mead et al., 1999), indicate the systems are not perfect, but they undoubtedly make some contribution. Fourth, through experience, sensory cues acquire predictive power for the metabolic consequences of ingesting specific foods. This provides a basis for deciding on the types and amounts of items to be consumed (Tepper and Farkas, 1993). Olfactory cues contribute substantially to flavor perception, but the prevailing view holds that information about the macronutrient content of foods is primarily obtained through gustatory input. The degree to which sensory cues for each energy-yielding nutrient and sodium are inherent and modifiable is fundamental to understanding the importance of taste to nutrition since these food constituents have been implicated in many diet-related chronic disorders (e.g., obesity, cancer, hypertension, diabetes). A.

Carbohydrate

There is strong evidence that humans possess an innate liking for sweetness. Fetal drinking is stimulated after injection of sodium saccharine into the amniotic fluid (DeSnoo, 1937). This may be due to general arousal, but is

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interpreted as a positive hedonic response. Comparable treatment with an unpleasant oral stimulus, as rated by children, inhibits fetal drinking (Liley, 1972). Stronger and more frequent sucking is observed upon initial oral exposure to glucose or sucrose in preterm infants with little or no extrauterine taste experience (Maone et al., 1990; Tatzer et al., 1985). Full-term newborns can discriminate between sugars and concentrations of a single sugar (Desor, 1975). They also exhibit more vigorous sucking as well as “positive” mimetic (e.g., smiling) (Bergamasco and Beraldo, 1990; Steiner, 1977) and autonomic (e.g., increased heart rate) (Crook, 1979) responses following oral exposure to the sweet taste. Recent evidence indicates that early sucking responses are predictive of weight gain at one year of life (Stunkard et al., 1999). Although concern about entraining a preference for sweet, high-sugar foods through early exposure is commonly expressed (Jerome, 1977; LeMagnen, 1977), few studies have empirically addressed this issue. The limited data available do not, in fact, support such a notion. For example, in one study, equivalent preference ratings were observed for a fruit-flavored beverage in (1) children not regularly exposed to sugar water during the first 2 years of life, (2) children exposed to sugar water, but only up to 6 months of age, and (3) children receiving exposure for greater than 6 months (Beauchamp and Moran, 1984). The frequency of consumption of sweet foods was also similar among these groups. The nonexposed group did consume more of a model sugar solution, but the dietary relevance of this stimulus is unclear. Clinically significant chronic energy deficiency early in life, which could be hypothesized to promote a conditioned preference or craving for energy-yielding nutrients, also has not been associated with heightened intake under controlled testing conditions relative to healthy peers (Vazques et al., 1982). This is not attributable to an inability to detect the sweet stimulus since both healthy and protein-calorie malnourished infants prefer a sweet solution to one that is unsweetened. Evidence that reduction of body weight intensifies sensory responsiveness and may, thereby, contribute to rebound weight gain is mixed (Kleifield and Lowe, 1991; Rodin, 1975; Rodin et al., 1976). The positive data may simply be attributable to an effect of hunger, which may increase the preference for sweets. Limited data indicate that the palatability of more concentrated sweet solutions is diminished in adults relative to adolescents (Desor et al., 1975), although the mechanism is uncertain. Furthermore, studies comparing infrequent and regular users of sweeteners and sweet foods have yielded mixed findings on the effects of exposure frequency on palatability ratings for sweetness. Only nonsignificant positive trends were noted between

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self-reported frequency of intake of 32 sweet foods and ratings for the pleasantness of self-optimized beverages or a set of experimentally prepared samples in 51 young adults (Pangborn and Giovanni, 1984). The low, medium, and high consumers of sweet foods assigned comparable intensity ratings for prepared samples indicating there was no effect of exposure frequency on ability to assign relative intensity ratings to graded samples. Other work revealed significant associations (rs
0.46–0.55) between use of sweeteners and peak hedonic ratings for fruit-flavored solutions with graded levels of sucrose, fructose, or aspartame in women with gestational diabetes mellitus (Tepper et al., 1996). Nonsignificant positive correlations were observed in healthy controls. Controlled studies of the effects of restricted nutritive sweetener use on the preferred sweetness level of foods are lacking (i.e., acclamation to a sweetener-restricted diet). Ordinarily, such data would be available from dietary intervention studies, but the widespread availability of reasonably acceptable high-intensity sweeteners makes reduction of sweet foods and beverages unnecessary where use of sweeteners is undesirable. Thus, the extent to which the preference for sweetness can be purposefully modified is not known. B. Protein Preliminary evidence suggests that humans are able to detect sensory cues from amino acids during the perinatal period and find them pleasant (Steiner, 1992). In light of evidence that maternal diet during pregnancy may influence protein selection by rat pups (Leprohon and Anderson, 1980; Morris and Anderson, 1986), it is not clear if this is a learned or inherent behavior. Ingestion of an adequate quantity and balance of amino acids is central to survival, as protein synthesis will cease when any single essential amino acid is lacking. There is evidence that chemosensory cues from foods and beverages may be used to prevent or correct such a situation. One possible mechanism may entail associative learning (Gibson and Booth, 1986; Gietzen et al., 1992; Naito-Hoopes et al., 1993). According to this hypothesis, the sensory properties of foods supporting adequate nutrition acquire a positive hedonic valence, whereas those that fail to provide adequate nutrition and lead to malaise take on a negative hedonic valence. Subsequent food choices are then guided by the acquired hedonic information. This appears to be the mechanism by which rats correct thiamine deficiency (Rozin, 1967). Another, but not necessarily mutually exclusive, mechanism is that sensory cues from proteins or amino acids are detected directly, perhaps as a result of innate mechanisms, and serve to modify ingestive behavior.

Mattes

In rats, for example, elimination of a single essential amino acid elicits a prompt shift of ingestive behavior to dietary sources that may correct the imbalance (Leung and Rogers, 1986; Leung et al., 1968). The speed of the corrective dietary shift suggests learning is not involved (Deutsch et al., 1989). Evidence for a similar phenomenon in humans comes from observations that proteinenergy malnourished (PEM) Mexican infants exhibit a heightened preference for novel soups supplemented with casein hydrolysate relative to an unsupplemented version (Vazquez et al., 1982). This is not apparent in protein replete infants. Further, the responses of the infants with PEM appear to be specific to the protein-supplemented stimuli. Elderly individuals with marginally adequate protein status have also been found to assign higher hedonic ratings to soups supplemented with casein hydrolysates during their first exposures than to unsupplemented soups (Murphy and Withee, 1987). However, substantive questions remain about an inherent chemosensory-based compensatory mechanism. Individual amino acids and proteins have complex tastes that encompass all of the basic qualities, and many have the same predominant tastes (Kato et al., 1989; Kemp and Birch, 1992; Schiffman and Dackis, 1975; Schiffman et al., 1981). This would complicate discrimination unless additional information is gleaned from side tastes, odors, textures, and temporal cues (Kemp and Birch, 1992). Nongustatory sensory attributes of amino acids exist, but have not been well characterized in humans (Schiffman and Dackis, 1975). A cultural component to perception of amino acids also has been documented, raising the possibility that preferences may be modifiable through exposure frequency. Thus, it has been reported that the Japanese, relative to Australians, have lower threshold sensitivity for, and give higher hedonic ratings to, monosodium glutamate and 5 nucleotides (Ishii et al., 1992; Schiffman et al., 1994; Prescott et al., 1992). These compounds are used more extensively in Asian foods. This pattern is not observed for sweetness, saltiness, or bitterness. Whether these cultural patterns reflect true cultural differences of gustatory function or simply attention to the sensation is not known. C.

Fat

The prevailing view is that fats are perceived by their textural properties (Mela et al., 1994). However, accumulating evidence suggests there may be an olfactory or gustatory component. Electrophysiological studies have yielded the intriguing observation that taste receptor cells are activated by essential fatty acids (Gilbertson, 1998; Tsuruta et al., 1999). Further, oral stimulation with dietary

Nutritional Implications of Taste and Smell

fat alters lipid metabolism, whereas fat replacers with indistinguishable textural properties are not effective (Mattes, 2001). Human data are not highly supportive of an inherent preference for dietary fat. Although fetal drinking is inhibited by injection of iodinated poppy seed oil into the amniotic fluid (Liley, 1972), it is not known if the fat component is responsible. If the fat was appealing, it appears not sufficiently so to override the negative component. Infants provided breast milk or formula varying in fat content do not differentially consume them (Chan et al., 1979; D. J. Mela, personal communication; Nysenbaum and Smart, 1982; Woolridge et al., 1980). One study noted a stronger sucking response to a higher fat formula (Nysenbaum and Smart, 1982), but this has not been consistently replicated (Drewett, 1982; Woolridge et al., 1980). It is presently not possible to determine whether the lack of preference for higher fat stimuli in infants is due to a lack of perceptual ability or differential palatability. Early exposure to a high-fat diet results in a stable preference for the diet in rats (Warwick et al., 1990). No data are available on the effects of early exposure on longterm preferences in human newborns. However, preferences can be modified through dietary experience in adults. This is widely experienced by individuals who switch from whole to low-fat milk for health reasons and develop a true preference for the sensory properties of the latter. Such a hedonic shift has also been demonstrated more generally among individuals required to restrict sensory exposure to dietary fats or replacers for 12 weeks (Mattes, 1993). No differences were observed in ability to discriminate fat levels in foods, indicating the hedonic shift is not attributable to altered perceptual ability. Less tightly controlled restrictions have yielded equivocal effects on the hedonics of dietary fats (Pangborn and Giovanni, 1984; Pangborn et al., 1985). Another study that explored the effects of dietary restriction to specific high-fat items and hedonic ratings for them found no significant effects. Differences in the discriminability of fat in individual foods and motivation for consuming them (e.g., health, sensory appeal) may account for these seemingly inconsistent findings (Mela et al., 1993). D.

Ethanol

In rats, in utero exposure to ethanol selectively alters responsivity to its odor postnatally (Dominguez et al., 1996). Similar studies have not been conducted in humans, but breastfeeding infants are able to detect ethanol in milk (Mennella, 1997). Its presence leads to increased sucking and decreased consumption, the latter effect possibly reflecting reduced milk availability in ethanol-drinking

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mothers (Mennella, 1997). The increased sucking could be due to an inherent liking for the odor/taste of the stimulus, the rewarding properties of sensory variety, or familiarity through repeated exposure in utero. The former seems unlikely since bitter stimuli are typically rejected by newborns (Rosenstein and Oster, 1990); however, this may not be the quality perceived by newborns at the low levels present in milk. There are marked species differences in the perceived quality of ethanol. Although other factors are also involved, some animals (e.g., C57BL/6ByJ mice, Roman high-avoidance rats) consume high levels of ethanol when available, whereas others (e.g., 129/J mice, Roman lowavoidance rats) tend to reject it on the basis of its taste (Razafimanalina et al., 1996; Stewart et al., 1994). This raises questions about the suitability of animal models for understanding human taste responsiveness. Human data are available supporting the predominance of both sweetness and bitterness of ethanol at threshold concentrations (Diamant et al., 1963; Martin and Pangborn, 1970; Wilson et al., 1973). Recently we found that approximately 60% of 50 nonalcoholic adults rated ethanol as predominantly bitter at threshold, whereas only 6% reported sweetness. The proportions were 75% and 5% one dilution step above threshold (a doubling of concentration). At higher concentrations, ethanol is also perceived as bitter and enhances the bitterness of other bitter compounds in foods (Noble, 1994). Studies on the palatability of ethanol are difficult to interpret because of the strong cultural influences on its acceptability and its pharmacological effects. Selective breeding studies in rats clearly highlight a genetic component that may be expressed through varying behavioral responses to its sensory qualities (Bachmanov et al., 1996a, 1996b, Kiefer et al., 1995; Razafimanalina et al., 1996). A genetic component has also been proposed in humans. For example, the genetically determined sensitivity to phenylthiocarbaminde (PTC) or propylthiouracil (PROP) has been associated with ethanol sensitivity (Bartoshuk, 1993), and there is evidence of a higher proportion of PROP nontasters among the children of alcoholics than nonalcoholics (Pelchat and Danowski, 1992). However, conflicting data have also been published (Kranzler et al., 1996; Noble, 1994). The importance of the sensory properties of ethanol on ingestion by humans has not been established. Among nonalcoholics, dislike of the sensory properties of ethanol is a commonly cited reason for abstinence (Moore and Weiss, 1995). In one study, adults asked to indicate the primary reason for alcohol use ranked flavor eighth out of 13 volunteered explanations. Preceding flavor were explanations related to social status, physiological effects,

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and psychological properties. Mixed findings have been reported on an association between taste thresholds for ethanol in model solutions and ingestion of the compound (Kranzler et al., 1996; Mattes, 1994). This may reflect a pharmacological or exposure effect since abstinence for 3 weeks results in a reduction of threshold sensitivity (Settle, 1978). Chronic exposure to ethanol leads to increased acceptance in rats (Kiefer et al., 1994). A pharmacological basis for differences in intake has been proposed as well (DeWit et al., 1989; Mattes 1994), but may ultimately relate back to sensory factors if the rewarding properties serve to condition a preference to the chemosensory characteristics of ethanol (Settle, 1978). Chronic alcohol abuse is associated with olfactory impairment (i.e., elevated thresholds, reduced scaling, and identification performance), as well as reduced gustatory intensity ratings that may persist even after detoxification (DiTraglia et al., 1991; Jones et al., 1975, 1978). E.

Sodium Chloride

Human neonates show similar ingestive and facial responses to saline and water, suggesting they are either insensitive or indifferent to the taste of salt (Beauchamp et al., 1986; Desor et al., 1973; Rosenstein and Oster, 1990). However, there is clear evidence of sensitivity and positive hedonic responsiveness to saline solutions by 4 months of age that continues through 2 years of age (Beauchamp et al., 1986). Despite the delay in overt acceptability, it has been proposed that the liking for saltiness is inherent (Beauchamp et al., 1991). In rats and sheep, the developmental lag in salt taste sensitivity coincides with maturation of the gustatory system for this quality (Hill and Mistretta, 1990). Knowledge of the developmental aspects of salt taste in humans is incomplete. A learning effect is also possible and clearly contributes to affective responses by 31-month-old infants. Such infants reject saline solutions that have questionable dietary relevance, but prefer salty foods to unsalted versions (Desor et al., 1973). There has been considerable concern regarding the influence of early exposure to the taste of salt on salt preferences throughout the life cycle. Short-term acceptability ratings of foods with varying salt content have been associated with salt exposure in 6-month-olds, but may simply reflect recent consumption (Harris and Booth, 1985). A longer-term effect has yet to be established. One study reported 3-month-olds that ingested formula containing 4.78 or 21.5 M Na/kJ for 5 months did not have higher salt use or preferences at eight years of age (Whitten and Stewart, 1980). Another study reported an association between salt use at 2 and 4 years of age

(Yeung et al., 1984), but it is not clear whether this was indicative of the establishment of a specific salt preference or familial salt use. Marked early sodium depletion has been associated with heightened preference for and intake of salt later in life. This has been demonstrated in infants experiencing dietary chloride deficiency (Stein et al., 1996), in preterm neonates treated with diuretics (Leshem et al., 1998), and in infants with vomiting and diarrhea or whose mothers experienced severe vomiting while they were in utero (Leshem, 1998). Dietary experience can also modify the preferred level of salt in foods. Chronic (e.g., 8–12 weeks) reduction of dietary sodium consumption elicits a hedonic shift where the preferred salt content of foods is reduced (Beauchamp et al., 1987; Bertino et al., 1982; Blais et al., 1986; Elmer et al., 1987; Jeffery et al., 1984; Mattes, 1990b; Thaler et al., 1982; Witschi et al., 1985). Increased exposure leads to preferences for higher salt levels in foods (Bertino et al., 1986). The shift is not attributable to a change of taste sensitivity or taste intensity scaling ability (Bertino et al., 1982; DiNicolantonio et al., 1984; Mattes, 1990b). The shift occurs when total sodium intake is unchanged but sensory exposure is curtailed (Mattes, 1990b) and does not occur under the reverse conditions (Beauchamp et al., 1987). These observations suggest that exposure frequency is prepotent over metabolic factors in eliciting the shift. It is also salt specific (Bertino et al., 1981; Mattes, 1990b).

IV. CHEMOSENSORY INFLUENCES ON NUTRIENT UTILIZATION Sensory, especially chemosensory, stimulation elicits an array of physiological responses, termed preabsorptive or cephalic phase responses, that mimic those occurring during food (nutrient) ingestion, digestion, absorption, and metabolism. They are neurally mediated (primarily through the parasympathetic nervous system) and occur within seconds to minutes following exposure to foods. They are transient and typically small in magnitude relative to responses occurring during actual food ingestion. Although there are many exceptions, there generally is a hierarchy in stimulus efficacy in eliciting these responses: swallowing  mastication  taste  smell  sight, audition, or thought. Further, concurrent stimulation of multiple sensory systems typically elicits a larger response than stimulation of a single system. The functional role of cephalic phase responses is largely unexplored, but the prevailing view is that they initiate and augment the efficacy of the normal digestive, absorptive, and metabolic processes associated with eating (Jordan, 1969; Nicolaidis, 1977). They have been hypothesized to influence food selection (Simon et al., 1986;

Nutritional Implications of Taste and Smell

Steffens et al., 1990) and have been implicated in varied diet-related disorders including insulin resistance and obesity (Ionescu et al., 1988; Konturek et al., 1994), eating disorders (LeGoff et al., 1988), and ulcers (Konturek et al., 1981). The effective stimuli and nutritional significance of the better-documented cephalic phases are described in this section. A.

Cephalic Phase Salivary Response

No study has explored salivary flow and composition following stimulation of each sensory system individually, but a rough ordering can be derived from the literature. Voluntary control over salivation has been documented in humans, but not consistently (Jenkins and Dawes, 1966; White, 1978). Visual stimulation elicits a 1.5-fold increment over resting or cognitive stimulation (Hayashi, 1968; Pangborn et al., 1979). The addition of olfaction and then taste leads to 2-fold and 14-fold higher salivary flow rates, respectively. Relative to viewing and smelling food, mastication leads to about a 5-fold increment in flow (Richardson and Feldman, 1986). Since both appealing and aversive stimuli can stimulate flow, palatability is not a straightforward predictor of response (Christensen and Navazesh, 1984; Klajner et al., 1981; Pangborn and Berggren, 1973; Sahakian et al., 1981). Potent sialagogues include items that are sour, bitter, physically sharp, or difficult to clear from the mouth (Guinard et al., 1998). Higher cephalic phase flow rates have been observed with exposure to foods that are potent sialagogues during mastication, consistent with a learning effect (Chistensen and Navazesh, 1984). However, this is not observed uniformly (Pangborn et al., 1979). Although salivary enzymes contribute to macronutrient digestion, protein, fat, and carbohydrate are not potent sialagogues (Christensen, 1986; Matsuo and Yamamoto, 1989; Meyers and Epstein, 1997), and their primary influence may be on salivary composition. Sucrose and fructose have been reported to prompt the release of amylase-rich saliva (Kemmer and Malfertheiner, 1983, 1985). Repeated olfactory and gustatory stimulation leads to reduced salivary flow (Epstein et al., 1996) due to adaptation to the sensory stimulus rather than gland fatigue (Epstein et al., 1996; Wisniewski et al., 1992). The nutritional significance of the cephalic phase component of salivary flow has not been documented, but saliva does play a number of key related roles. It facilitates swallowing, promotes oral health and functionality of taste receptor cells (Bodner, 1991; Cano and RodriguezEchandia, 1980; Matsuo et al., 1997; Thesleff et al., 1988), alters the sensory properties and palatability of foods, initiates macronutrient digestion, and may alter the gastric

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and intestinal phases of digestion (Layer et al., 1986; Lebenthal, 1987; Malhourta, 1967). B.

Cephalic Phase Gastrointestinal Response

The thought of foods can elicit a weak cephalic phase gastric response (Moor and Motoki, 1979), but progressively higher levels of secretion are observed with olfactory and gustatory stimulation. The taste and smell of foods stimulate the release of gastrin and gastric acid (Feldman and Richardson, 1986) at levels up to a third of those occurring with food ingestion. Food palatability has a positive effect (Janowitz et al., 1950). Sensory stimulation, including differential effects by the macronutrients, influences additional aspects of gastrointestinal function as well. These include (1) release of digestive enzymes (trypsin, lipase, and chymotrypsin are elevated by 35–66, 16–50, and 25–60%, respectively, during modified sham feeding) (Novis et al., 1971), (2) secretion gut peptides (e.g., cholecystokinin, somatostatin, neurotensin) involved with intestinal absorption and satiety (Konturek et al., 1994; Wisen et al., 1992) (3) motility, and (4) gastric emptying (Cecil et al., 1999; Katschinski et al., 1992; Nagao et al., 1998; Rogers et al., 1993). Recent studies suggest oral exposure to dietary fat augments and prolongs the postprandial triacylglycerol concentration (Mattes, 1996), possibly through enhanced fat absorption (Ramirez, 1985) or decreased clearance. Given the evidence that postprandial lipemia is a risk factor for atherogenesis (Nafziger, 1994; Zilversmit, 1979), sensory exposure to fats may contribute to this problem. The cephalic phase gastrointestinal response has also been implicated in the etiology or severity of duodenal ulcers (Feldman and Richardson, 1986; Konturek et al., 1978; Thirlby and Feldman, 1984), irritable bowel syndrome, ulcerative colitis, and colonic diverticular disease (Rogers et al., 1993). C.

Cephalic Phase Pancreatic Exocrine Response

Combined visual, tactile, and olfactory stimulation elicit a pancreatic exocrine response, but not as large as that following gustatory stimulation (Sarles et al., 1968). Protein levels are selectively increased (Novis et al., 1971) and under certain conditions, lipase and amylase secretion rates can exceed those obtained with pharmacological agents (Sarles et al., 1968). Studies in dogs indicate the response is enhanced by palatable (i.e., sweet) relative to less palatable (sour, bitter) stimuli (Ohara et al., 1988). Food acceptability also modulates the response in humans (Sarles et al., 1968). The sensory-mediated release of an enzyme-rich pancreatic secretion could enhance digestive

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Mattes

processes, but its importance is uncertain due to the large reserve capacity for enzyme secretion by this organ (Brannon, 1990). D.

Cephalic Phase Pancreatic Endocrine Response

Insulin, pancreatic polypeptide, and glucagon (Teff and Engelman, 1996a; Teff et al., 1991) are all released following appropriate sensory stimulation, although only studies using sham feeding are available for the latter two hormones, thereby precluding identification of the independent effects of different stimuli (Glasbrenner et al., 1995; Schwartz et al., 1979). The relative potency of different sensory stimuli has also not been established. Early work indicated plasma insulin could be elevated by the thought of preferred foods (Johnson and Wildman, 1983), as well as visual, olfactory, and gustatory stimulation (Johnson and Wildman, 1983; Louis-Sylvestre and LeMagnon, 1980). Levels approximated 10–15% of concentrations observed 2 hours after an oral glucose load. More recent studies indicate that the perception of sweetness is not a sufficient stimulus and that mastication may be required (Abdallah et al., 1997; Teff et al., 1991, 1995). Moreover, carbohydrate is not necessary for the response (LeBlanc et al., 1996; Niijima et al., 1990). The role of palatability has also been called into question (Teff and Engelman, 1996b). Although effects remain to be confirmed, it appears that individual characteristics may modulate the response where greater insulin release occurs in restrained (i.e., individuals cognitively controlling food intake), as compared to nonrestrained, females (Teff and Engelman, 1996b) and African Americans relative to Caucasians (Saad et al., 1990). Across studies, 10–15% of subjects exhibit no response to sensory stimulation, suggesting there may be a subgroup of nonresponders, but this has not been established. The limited available data suggest cephalic stimulation may lead to a doubling of pancreatic polypeptide release and a 10–15% increment in glucose secretion. Accumulating evidence supports a functional role for the cephalic phase insulin response. Glucose tolerance is improved when sham feeding accompanies a glucose infusion (Lorentzen et al., 1987; Teff and Engelman, 1996a). Somatostatin inhibition of early insulin release leads to hyperglycemia and hyperinsulinemia (Calles-Escandon and Robins, 1987). Concurrent administration of insulin and meals leads to a twofold decrease in incremental blood glucose among insulin-dependent diabetics (Kraegen et al., 1981), and a 33% lower glycemic response among non-insulin-dependent diabetics (Bruce et al., 1988), as compared to responses when meals (and sensory stimulation) were delayed by only 30 minutes. To the extent that

small transient changes in plasma glucose prompt hunger sensations and meal initiation (Smith and Campfield, 1993), the cephalic phase insulin response may also influence food selection and ingestion. Insulin, pancreatic polypeptide, and glucagon also influence aspects of the gastric and intestinal phases of digestion. E.

Cephalic Phase Thermogenic Response

Exposure to the sight and smell of food leads to a thermogenic response in dogs that is comparable to the level following food ingestion (Diamond and LeBlanc, 1985). Work in humans indicates the response is positively related to food palatability (LeBlanc and Brondel, 1985; LeBlanc and Cabanac, 1989; Soucy and LeBlanc, 1999; Westrate et al., 1990), especially in lean individuals (Hashkes et al., 1997). Intragastric feeding is not associated with a rise in thermogenesis, and blocking the cephalic component with somatostatin or atropine infusion (Calles-Escandon and Robbins, 1987) diminishes the postprandial increment in heat production. The thermic effect of food represents about 8–10% of total energy expenditure, but can be greater with imbalanced diets (Stock, 1999). Consequently, this response may contribute to energy balance and body weight. F.

Cephalic Phase Cardiovascular Response

Cardiac output and stroke volume transiently decline by 11–13% and heart rate increases by 5–10% to the sight and smell of food (Andersen et al., 1992; Fagius and Berne, 1994). Macronutrient specificity has also been reported, with protein  carbohydrate  fat. A 31% increase in mesenteric resistance has been noted in dogs permitted only to view and smell food (Vatner et al., 1970). While nutrient absorption from the gastrointestinal tract is very efficient, it is directly related to blood flow (Winne, 1979). Consequently, the cephalic phase hemodynamic effects may influence the kinetics of nutrient absorption. This, in turn, may alter endocrine responses (e.g., insulin and vasopressin release are largely determined by the rate of glucose and ethanol absorption, respectively). G.

Cephalic Phase Renal Response

Oral exposure to graded saline solutions alters the acute diuretic and natriuretic response in a stepwise fashion (Akaishi et al., 1991). Osmolality decreases or increases by 10–20% with exposure to hypotonic and hypertonic solutions, respectively. No response occurs following isotonic saline exposure. Comparable responses are observed with sodium chloride and mannitol challenges,

Nutritional Implications of Taste and Smell

suggesting the salient stimulus attribute is osmolality (Gebruers et al., 1985). However, palatability also alters the response. Decreased diuresis is observed with ingestion of a less palatable beverage (Nagao et al., 1999). The nutritional significance of this response is not clear, but it may contribute to fluid and electrolyte balance. V. EFFECTS OF PRIMARY CHEMOSENSORY DISORDERS ON FOOD ACCEPTABILITY, DIET, AND NUTRITIONAL STATUS Data on the dietary and nutritional implications of specific primary chemosensory disorders are available from several chemosensory clinical research centers. However, the number of patients in specific diagnostic groupings is often limited, and evaluation and assessment measures differ between centers. In addition, patient characteristics may vary between clinics and account for apparent discrepancies in reported findings. Thus, while the following summary represents the current state of knowledge on this issue, the findings must be interpreted cautiously. It must also be noted that the numerous published reports of chemosensory abnormalities associated with specific pathologies are not reviewed here since many of the dietary and nutritional complications observed in these patients may be attributable to aspects of their pathology and not directly related to their chemosensory complaint. The reader is referred to Chapter xx for more specific information on this point. A.

Anosmia

A total loss of the ability to smell is one of the most common complaints of patients presenting for treatment to chemosensory clinical research centers (Deems et al., 1991; Ferris et al., 1986; Goodspeed et al., 1987; Mattes and Cowart, 1994). Findings from several centers indicate that approximately 50% of such patients express food complaints (Ferris et al., 1985; Mattes and Cowart, 1994). Twenty to forty percent of patients with noncongenital anosmia report an increased use of sugar and seasonings (Ferris et al., 1985; Mattes and Cowart, 1994). While this dietary adjustment may enhance the appeal of foods and eating, it also poses a potential problem for individuals who must control their intake of sugar (e.g., diabetics) and sodium (e.g., patients with salt-sensitive hypertension). It is noteworthy that one study found that the prevalence of food complaints is inversely related to the duration of the sensory loss and is lowest in congenital anosmics whose responses resemble control subjects (Ferris et al., 1985). These patients apparently learn to compensate for their

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dysfunction by accentuating other aspects (i.e., taste, temperature, texture) of foods (Davidson et al., 1987). This may explain, in part, a report that the food preference ratings of anosmics were only slightly lower than those of healthy controls. Differences that were noted varied by food classes, with the largest discrepancy occurring for homogeneous items (e.g., puddings, sherbets) (Doty, 1977). Mean energy and macronutrient intake, as estimated by diet records, are similar in patients with anosmia and healthy controls (Ferris et al., 1985; Mattes and Cowart, 1994). Body weight, body mass index, and selected anthropometric indices (i.e., triceps skinfold, midarm circumference) are also comparable and within normal ranges. However, approximately 14% of anosmic patients experience a gain of body weight exceeding 10% of their predisorder weight and about 6.5% lose at least this amount. A weight change of this magnitude would not be expected in a healthy nondieting individual. At present, there is no predictive index of nutritional risk for patients with recent onset of symptoms. Individuals who experience marked changes of body weight do not differ by age, gender, duration of disorder, or etiology (Mattes and Cowart, 1994). B.

Hyposmia

Estimates of the prevalence of food complaints in hyposmic patients range from 31 to almost 80% (Ferris et al., 1986; Mattes and Cowart, 1994). This wide range in prevalence estimates may be attributable to variations in sample sizes, differences in diagnostic criteria or patient characteristics. For example, a diagnosis of hyposmia may include patients with small reductions in sensitivity to a limited number of odors as well as individuals who are nearly anosmic. Twenty to fifty percent of patients with hyposmia report altering their eating patterns and use of seasonings. Decreased appetite is reported by 10–20% of such patients and a small proportion experience an increase. Mean nutrient intake levels and biochemical indices of nutritional status of hyposmic patients are comparable to healthy controls. Over 85% of such patients maintain a stable body weight subsequent to the onset of their disorder. Among those who do experience excursions exceeding 10% of their predisorder weight, there is a tendency towards weight loss; 10.6% have a loss of this magnitude or greater whereas 1.5% of patients experience such an increase. No reliable predictive index of nutritional risk has been identified for this group of patients. C.

Dysosmia

In one study, 24% of patients diagnosed as dysosmic (i.e., having distorted smell sensations) reported a

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disorder-related decrease of appetite and 83% indicated that they enjoyed food less (Mattes and Cowart, 1994). Approximately 60% stated that they had altered their eating patterns and use of seasonings. The majority of patients with dysosmia maintain adequate nutritional status, but individual responses may warrant aggressive dietary intervention. At present there are no means to identify patients at high nutritional risk. D.

Ageusia

Complete loss of taste is rare, accounting for less than 1% of patients reporting to chemosensory clinical research centers (Deems et al., 1991; Ferris et al., 1986; Goodspeed et al., 1987; Mattes and Cowart, 1994). With available data, it is impossible to draw any general conclusions about the effect of this sensory disorder on diet and nutritional status. Loss of appetite has been observed (Ferris et al., 1986), but increased and decreased body weight have also been reported. E.

Hypogeusia

The incidence of this taste disorder, independent of other chemosensory abnormalities, is low. Some, but not all, report decreased enjoyment of food and decreased appetite (Ferris et al., 1986). When present in combination with hyposmia or anosmia, hypogeusia leads to only a slightly higher incidence of food complaints relative to patients with hyposmia or anosmia alone (Ferris et al., 1986). When concurrently present with dysgeusia, hyposmia does not exacerbate the changes in dietary intake noted in patients with dysgeusia alone (Mattes-Kulig and Henkin, 1985). F.

Dysgeusia

The only data on patients with a single diagnosis of dysgeusia indicate the incidence of decreased appetite is approximately 30% and decreased enjoyment of food is about 70% (Mattes and Cowart, 1994). Roughly 60% of such patients alter their eating patterns, and 40% modify their use of seasonings. Based upon reports from patients who may have had concurrent diminutions of sensory function, the foods most distorted in taste are meats, fresh fruits, coffee, eggs, and carbonated beverages (Markley et al., 1983). However, this is highly idiosyncratic, and affected items are not necessarily consumed in lesser amounts. A significant decline in fruit and vegetable intake has been observed, especially for individuals with the most severe dysgeusic symptoms. Mean nutrient and energy intake among dysgeusics with no additional sensory disorders were within normal ranges. They were comparable to control subjects in one report (Mattes and Cowart,

1994) and reduced in another (Mattes-Kulig and Henkin, 1985). In neither of these studies was energy intake related to the duration of symptoms. Approximately 15–20% of dysgeusic patients experience a clinically significant change of body weight and/or body composition (Mattes and Cowart, 1994; Mattes-Kulig and Henkin, 1985). In summary, a high proportion of patients with a primary complaint of a taste or smell disorder experience a loss of appetite and decreased enjoyment of food. Nutrient and energy intake is generally maintained at adequate levels, but individual patients with any of the above diagnoses may experience a marked increase or decrease of body weight. Increases may occur if patients are attempting to achieve a missed level of sensory stimulation or to mask an unpleasant sensation. A decline in intake often occurs if foods contribute to an unpleasant sensation or because of frustration with the lack of appeal of foods. Questioning patients about their compensatory dietary response may provide the best prognostic index of dietary change. Patients reporting increased or decreased intake are disproportionately represented in the subgroup of individuals experiencing shifts of body weight exceeding 10% of their predisorder weight. In a study of 396 patients (Mattes and Cowart, 1994), 34 individuals (8.7% of the sample) reported a compensatory increase in intake and represented 35.3% of the group that gained more than 10% of their predisorder weight. The 47 patients (12.0% of the sample) who reported decreased intake constituted 38.3% of the subgroup losing at least this amount of weight. VI. NUTRITIONAL IMPLICATIONS OF CHEMOSENSORY ABNORMALITIES IN PATIENTS WITH SELECTED CHRONIC DISORDERS A.

Cancer

There is a high incidence of chemosensory complaints among patients with untreated cancers (Gallagher and Tweedle, 1983; e.g., Williams and Cohen, 1978). This is especially true for those with more advanced disease (DeWys, 1974), but such symptoms are not a reliable prognostic index (DeWys, 1974; Walsh et al., 1982). The nature of reported complaints is highly variable, even among patients with similar pathologies. Reports range from enhanced or diminished sensitivity to specific taste qualities to generalized alterations (Trant et al., 1982). There is no definitive evidence implicating chemosensory disorders with diminished appetite, altered food preferences or loss of body weight in untreated cancer patients (Bruera et al., 1984; Carson and Gormican, 1977; Trant et al., 1982).

Nutritional Implications of Taste and Smell

External radiation therapy that damages sensory and supporting tissues (e.g., salivary glands) can lead to dysgeusia and/or hypogeusia (Conger and Wells, 1969; Cooper, 1968; Mossman, 1986; Ophir et al., 1988). When partial loss occurs, salt and bitter are the most severely affected qualities. Taste impairment is generally noted during the second week of treatment (Mossman, 1994). Because the recovery rates for different aspects of gustatory function vary and individual awareness of these discrepancies differs, it is difficult to counsel patients regarding the expected duration of symptoms. Typically, the ability to detect or recognize low concentrations of a taste quality recovers within several months (Conger, 1973; Kalmus and Farnsworth, 1959), whereas suprathreshold sensations may require a year or more to normalize (Mossman et al., 1982). Olfactory loss also occurs and is persistent (Ophir et al., 1988). Chemotherapy regimens may produce gustatory disturbances as well (Carson and Gormican, 1977; Mulder et al., 1983), but the problem is generally less common and less severe than that noted in patients receiving radiotherapy to the head and neck regions. Reduced food intake and loss of body weight are more common in patients experiencing radiotherapy-related chemosensory disturbances than in those with alterations attributable to the pathology alone (Bolze et al., 1982; Mossman and Henkin, 1978). Dysgeusia has also been linked to reduced energy intake and a higher incidence of food aversions, especially for meats (Markley et al., 1983). However, it cannot be assumed that this is primarily attributable to the chemosensory disturbance. Other complications of the disease or treatment (e.g., oral lesions, difficulty swallowing) may contribute to a reduction of appetite. Some efficacy of zinc sulfate treatment in managing treatment-related taste disorders in patients with low zinc levels has been reported, but changes in taste were not associated with changes of body weight (Ripamonti et al., 1998). B.

HIV Infection

In one study, 72% of HIV-infected patients reported some chemosensory abnormality (Mattes et al., 1995). Assessment of olfactory ability in infected, but healthy, patients reveals little or no decrement (Lehrner et al., 1995; Mattes et al., 1995). However, the severity of olfactory complications increases with progression of disease (Lehrner et al., 1995). The most marked olfactory changes occur in patients with AIDS dementia complex (Brody et al., 1991). Taste is less affected (Mattes et al., 1995), and where complaints are noted, they may be treatment related. The mechanisms responsible for the loss of appetite often

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accompanying HIV infection have not been fully characterized, but the limited available data do not support a substantive role for chemosensory disturbances. C.

Hypertension

Various aspects of salt taste have been evaluated in selected subgroups of hypertensive patients. Detection thresholds are similar in hypertensive and normotensive individuals. In the subset of studies noting a discrepancy between patients and controls for recognition thresholds, values were higher for the former group (Mattes, 1984). Interestingly, hypertensive patients have normal or reduced salivary sodium levels (Neidermeier et al., 1956), which should enhance sensitivity to external stimulation by salt. No explanation for the conflicting evidence is apparent. Thiazide diuretics have been reported to increase salt taste sensitivity (Langford et al., 1976), although more recent studies have failed to confirm this effect (Mattes and Engelman, 1992; Mattes et al., 1988). Comparable intensity ratings for suprathreshold salt stimuli have been obtained from healthy controls and (1) unselected samples of hypertensive patients (Little and Brinner, 1985; Mattes et al., 1983; Pikielna et al., 1985), (2) patients with low normal plasma renin activity (Bernard et al., 1980), and (3) individuals classified as saltsensitive (Mattes and Falkner, 1989; Mattes et al., 1999). There is suggestive evidence that hypertensive subjects show larger shifts in salt intensity ratings when on a sodium-restricted diet as compared to controls (Zumkley et al., 1987), but the reliability and implications of this observation have not been determined. While a heightened preference for salt has been noted in several studies of hypertensive patients, the preponderance of evidence indicates hypertensive and normotensive individuals do not differ on this attribute (Little and Brinner, 1985; Mattes, 1984). Similar hedonic ratings have also been observed between salt-sensitive and non–salt-sensitive subjects (Mattes and Falkner, 1989; Mattes et al., 1999), although the latter group shows a greater increment in acceptability of reduced salt foods when on a salt-restricted diet (Mattes et al., 1999). There is no evidence of salt cravings in untreated or treated patients with hypertension. Dysgeusia and or taste loss have been associated with the use of several antihypertensive medications (e.g., captopril, spironolactone, ethacrynic acid, diazoxide) (Clee and Burrow, 1983; Coulter, 1988; Schiffman, 1983), but the problem generally resolves when the medication is eliminated (e.g., Boyd, 1993). The sensory changes are not specific to the salty taste.

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D.

Mattes

Hypothyroidism

Case histories indicate that hypothyroidism, hyperthyroidism, or pharmacological treatment of these conditions can result in alterations of olfactory sensitivity and odor recognition (Lewitt et al., 1989; Mackay-Sim, 1991; McConnell et al., 1976). Olfactory thresholds were elevated for some (e.g., pyridine, nitrobenzene), but not all (2-phenylethanol), test stimuli, suggesting the impairment is not generalized. Thresholds improve following hormone-replacement therapy (Deems et al., 1991), but patient complaints about sensation may persist. The mechanism(s) underlying olfactory changes in hypothyroidism have not been determined, but may include altered secretions in the nasal cavity, changes of the olfactory epithelium, and /or modified function of the olfactory bulb. The incidence of taste dysfunction in hypothyroid patients is not known, but estimates range up to 83% (Mackay-Sim, 1991; Mattes et al., 1986; McConnell et al., 1976). Patients with hypothyroidism express little interest in eating, but available evidence does not implicate a decrement in hedonic responses to foods (Lewitt et al., 1989; Mattes et al., 1986). A major problem for hypothyroid patients is dysgeusia, which may occur in as many as 39% of cases. Consistent with studies in rats showing no change in taste sensitivity coincident with propylthiouracil treatment (Brosvic et al., 1992), taste thresholds of patients with recent onset of hypothyroidism are comparable to controls (Mattes et al., 1986). In contrast, longer-standing deficiency may be associated with measurable changes (McConnell et al., 1976). Alterations of suprathreshold function appear to be impaired before changes of threshold sensitivity are manifest (Mattes et al., 1986). The degree of taste impairment is not correlated with disease severity (Pittman and Bischi, 1967). Ambiguous results have been obtained on measures of taste preferences, as different tests yield discrepant findings. Studies in rats have also noted inconsistent effects of hypothyroidism on hedonic responses (Brosvic et al., 1992; Rivlin et al., 1977). Restoration of normal hormone status leads to a normalization of threshold sensitivity and suprathreshold function as well as taste preferences (Deems et al., 1991; Mattes et al., 1986). However, burning mouth syndrome and dysgeusia have been observed in 23.1% and 20.5% of patients treated with levothyroxine, respectively (Deems et al., 1991). Burning mouth syndrome has been associated with decreased body mass index (Deems et al., 1991). Treatment of hyperthyroid patients with either methimazole or methylthiouracil can also impair taste sensitivity, but normal function returns upon cessation of treatment. The mechanism(s) underlying the gustatory changes associated with hypothyroidism have not been

elucidated but may include alterations of salivary secretion, central or peripheral neural function, cellular metabolism, and /or epithelial integrity. E.

Obesity

Studies of the sensory function of the obese have largely focused on responses to sweets and fats. Threshold sensitivity and suprathreshold intensity judgments of sweet stimuli are similar in the lean and obese (Drewnowski, 1987; Frijters and Rasmussen-Conrad, 1982; Grinker, 1978; Witherly et al., 1980). Reported hedonic responses to sweetness are more varied. Both heightened and depressed pleasantness ratings have been noted in obese individuals (Drewnowski, 1987; Rodin et al., 1976), but the preponderance of evidence indicates there is no association between such ratings and body weight (Frijters and Rasmussen-Conrad, 1982; Spitzer and Rodin, 1981). A decrease in the palatability of sweet foods has been observed following therapeutic jejunoileostomy in the obese (Rodin, 1980) and may contribute to the reduction of food intake following this procedure. However, changes in intestinal absorption and /or distention may also contribute (Sclafani and Koopmans, 1981). In contrast, hedonic ratings of sweetness reportedly rise during restriction of energy intake (Cabanac, 1971; Rodin et al., 1976). Whether this hedonic shift influences adherence to energy restricted diets is unknown. The obese have exhibited a greater preference for high-fat foods than for low-fat items compared to lean controls in some (Drewnowski et al., 1985), but not all, studies (Pangborn et al., 1985; Warwick and Schiffman, 1990). These apparently conflicting findings may be attributable to the types of stimuli used in testing. Hedonic responses to fat by the lean and obese may be differentially influenced by other characteristics of the stimulus such as sweetness and rheology (Drewnowski et al., 1985, 1989). In addition, sensory responsiveness may vary with the timing and duration of obesity (Drewnowski et al., 1990). Recently, a novel approach for weight reduction involving regulation of food intake based on hedonic shifts associated with eating has been proposed (Poothullil, 1999). Four of six patients instructed to terminate meals when palatability declined had lost more than 5 pounds after a year’s trial. F.

Diabetes

Disturbances of taste and smell occur in over 60% of patients with diabetes (Settle, 1991). The most consistent sensory change is an elevated glucose taste threshold in patients with glucose intolerance and in individuals with

Nutritional Implications of Taste and Smell

a family history of the disease. Taste perception of other qualities remains largely intact (Perros et al., 1996). The mechanisms have not been identified but may involve nerve degeneration, alterations in salivary glucose levels, and various biochemical changes. The specificity of changes and apparent genetic influence suggest an abnormality of glucose receptors in some cases, but evidence that the severity of gustatory symptoms increases with progressing neuropathy (Abbasi, 1981) supports a neural explanation in others. There are also reports of olfactory deficits associated with degenerative complications (LeFloch et al., 1993; Weinstock et al., 1993). The influence of sensory changes associated with diabetes on food selection has not been determined, but there is one report of heightened sweet preference and sweet food intake among women with gestational diabetes (Tepper and Seldner, 1999). G.

Renal Disease

An elevation of taste recognition thresholds has been observed in patients with renal disease. However, the qualities affected have varied across studies and patient groups. Some indicate sweet and sour are the most severely affected (Anon., 1981; Burge et al., 1979), whereas others observed greater impairment for bitter and salty tasting stimuli (Fernstrom et al., 1996; Middleton and AllmanFarinelli, 1999). Dialysis leads to an improvement of function (Anon., 1981; Burge et al., 1984). Renal transplantation has also normalized taste sensitivity but was not apparent for 12–18 months following surgery (Mahajan et al., 1984). Early studies suggested the impairment in taste sensitivity was related to reduced zinc status (Mahajan et al., 1980). The preponderance of data do not support this claim, although it may hold merit in the subgroup of patients with a documented marked deficiency (Anon., 1981; Burge et al., 1984). An explanation for the shift in threshold sensitivity has not been identified. In contrast, intensity ratings for suprathreshold concentrations of taste stimuli are similar in uremic patients and controls and within patients over the course of a dialysis treatment (Shapera et al., 1986; Shepherd et al., 1987). Preference ratings also are not markedly affected by this treatment. The interest in taste function in renal disease stems, in part, from its role in food selection, which must be controlled in this patient population. The failure to note an impact of renal disease on taste intensity judgments and preferences raises questions about the dietary significance of the noted pathology-related shifts in threshold sensitivity measures. Olfactory quality recognition performance is lower in renal patients than in healthy controls (Conrad et al., 1987;

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Schiffman et al., 1978), and in one study (Schiffman et al., 1978) patients rated food odors as less pleasant. Dialysis exerted no effect on performance in one study (Schiffman et al., 1978), but a further 5% decrement in another (Conrad et al., 1987). These observations on dialyzed patients indicate that uremic toxins do not account for the olfactory disturbances. The mechanism remains unknown, as does the influence of olfactory changes on food habits. H.

Liver Diseases

Decreased taste and smell threshold sensitivity occur in patients with cirrhosis of the liver (Burch et al., 1978; Garrett-Laster et al., 1984; Weismann et al., 1979) and acute viral hepatitis (Smith et al., 1976). Liver transplantation results in quality-specific (salt, phenethyl alcohol) improvement of these disorders (Bloomfeld et al., 1999). While correlations between chemosensory function and plasma levels of zinc, vitamin A, and protein have not been observed, supplementation with these nutrients may result in a normalization of sensation. Whether alterations of chemosensory function contribute to the reduction of appetite and changes of food preferences associated with hepatic pathology is unknown (Deems et al., 1993). It has also been hypothesized that taste disorders accompanying liver disease have a neural origin (Bergasa, 1998). In summary, chemosensory abnormalities have been documented in patients with a variety of health disorders. The extent to which a change of taste and/or smell alters food intake and thereby contributes to the etiology or sequelae of these diseases or hampers recovery by adversely influencing adherence to therapeutic diets or recuperative processes remains unknown.

VII. GUIDELINES FOR THE DIETARY MANAGEMENT OF PATIENTS WITH CHEMOSENSORY DISORDERS Because no predictable dietary response has been identified for any chemosensory abnormality, the dietary management of patients must be individualized. The approach used must be based upon sound nutritional principals (i.e., balance, variety, and moderation) since for many patients’ adherence will be required for extended periods of time. To be effective, it is necessary to address the full range of environmental and physiological factors influencing ingestive behavior. To date, no specialized dietary management approach has been developed and tested, but several recommendations may be made based upon the available data. One important issue concerns the use of nutrient supplements. It is preferable to obtain all

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nutrition from foods as this route is most likely to provide nutrients in biologically available forms in reasonably balanced proportions. If this is not feasible, a supplement that provides up to 100% of the recommended dietary allowances for the individual may be appropriate. Patients should be advised that ingestion of high levels of specific nutrients, even water-soluble vitamins, may be harmful and exacerbate their problem. Peripheral neuropathy has been reported following excessive intake of pyridoxine (Krinke et al., 1980; Schaumberg et al., 1983), and elevated taste preference thresholds have been observed in mice ingesting high levels of heavy metals (Iwasaki and Sato, 1984; Kasahara et al., 1987). Evidence that zinc deficiency may lead to chemosensory abnormalities has prompted recommendations to treat such disorders with high levels of this nutrient. However, this practice may only be effective in individuals where a frank zinc deficiency is the underlying basis for the sensory disorder. Double-blind studies indicate that for the majority of patients, zinc supplements will offer no therapeutic benefit (Gibson et al., 1989; Greger and Geissler, 1978; Henkin et al., 1976), and studies of patients with chemosensory dysfunction taking or not taking zinc supplements reveal no differences (Deems et al., 1991). Indeed, at the doses often recommended, other adverse effects such as gastric distress, anemia, neutropenia, and impaired immune function may occur (Fosmire, 1990). Fortification of foods with natural food odors and tastes has been proposed as a means for enhancing the sensory appeal of items for individuals with diminished chemosensory ability (Schiffman, 2000). In a healthy elderly population, this technique enhanced hedonic ratings and stimulated intake of selected foods (Schiffman and Warwick, 1993). However, total nutrient intake was unaffected. If a decrement of intake is not attributable to a decline of sensory function, this approach may not be effective (e.g., some patients with cancer). It would also be ineffective with anosmic patients and those with chemosensory distortions. Flavor amplification can reduce the appeal of foods to individuals not experiencing an impairment of olfactory sensitivity. Determination of the optimal level of flavor amplification for individual patients may be problematic, but the concept warrants further consideration. One study reported that individualized administration to hospitalized elderly patients did lead to greater short-term intake and several indices of nutritional status (Schiffman, 1998). Rather than amplifying a flavor component that is not perceived normally by a patient, it may be useful to emphasize the other sensory characteristics of foods. Thermal, textural, irritant, and visual characteristics of foods are important determinants of acceptability and can

Mattes

be accentuated to provide greater variety and appeal to foods that cannot be tasted or smelled. However, this approach must be implemented under appropriate guidance since increased use of certain seasonings and food constituents (e.g., salt, sugar, fiber) can have adverse nutritional effects in selected individuals.

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901 Rodin, J., Moskowitz, H. R., and Bray, G. A. (1976). Relationship between obesity, weight loss, and taste responsiveness. Physiol. Behav. 17:591–597. Rogers J., Raimundo A. H., and Misiewicz J. J. (1993). Cephalic phase of colonic pressure response to food. Gut 34:537–543. Rogers, P. J., and Schutz, H. G. (1992). Influence of palatability of subsequent hunger and food intake: A retrospective replication. Appetite 19:155–156. Rolls, B. J. (1986). Sensory-specific satiety. Nutr. Rev. 44: 93–101. Rolls, B. J., Hetherington, M., Burley, V. J., and van Duijvenvoorde, P. M. (1986). Changing hedonic responses to foods during and after a meal. In Interaction of the Chemical Senses with Nutrition, Kare, M. R., and Brand, J. G., (Eds.). Academic Press, New York, pp. 247–268. Rolls, E. T. (1997). Taste and olfactory processing in the brain and its relation to the control of eating. Crit. Rev. Neurobiol. 11(4):263–287. Rolls, E. T., Rolls, B. J., and Rowe, E. A. (1983). Sensory-specific and motivation-specific satiety for the site and taste food and water in man. Physiol. Behav. 30:185–192. Rosenstein, D., and Oster, H. (1990). Differential facial responses to four basic tastes in newborns. Child Dev. 59:1555–1568. Rozin, P. (1967). Specific aversions as a component of specific hungers. J. Comp. Physiol. Psychol. 64:237–242. Saad, M. J. A., Monte-Alegre, S., and Saad, S. T. O. (1990). Differences in first-phase insulin release between normal blacks and whites. Brazil. J. Med. Res. 23:655–657. Sahakian, B. J., Lean, M. E. J., Robbins, T. W., and James, W. P. T. (1981). Salivation and insulin release in response to food in nonobese men and women. Appetite 2:209–216. Sarles H., Dani R., Prezelin G., Souville C., and Figarella C. (1968). Cephalic phase of pancreatic secretion in man. Gut 9: 214–222. Schaumburg, H., Kaplan, J., Windebank, A., Vick, N., Rasmus, S., Pleasure, D., and Brown, M. J. (1983). Sensory neuropathy from pyridoxine abuse. N. Engl. J. Med. 309:4 45–448. Scherr, S., and King, K. R. (1982). Sensory and metabolic feedback in the modulation of taste hedonics. Physiol. Behav. 29:827–832. Schiffman, S. S. (1983). Taste and smell in disease (Part I). N. Engl. J. Med. 308:1275–1279. Schiffman, S. S. (1998). Sensory enhancement of foods for the elderly with monosodium glutamate and flavors. Food Rev. Int. 14:321–333. Schiffman, S. S. (2000). Intensification of sensory properties of food for the elderly. J. Nutr. 130:927S–930S. Schiffman, S. S., and Dackis, C. (1975). Taste of nutrients: amino acids, vitamins, and fatty acids. Percept. Psychophys. 17:140–146. Schiffman, S. S., and Warwick, Z. S. (1993). Effect of flavor enhancement of foods for the elderly on nutrition status: Food intake, biochemical indices and anthropometric measures. Physiol. Behav. 53:395–402. Schiffman, S. S., Nash, M. L., and Dackis, C. (1978). Reduced olfactory discrimination in patients on chronic hemodialysis. Physiol. Behav. 21:239–242.

902 Schiffman, S. S., Sennewald, K., and Gagnon, J. (1981). Comparison of taste qualities and thresholds of D- and Lamino acids. Physiol. Behav. 27:51–59. Schiffman, S. S., Sattely-Miller, E. A., Zimmerman, I. A., Graham, B. G., and Erickson, R. P. (1994). Taste perception of monosodium glutamate (MSG) in foods in young and elderly subjects. Physiol. Behav. 56:265–275. Schwartz, T. W., Stenquist, B., and Olbe, L. (1979). Cephalic phase of pancreatic-polypeptide secretion studied by sham feeding in man. Scand. J. Gastroent. 14:313–320. Sclafani, A., and Koopmans, H. S. (1981). Intestinal bypass surgery produces conditioned taste aversion in rats. Int. J. Obes. 5:497–500. Scott, T. R. (1992). Taste: The neural basis of body wisdom. Nutritional triggers for health and in disease. World Rev. Nutr. Dietet. 67:1–39. Settle, R. G. (1978). The alcoholic’s taste perception of alcohol: preliminary findings. In Currents in Alcoholism, Vol. 5, Galanter, M. (Ed.). Grune and Stratton, New York, pp. 257–267. Settle, R. G. (1991). The chemical senses in diabetes mellitus. In Smell and Taste and Disease, T. V. Getchell, L. M. Bartoshuk, R. L. Doty, and J. B. Snow (Eds.). Raven Press, New York, pp. 829–843. Shapera, M. R., Moel, D. I., Kamath, S. K., Olson, R., and Beauchamp, G. K. (1986). Taste perception of children with chronic renal failure. J. Am. Dietet. Assoc. 86:1359–1365. Shepherd, R., Farleigh, C. A., Atkinson, C., and Pryor, J. S. (1987). Effects of haemodialysis on taste and thirst. Appetite 9:79–88. Simon, C., Schlienger, J. L., Sapain, R., and Imler, M. (1986). Cephalic phase insulin secretion in relation to food presentation in normal and overweight subjects. Physiol. Behav. 36:465–469. Smith, F. J., and Campfield, L. A. (1993). Meal initiation occurs after experimental induction of transient declines in blood glucose. Am. J. Physiol. 34:R1423–R1429. Smith, F. R., Henkin, R. I., and Dell, R. B. (1976). Disordered gustatory acuity in liver disease. Gastroenterology 70:568–571. Soucy, J., and LeBlanc, J. (1999). Protein meals and postprandial thermogenesis. Physiol. Behav. 65(415):705–709. Spitzer, L., and Rodin, J. (1981). Human eating behavior: A critical review of studies in normal weight and overweight individuals. Appetite 2:93–329. Steffens, A. B., Strubbe, J. H., Balkan, B., and Scheurink, A. J. W. (1990). Neuroendocrine mechanisms involved in regulation of body weight, food intake and metabolism. Neurosci. Biobehav. Rev. 14:305–313. Stein, L. J., Cowart, B. J., Epstein, A. N., Pilot, L. J., Laskin, C. R., and Beauchamp, G. K. (1996). Increased liking for salty foods in adolescents exposed during infancy to a chloride-deficient feeding formula. Appetite 27:65–77. Steiner, J. E. (1977). Facial expressions of the neonate infant indicating the hedonics of food-related chemical stimuli. In Taste and Development: The Genesis of Sweet Preference,

Mattes J. M. Weiffenbach (Ed.). U.S. Government Printing Office, Washington, DC, pp. 173–189. Steiner, J. E. (1992). XI International Congress of Dietetics. Jerusalem, Israel. Stellman, S. D., and Garfinkel, L. (1986). Artificial sweetener use and one-year weight changes among women. Prev. Med. 15:195–202. Stewart, R. B., Russell, R. N., Lumeng, L., Li, T., and Murphy, J. M. (1994). Consumption of sweet, salty, sour, and bitter solutions by selectively bred alcohol-preferring and alcoholnonpreferring lines of rats. Alcohol Clin. Exp. Res. 18:375–381. Stock, M. J. (1999). Gluttony and thermogensis revisited. Int. J. Obes. 23:1105–1117. Stunkard, A. J., Berkowitz, R. I., Stallings, V. A., and Schoeller, D. A. (1999). Energy intake, not energy output, is a determinant of body size in infants. Am. J. Clin. Nutr. 69:524–530. Tatzer, E., Schubert, M. T., Timischl, W., and Simbruner, G. (1985). Discrimination of taste and preference for sweet in premature babies. Early Hum. Dev. 12:23–30. Teff, K. L., and Engelman, K. (1996a). Oral sensory stimulation improves glucose tolerance in humans: effects on insulin, C-peptide, and glucagon. Am. J. Physiol. 27:R1371–R1379. Teff, K. L., and Engelman, K. (1996b). Palatability and dietary restraint: Effect on cephalic phase insulin release in women. Physiol. Behav. 50:567–573. Teff, K. L., Mattes, R. D., and Engelman, K. (1991). Oral sensory stimulation improves glucose tolerance in humans: Effects on insulin, C-peptide and glucagon. Am. J. Physiol. 261:E430–E436. Teff, K. L., Devine, J., and Engelman, K. (1995). Sweet taste: Effect on cephalic phase insulin release in men. Physiol. Behav. 57:1089–1095. Tepper, B. J., and Farkas, B. K. (1994). Reliability of the sensory responder classification to learned flavor cues: a test-retest study. Physiol. Behav. 56(4):819–824. Tepper, B. J., and Seldner, A. C. (1999). Sweet taste and intake of sweet foods in normal pregnancy and pregnancy complicated by gestational diabetes mellitus. Am. J. Clin. Nutr. 70:277–284. Tepper, B. J., Hartfiel. L. M., and Schneider, S. H. (1996). Sweet taste and diet type II diabetes. Physiol. Behav. 60:13–18. Thaler, B. I., Paulin, J. M., Phelan, E. L., and Simpson, F. O. (1982). A pilot study to test the feasibility of salt restriction in a community. N. Z. Med. J. 95:839–842. Thesleff, I., Viinikka, L., Saxen, L., and Perheentupa, J. (1988). The parotid gland is the main source of human salivary epidermal growth factor. Life Sci. 43:13–18. Thirlby, R. C., and Feldman, M. (1984). Effect of chronic shamfeeding on maximal gastric acid secretion in the dog. J. Clin. Invest. 73:566–569. Tordoff, M. G. (1988). How do nonnutritive sweeteners increase food intake? Appetite 11:5–11. Tordoff, M. G., and Alleva, A. M. (1990). Oral stimulation with aspartame increases hunger. Physiol. Behav. 47:55–559. Trant, A. S., Serin J., and Douglass, H. O. (1982). Is taste related to anorexia in cancer patients? Am. J. Clin. Nutr. 36: 45–58.

Nutritional Implications of Taste and Smell Tsuruta, M., Kawad, T., Fukuwatari, T., and Fushiki, T. (1999). The orosensory recognition of long-chain fatty acids in rats. Phys. Behav. 66(2):285–288. Vatner, S. F., Franklin, D., and Van Critters, R. L. (1970). Mesenteric vasoactivity associated with eating and digestion in the conscious dog. Am. J. Physiol. 219:170–174. Vazquez, M., Pearson, P. B., and Beauchamp, G. K. (1982). Flavor preferences in malnourished Mexican infants. Physiol. Behav. 28:513–519. Walsh, T. D., Bowman, K., and Jackson, G. P. (1982). Taste changes in advanced cancer. Proc. Nutr. Soc. 41(3):108A. Warwick, Z. S., and Schiffman, S. S. (1990). Sensory evaluations of fat-sucrose and fat-salt mixtures: Relationship to age and weight status. Physiol. Behav. 48:633–636. Warwick, Z. S., Schiffman, S. S., and Anderson, J. J. B. (1990). Relationship of dietary fat content to preferences in young rats. Physiol. Behav. 48:581–586. Weinstock, R. S., Wright, H. N., and Smith, D. U. (1993). Olfactory dysfunction in diabetes mellitus. Physiol. Behav. 53:17–21. Weismann, K., Christensen, E., and Dreyer, V. (1979). Zinc supplementation in alcoholic cirrhosis. Acta Med. Scand. 205:361–366. Westrate, J. A., Dopheide, T., Robroch, L., Deurenberg, P., and Hautvast, J. G. (1990). Does variation in palatability affect the postprandial response in energy expenditure? Appetite 15: 209–219. White, K. D. (1978). Salivation: The significance of imagery in its voluntary control. Psychophysiology 15(3):196–203. Whitten, C. F., and Stewart, R. A. (1980). The effect of dietary sodium in infancy on blood pressure and related factors. Acta Paediatr. Scand. Suppl. 279:1–17. Williams, L. R., and Cohen, M. H. (1978). Altered taste thresholds in lung cancer. Am. J. Clin. Nutr. 31:22–125.

903 Wilson, C. W. M., O’Brien, C. O., and MacAirt, J. G. (1973). The effect of metronidazole on the human taste threshold to alcohol. Br. J. Addict. 68:99–110. Winne, G. I. (1979). Influence of blood flow on intestinal absorption of drugs and nutrients. J. Pharmacol. Exp. Ther. 6:333–393. Wisen, O., Bjorvell, H., Canto, P., Johansson, C., and Theodorsson, E. (1992). Plasma concentrations of regulatory peptides in obesity following modified sham feeding (MSF) and a liquid test meal. Reg. Peptides 39:43–54. Wisniewski, L., Epstein, L. H., and Caggiula, A. R. (1992). Effect of food change on consumption, hedonics, and salivation. Physiol. Behav. 52:21–26. Witherly, S. A., Pangborn, R. M. and Stern, J. S. (1980). Gustatory responses and eating duration of obese and lean adults. Appetite 1:53–63. Witschi, J. C., Ellison, R. C., Doane, D. D., Vorkink, G. L., Slack, W. V. and Stare, F. J. (1985). Dietary sodium reduction among students: feasibility and acceptance. J. Am. Dietet. Assoc. 85:816–821. Woolridge, M. W., Baum, J. D., and Drewett, R. F. (1980): Does a change in the composition of human milk affect sucking patterns and milk intake? Lancet 2:1292–1294. Yeung, D. L., Leung, M., and Pennell, M. D. (1984). Relationship between sodium intake in infancy and at 4 years of age. Nutr. Res. 4:553–560. Young, P. T. (1959). The role of affective processes in learning and motivation. Psychol. Rev. 66:104–125. Zilversmit, D. B. (1979). Atherogenesis: a postprandial phenomenon. Circulation 60:473–85. Zumkley, H., Vetter, H., Mandelkow, T., and Spieker, C. (1987). Taste sensitivity for sodium chloride in hypotensive, normotensive and hypertensive subjects. Compar. Stud. Hyperten. 47:132–134.

43 Conditioned Taste Aversions Kathleen C. Chambers University of Southern California, Los Angeles, California, U.S.A.

Ilene L. Bernstein University of Washington, Seattle, Washington, U.S.A.

I.

INTRODUCTION

alone, and a group exposed to both the taste and illness, but in an unpaired fashion. Learning typically is expressed as a substantial reduction in the consumption of the food and an increase in the display of aversive orofacial responses in experimental, as compared to control, groups. A detailed review of the extensive taste aversion literature is beyond the scope of this chapter (for additional reviews not discussed in this chapter, see Barker et al., 1977; Braveman and Bronstein, 1985; Bures et al., 1998; Domjan, 1980; Grant, 1987; Logue, 1979; Milgram et al., 1977). The goals of this chapter are to discuss the following topics: (1) conditioned taste aversions in the context of feeding systems; (2) conditioned taste aversions in the context of illness systems; (3) the type of learning that conditioned taste aversions represent; (4) the neural mechanisms that mediate taste aversion learning; and (5) the role of estrogen in conditioned taste aversions.

The main focus of this chapter is on the contribution of learning to the rejection, avoidance, and dislike of certain foods. Learned taste aversions are those that develop on the basis of experience with a food or drink. The experience on which such aversions are based is often the consumption of a food or drink prior to a bout of illness. The illness can be a consequence of ingestion or it can be coincidentally associated with consumption of a food or drink, such as happens when one acquires aversions during a bout of flu. Aversions tend to be idiosyncratic; that is, individuals differ from one another with respect to the targeted food or drink that becomes associated with the illness. There also is a strong propensity for humans and other animals to “blame” any illness on some ingested food (Garb and Stunkard, 1974). Aversions can be reduced or extinguished if the individual experiences the food without subsequent illness. In the laboratory, taste aversions are most commonly induced by administering a chemical agent or drug after consumption of a food or drink that has a novel taste. Aversions develop more readily when tastes are novel than when they are familiar (Kalat and Rozin, 1973). Control groups are essential in establishing that the observed behavior change is learned and not due to some nonspecific effects of the treatment, such as lingering drug effects. The control groups usually employed include a group exposed to the taste alone, a group exposed to the illness

II.

CONDITIONED TASTE AVERSIONS IN THE CONTEXT OF FEEDING SYSTEMS

A.

Innate and Learned Preferences and Aversions

Food and some drinks provide nutrients and are a source of calories necessary for survival. For omnivores such as humans, a wide range of substances potentially can serve as food. Some of these substances have nutritive and caloric value, some have no food value, and some are harmful. Particular characteristics associated with the 905

906

substance, such as taste, odor, texture, and appearance, serve as identifiers and become signals of the consequences of ingestion. For most mammals, taste or flavor, that is, a combination of taste, smell, and texture, appears to be the primary cue used as an identifier. Evidence suggests that certain tastes evoke innate ingestive and rejective reactions. For example, premature and full-term neonatal humans and neonatal rats show ingestive orofacial responses to sweet tastes and rejective orofacial responses to bitter tastes (Ganchrow et al., 1986; Steiner, 1973, 1979). These individuals had no prior experience with these tastes. Teleological explanations of why reactions to these particular tastes should be innate include the observation that foods that provide energy and nutrients and are nontoxic often have a sweet taste, whereas foods that are toxic usually taste bitter. Innate preferences (approach, ingestion, etc.) for the taste of salt (Mattes, 1997; Moe, 1986), starch (Sclafani, 1987), and fat (Ackroff et al., 1990) have been observed, and an innate aversion to the flavor of chili peppers also has been reported (Rozin and Schiller, 1980).* It is interesting to note that seemingly innately preferred tastes, but not aversive tastes, apparently elicit an endogenous opioid response. When sweet-tasting saccharin or sodium chloride solutions are given intraorally to young rat pups, they show decreases in ultrasonic vocalizations under conditions of isolation and an increased analgesic response to heat (Kehoe and Sakurai, 1991). These effects are eliminated with opioid antagonist treatment. Beyond these initial predispositions, postnatal experience plays a critical role in food selection. Predispositions can be strengthened or weakened and new preferences and aversions can be established through experience with different foods. There appear to be two processes that contribute to these changes. One is based on feedback processes that can inform an animal whether a given substance provides nutrients and calories (or some general positive benefit to the body) or is toxic (or has some negative effect on the body). The other process is based on a hedonic process. Innate taste preferences can be strengthened and preferences for the tastes of new foods can be established if consumption of the food is followed by caloric or nutrient repletion. These learned preferences are referred to as conditioned taste preferences. Rats learn enhanced preferences *If

is often difficult to disentangle “innate” behaviors from those learned in utero (see Chapters 15, 17, and 39). Throughout this chapter, we use the term “innate” in a general sense, i.e., not due to identifiable postnatal learning processes, with the realization that at least some “innate” responses may, in fact, be learned or modified via learning prepartum.

Chambers and Bernstein

for the taste or flavor of substances that provide no calories or nutrients when these substances are associated with carbohydrates, fats, and proteins. For example, the innate preference for a saccharin solution is enhanced in foodrestricted rats when that solution is mixed with polycose, a form of hydrolyzed cornstarch (Elizalde and Sclafani, 1988). However, enhanced preferences fail to develop when acarbose, a drug that inhibits starch digestion, is added to the saccharin-polycose mixture. Food-restricted rats given intragastric fat infusions during consumption of a specific flavored solution develop an enhanced preference for the taste, whereas those rats given intragastric water infusions do not (Lucas and Sclafani, 1989). Finally, rats deprived of the essential amino acid threonine show learned preferences for a flavored solution paired with repletion of this amino acid (Gietzen et al., 1992). A hedonic mechanism, independent of the nutritive/ caloric feedback mechanism, also seems to play a role in the learning of food preferences (Mook and Cseh, 1981). It has been suggested that preferences for innately preferred tastes can be strengthened simply on the basis of repeated exposure. For example, rats will increase their consumption of sucrose solutions that provide calories, but also sweet-tasting saccharin-flavored substances even though saccharin provides no calories or nutrients (Booth, 1985). Other explanations have been suggested to account for this effect, however. These include inhibition of a neophobia process and learning safety through a feedback process (Kalat and Rozin, 1973; Vogt and Rudy, 1984). Enhanced preferences can be established for new tastes that have no food value by associating them with a taste solution that has hedonic value but no food value. Bolles (1985) found that when orange flavoring is placed in a saccharin solution, rats will begin to prefer the orange flavoring when it is offered alone in water to just plain water. Thus, the hedonic quality of innately preferred sweet tastes can serve as a reinforcer for establishing preferences for different flavored substances. However, greater preferences for flavors are established by associating them with innately preferred sweet taste solutions that have food value than with those that do not. Lambs developed greater preferences for orange or grape flavors paired with a glucose solution than with a saccharin solution even though they showed no difference in the initial preference for the two solutions (Burritt and Provenza, 1992). Innate preferences for sweet tastes can be reversed or changed to aversions if consumption of these tastes is followed by illness (Garcia et al., 1955). These reversals, referred variously as conditioned taste, flavor, or food aversions, have been found in most mammals that have been studied, and they have been established for a wide range of tastes (Garcia et al., 1977; see also Riley and

Conditioned Taste Aversions

Baril, 1976). Animals that have learned a taste aversion show both avoidance and a palatability shift, as indicated by a change from displaying ingestive to aversive orofacial responses. There are some types of internal discomfort that when associated with tastes produce avoidance but not palatability shifts. These are thought to be mediated by different neural circuitry than conditioned taste aversions (Parker, 1995) (see Sec. V.B). In contrast, reversals of aversions are rare in animals, although they are frequently found in humans (Rozin et al., 1979). A flavor that has been found to be highly unpalatable when first encountered is chili peppers. Chili peppers stimulate a sensation of burning. In rats, prolonged exposure to chili flavoring does not alter the aversion and repeated pairings of a chili-flavored food with recovery from thiamine deficiency attenuates, but does not reverse, the innate aversion to the taste of chili (Rozin et al., 1979). However, millions of people in many parts of the world have come to prefer the taste of chili peppers and use chili peppers or chili pepper seasoning as an essential flavoring ingredient. The burning sensation comes from the chemical capsaicin found in chili peppers. Capsaicin stimulates release of the neuropeptide transmitter substance P from sensory neurons. Shortly after stimulation, the sensory nerves become unexcitable by capsaicin, but levels of transmitter in peripheral terminals are unaffected. This is followed by depletion of sensory transmitters in both central and peripheral terminals of sensory neurons (Buck and Burks, 1986; Szoicsanyi, 1984, 1985). However, these physiological changes and their associated desensitization cannot account for the large affective shift that occurs in individuals who are frequent chili eaters (Rozin and Schiller, 1980, Rozin et al., 1981). Although these individuals show slightly higher absolute thresholds for detection of the burning sensation than individuals who rarely or never eat chili peppers, they still experience the burning sensation and report that they come to like it. Rozin and Schiller (1980) have suggested that exposure to increasing levels of chili in food is a sufficient condition for the reversal of the innate aversion to chili pepper in humans. However, the possibility that this innate aversion reversal is mediated by a nutritive/caloric feedback process or by a hedonic associative process has not been ruled out. Food selection, then, involves a complex interaction of innate predispositions and experience. Rozin (1976) has suggested that in omnivores, food choice is influenced primarily by learning mechanisms rather than innate predispositions or genetic factors. In one study he found no greater resemblance in the food preferences of monozygotic than same-sex dizygotic twin pairs or in the variety of foods eaten by each twin type (Rozin and Millman, 1987). Interestingly, however, there was a greater resem-

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blance among monozygotic than dizygotic twins for the preferred degree of hotness from chili peppers. B.

Allesthesia

The hedonic value of established preferences and aversions does not remain constant. Preferred foods can become less positive and aversive foods can become less aversive as the internal state of the individual changes (Cabanc, 1971, 1979; Fantino, 1984). The shift in hedonic value is temporary because when the body state is restored, the hedonic value also is restored. These temporary shifts in hedonic value are referred to as allesthesia. Under most circumstances, a sweet taste will elicit ingestive responses, but when the individual is satiated, that taste will elicit aversive responses. In one experiment, Cabanac (1971) gave subjects sweet solutions to taste and asked them to rate them from very pleasant to very unpleasant. When subjects did not swallow the solutions after tasting them, they rated the tastes as pleasant throughout the experiment. However, when they swallowed and ingested the solutions after tasting them, their ratings changed from pleasant to unpleasant as the experiment progressed. These same results have been obtained with rats (Grill and Berridge, 1985). Taste reactions to a sucrose solution change as an animal goes from a food-depletion state to a food-repletion state. The number of ingestive responses, e.g., tongue protrusions, lateral tongue movements, and paw licks, decreases, and the number of aversive responses, e.g., head shakes, paw shakes, and gapes, increases as meal termination is approached. Tastes that are usually unpleasant also can shift to pleasant under certain internal states. The taste reactions of rats to hypertonic salt solutions will shift from aversive to ingestive during sodium depletion (Berridge et al., 1984). This hedonic shift is dependent on the deprivation state at the time of preference testing, although exposure to a highsodium diet early in life can increase the consumption of sodium-enriched diets when sodium deficient (Grimsley, 1994). Sodium preferences also can increase when rats are placed on a low-protein diet (Okiyama et al., 1996). C.

Conditioned Reactions to Tastes Across the Life Span

The ability to acquire conditioned taste aversions is present from fetal life through old age (Chambers et al., 1997; Kehoe and Blass, 1986; Stickrod et al., 1982). The learning and expression of aversions in preweanling rats, however, are modulated by stimuli associated with the home environment. Preweanlings exhibit weaker taste avoidance when conditioned in their home environment than in

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a novel location (Spear et al., 1985). Preweanlings also do not exhibit avoidance of novel tastes received while engaged in suckling. This appears to be a difficulty in expression rather than learning since the avoidance acquired while on the nipple is expressed when the pups are older and an acceptable food alternative to mother’s milk is available (Gubernick and Alberts, 1984). Adult members of a species, in particular parents, play an important role in conveying information about what constitutes acceptable foods. A mother’s milk appears to contain gustatory cues that reflect the taste of her diet, and these cues can influence food preferences during weaning (Galef, 1977). When rat pups are given their first meal of solid food in isolation, they will preferentially ingest the diet that their mother or surrogate mother ate while nursing them (Galef and Henderson, 1972). Rat pups will show avoidance of a diet during weaning if they were poisoned as nurslings after ingestion of milk from a female rat eating that diet (Galef and Sherry, 1973). It has been suggested that weaning may be facilitated by a learned aversion to milk, but it is unlikely that an aversion process is involved (DiBattista, 1990). Adult rats have low levels of the intestinal enzyme lactase and will learn to avoid foods paired with lactose ingestion, but they do not show negative palatability shifts (DiBattista, 1990; Pelchat et al., 1983). Adults usually introduce weanlings to a subset of acceptable foods. Species differ in the means by which they accomplish this. The physical presence of adult rats at feeding sites attracts pups to these sites (Galef and Clark, 1971). Mother hens use specific calls to attract their young to foods that are not innately preferred (Hogan, 1966), and humans use behavioral modeling and verbalizations (Capretta et al., 1973). It has been suggested that the young come to prefer these foods, in part, because of their familiarity (Galef, 1977). As young mammals gain more independence, they begin to sample new and different foods that are present in their environment. They eventually develop a relatively stable pattern of food selection that, while based primarily on learning mechanisms, also reflects their species’ typical palatability hierarchy (Bronson, 1966; Warren and Pfaffmann, 1959). They rely on a number of strategic mechanisms to guide their selection of foods. Opportunistic omnivores such as rats exhibit neophobia to new foods (Barnett, 1958; Domjan, 1977), as do nonhuman primates (Itani, 1958; Weiskrantz and Cowey, 1963). They will sample only small amounts of the food on first exposure, and when faced with a number of new foods at the same time, they will tend to try one at a time (Rozin, 1969). If the food is safe and has caloric and nutritional value, the rat will learn to prefer the food and will subse-

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quently increase its intake. If the food is toxic, cautious sampling might lead to illness but usually not death. Upon subsequent exposure to the toxic food, the rat will avoid the substance because of a learned aversion. The extent to which an animal exhibits neophobia can be increased by its experience with poisoned foods. Richter (1950, 1953) observed that after repeated poisoning, some rats became so suspicious of new foods that they chose not to eat but to starve rather than try additional novel foods. Studies in humans indicate that 2-year-olds show strong neophobia, and preferences for foods are an increasing function of exposure frequency (Birch and Marlin, 1982). Rats will exhibit neophilia if the foods they are eating are deficient in nutrients. This neophilia, however, is based on a learned avoidance of, and probable aversion to, the deficient food. Sodium is the only nutrient for which it is well established that animals have a specific detection system and an innate hunger. They are able to incidentally learn sources of sodium when not deficient and to use that information when deficient (Krieckhaus and Wolf, 1968). In contrast, there is no specific detection system or innate hunger for other nutrients such as thiamine. Animals use general taste characteristics to identify which foods provide positive or negative postingestional consequences. In a series of studies using thiamine-deficient diets, it was shown that rats developed a strong avoidance of the deficient diet when they were maintained on it long enough to become ill as a result of thiamine deficiency (Rodgers and Rozin, 1966; Rozin and Kalat, 1971). The thiaminedeficient rats displayed neophilia or an immediate preference for any new food offered to them, even if the new food also had no thiamine. If thiamine was added to the new diet, they continued to choose to eat this food and to avoid the deficient diet, even after they had recovered from the effects of the deficiency. The choice was not a preference for novelty, but for any food other than the thiamine-deficient diet. Thiamine-deficient rats that are given a choice between a novel food and a familiar food, which had not been deficient in the past, show a preference for the familiar food. Taken together, all of these studies indicate that food preference, avoidance, and aversion learning have adaptive roles in nutrient selection through the development of learned preferences for nutritionally adequate sources and learned avoidance of and aversion to nutritionally inadequate nutrient sources. Finally, social cues can play an important role in transmitting information about the possible consequences of ingestion of a particular food and about sources of acceptable foods. Mother rats will decrease consumption of a novel taste when ingestion of that substance is followed by the illness of her pups (Gemberling, 1984). In the case of lithium toxicosis, odors emitted by the pups serve as the

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cue that the pups are ill and are essential for the mother to learn the aversion. This learned aversion has adaptive significance since there are toxic substances that apparently do not have aversive effects on the mother but are probably transmitted to the offspring through the milk of the mother (Knowles, 1965). This aversion may also protect the mother from illness in the event that repeated and increased consumption of the substance affects her as well. When adults ingest a novel-flavored substance prior to illness in their adult cage partner, they also will subsequently reduce their consumption of the substance (the “poisoned-partner” effect) (Lavin et al., 1980). Adult rats can increase their knowledge about acceptable food sources by following other rats to these sources (Galef et al., 1987). During social interactions they can identify the diet a conspecific has recently eaten and will follow that conspecific to the food source if that food is known by the follower to be safe. They will not follow the conspecific if the follower has previously acquired a conditioned food aversion to the diet. Birch (1980b) studied preferences for a variety of foods among a group of preschool children and their parents. She found that the preferences of children show nonsignificant correlations with those of their parents. However, preschool children can be strongly influenced in their food choices by the preferences of their peers (Birch, 1980a). As children grow older, parental influence may begin to be more evident since similar studies with college-age children indicate that correlations in food preferences between parents and children are statistically significant (Pliner, 1983). III. CONDITIONED TASTE AVERSIONS IN THE CONTEXT OF ILLNESS SYSTEMS A.

Anorexia Associated with Systemic Infections and Diseases

Decreases in food intake are observed in a number of physical illnesses. It has been suggested that anorexia, i.e., hypophagia and aversion to food, is part of a homeostatic behavioral strategy that has evolved to facilitate survival of individuals suffering from a variety of systemic diseases, including illnesses caused by protozoa, bacteria, helminth, or viruses, and in some cases localized infections (Hart, 1988; Murray and Murray, 1981; Thompson, 1990). Animals exhibit a constellation of behavioral and physiological changes when they are infected with a wide range of illnesses and diseases (Hart, 1988; Kent et al., 1992). The behavioral changes include lethargy or reduced motor activity, depression, sleepiness, adipsia, hypophagia along with reductions in food-motivated behaviors, decreases in

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social exploration, reductions in body-care activities or grooming, and increases in slow-wave sleep; the physiological changes include increases in body temperature. Anorexia is thought to work in conjunction with physiological mechanisms to fight infection (Hart, 1988; Kent et al., 1992). The febrile response acts synergistically with reductions in plasma iron levels to inhibit the growth of some bacterial pathogens (Kluger and Rothenburg, 1979), amino acids and zinc influence the rate of multiplication and survival of certain infective agents (Murray et al., 1978), and the immune response is affected by intake of nutrients (Chandra, 1993). A number of hypotheses have been proposed to account for the function of anorexia in illness. It has been suggested that anorexia (1) reduces the chances of elevating nutrient levels that promote the viability of infectious agents, (2) promotes a more effective immune response, (3) facilitates conservation of calories for use in maintaining elevated body temperatures by reducing energy reserve loss through the energy expenditure and heat loss that would ensue during searches for food, and (4) promotes increased diet selectivity, which could maximize the possibility of finding foods that contribute to a decrease in infection (Hart, 1988; Kyriazakis et al., 1998; Lozano, 1991). Cancer is a disease for which there is substantial evidence of hypophagia. There are at least three factors that contribute to this hypophagia. First, alterations in peripheral metabolism or in the functioning of neural areas controlling eating behavior lead to early satiety signals. Early satiety, bloating, and symptoms of abdominal fullness are commonly found in cancer patients (DeWys, 1985; Grosvenor et al., 1989). Second, altered taste quality and reduced palatability have been reported in patients with cancer who have experienced weight loss (DeWys, 1985). Decreases in the palatability of a diet have been shown to lead to decreases in food intake and body weight (Peck, 1978). Finally, learned taste avoidance contributes to the decreases in food intake (Bernstein and Borson, 1986). In rats, implantation of experimental sarcoma results in suppressed food intake and decreased body weight (Bernstein and Sigmundi, 1980; Bernstein et al., 1985). In addition, when the diet available during tumor growth is novel, striking aversions develop to it which appear to contribute to anorexia. This is indicated by the observation that anorexia arises earlier and is more severe in tumor-bearing animals eating a novel diet than in those eating a familiar one. This reduced preference is not due to a nonspecific effect of tumor growth on taste preference since it is specific to the novel diet present during tumor growth. There is supporting evidence that learned food aversions contribute to hypophagia in humans as well. Thus symptoms of nausea and vomiting are common in cancer patients, especially

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those experiencing weight loss, and specific odor and food aversions are frequently reported (DeWys, 1985; Grosvenor et al., 1989; Nielsen et al., 1980). Various cancer treatments also can induce experiences of nausea and vomiting and can trigger the development of food avoidance and aversion (Bernstein, 1978; Mattes et al., 1992; Smith et al., 1984). It has been suggested that successful treatment to control the secondary effects of narcotic analgesics, such as nausea and vomiting, contributes to a reduction in the hypophagia of elderly patients with terminal cancer (Feuz and Rapin, 1994). Evidence that these symptoms lead to the learning of specific food aversions, which in turn contributes to the hypophagia present before successful symptom management, is equivocal. Some investigators have found no association between the experiences of nausea and vomiting and the incidence of food aversions after radiation therapy or chemotherapy (Mattes et al., 1992). Also, lung cancer patients who have greater loss of appetite and body mass index than breast cancer patients show no difference in the development of food aversions or in the food targets of these aversions during treatment (Mattes et al., 1992). However, aversions to specific foods consumed before treatment are learned in patients receiving gastrointestinally toxic chemotherapy, which suggests that under some conditions food aversions do contribute to the hypophagia already present in cancer patients (Bernstein, 1978; Bernstein and Webster, 1980). All of the data taken together strongly suggest that food aversions, learned as a consequence of the disease and the treatment, play a role in the existing hypophagia of cancer patients. This role may be more important in young patients since aversions develop more easily before the age of 20 (Garb and Stunkard, 1974; Logue et al., 1981). A technique has been developed that successfully reduces the treatmentassociated hypophagia and development of food aversions in children and adults (Broberg and Bernstein, 1987; Mattes et al., 1987). In this technique, a distinctive flavored food (such as coconut or root beer Lifesavers) is used as a target food, or a “scapegoat.” When given after consumption of a meal but before administration of chemotherapy, this target food interferes with the development of aversions to items in the meal. Thus, the impact of chemotherapy on preferences for normal meal items can be reduced. B.

Anorexia Associated with Toxins

Although toxins activate a different constellation of behavioral and physiological responses than infectious illnesses and diseases, they also produce many of the same responses. LiCl induces hypothermia and thus has an opposite effect on body temperature (Batson, 1983; Taukulis, 1982), but it decreases motor activity and stimulates prostration or lying-on-belly (Ladowsky and Ossenkopp, 1986; Meachum

and Bernstein, 1990), decreases general environmental responsiveness (Syme and Syme, 1974), increases EEG slow waves and sleepiness (Karniol et al., 1978; Schou, 1968), suppresses food intake (Bernstein and Goehler, 1983), and decreases activity in a social testing situation (Syme and Syme, 1973). In addition, it induces vomiting in species with this capacity (Schou, 1968; Stricker et al., 1988), induces diarrhea (Schou, 1968), decreases gastric motility and emptying (Flanagan et al., 1989; McCann et al., 1989), inhibits basal and food-stimulated gastrin secretion (Lauritsen et al., 1978), increases vasopressin in primates, which reduces intestinal blood flow through constriction of splanchnic blood vessels (Verbalis et al., 1987), increases mean arterial pressure (O’Connor et al., 1987), and decreases heart rate (O’Connor et al., 1987; Wilkin and Cunningham, 1982). Many of these responses are thought to reduce exposure to the toxin (Stricker and Verbalis, 1991). Conditioned food aversions contribute to the hypophagia induced by LiCl. When constant infusions of LiCl are initiated at the same time that rats are placed on a novel diet, they exhibit decreases in intake of the diet over an 8-day period and show decreases in preferences for the diet (Bernstein and Goehler, 1983). In addition, there is an increase in the number of aversive orofacial responses that are displayed toward substances that have been paired with LiCl (Parker, 1982). However, decreases in consumption are transient when rats are given a familiar instead of a novel diet and preferences for the familiar diet are not reduced (Bernstein and Goehler, 1983). Before the animals had been given the novel diet in this study, they had been maintained on one diet since weaning. Although conditioned taste avoidance of familiar substances, e.g., tap water, can be induced, it is more difficult to obtain conditioned avoidance of familiar substances (Elkins, 1974; Kalat and Rozin, 1973). Nothing is known about the impact of conditioned food aversions under conditions of continuous LiCl exposure in animals maintained on varied diets, a situation that is more typical of human eating patterns. This issue is important since it is possible that for individuals on varied diets, specific food items may not achieve the degree of familiarity found when individuals are maintained on one diet, and thus may be more subject to aversive conditioning.

IV. CONDITIONED TASTE AVERSIONS: WHAT KIND OF LEARNING? Although learned taste aversions have been of scientific interest since 1887 (Poulton, 1887; Rzoska, 1953; Steiniger, 1950), it was not until Garcia and his associates

Conditioned Taste Aversions

presented it as a challenge to existing theories of learning that the study of this topic mushroomed (Garcia and Koelling, 1966; Garcia et al., 1966). Three characteristics of conditioned taste aversions challenged traditional learning theories: (1) selective associations, i.e., a greater propensity for some food stimuli (e.g., taste) to be associated with illness than other stimuli (e.g., visual or auditory cues); (2) strong and persistent learning after a single pairing of the food stimulus and illness; and (3) learning after a long delay between the food stimulus and the illness (several hours or more). In subsequent analysis and study, selective associations and single trial learning have become less problematic to more traditional theories of learning, whereas the long delay learning continues to be challenging. Countless discussions of whether a conditioned taste aversion constituted a new kind of learning or whether it could be considered a form, albeit anomalous, of instrumental or classical conditioning have ensued. General consensus had placed conditioned taste aversions within the Pavlovian or classical conditioning framework. In classical conditioning paradigms, a stimulus (unconditioned stimulus, US) that elicits a response (unconditioned response, UR) is paired with another stimulus (conditioned stimulus, CS) that does not elicit the UR. Learning is inferred when presentation of the CS produces a response similar to the UR (conditioned response, CR). In conditioned taste aversions, an animal is thought to learn an association between a taste or flavor CS and an illness US. In the laboratory, the CS is usually a highly palatable solution such as sucrose or saccharin-flavored water and the US is often an injected drug that stimulates sensory systems mediating URs indicative of illness, such as lethargy, hypophagia, adipsia, and vomiting. The critical problem when thinking about conditioned taste aversions within the framework of classical conditioning is that behaviors that are generally regarded as the CRs, taste avoidance and aversive responses, are dissimilar to the URs, illness responses. Although the reduction in consumption of the CS appears similar to the hypophagia UR, it is specific rather than general. This led some investigators to suggest that conditioned taste aversion learning is different from many other forms of classical conditioning because the CR is entirely part of the repertoire of elicited responses for the taste CS (Chambers, 1990). It also led Garcia (1989, 1990) to revise his conceptualization of the kind of learning conditioned taste aversion represents. Although he originally designated taste as a CS and illness as a US, he now suggests that taste should be designated a US and illness a noncognitive feedback (FB). He further suggests that the US-FB learning in conditioned taste aversion is different from the CS-US learning in classical conditioning. Whereas US-FB

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learning involves an affective processing system that results in a hedonic shift or a modification of incentive, CS-US learning involves a cognitive processing system and results in the modification of behavior. Further analysis has led us to disagree with this conceptualization of Garcia. We suggest that there is only one kind of learning, the association between a taste and illness, and as a result of this association, a number of different changes in the kinds of responses the taste can evoke ensue. After acquisition of a conditioned taste aversion, the taste CS does elicit a response that is similar to the UR. Rats exhibit a distinctive posture called “lying-on-belly” (an UR) when administered the illness-inducing agent LiCl (Parker et al., 1984). When LiCl is paired with a saccharin solution, this response is exhibited after the rats are exposed to the saccharin (Meachum and Bernstein, 1990). Thus, after the CS is paired with the US, the UR, lying-onbelly, is elicited by the CS, saccharin, as well as the US, LiCl-induced illness. An elicitation of an illness response by the taste CS is not the only change that occurs after the CS has been paired with the US. Animals exhibit behavioral responses to the CS that resemble those exhibited after consumption of innately aversive bitter tastes such as quinine. Similar kinds of effects have been found in other classical conditioning situations. In some cases, the CR resembles the UR closely, although it generally does not become identical to the UR (Holland, 1984; Rescorla, 1988). It can lack the intensity and some of the response repertoire of the UR. In other cases, however, the CR produced by a given CS can include some responses that are not part of the UR and can even produce CRs that are opposite that of the UR, e.g., increases in heart rate elicited by a shock US and decreases in heart rate elicited by a tone CS (de Toledo and Black, 1966; Rescorla, 1988). These results have led Holland (1984) to suggest that the CR is composed of two behavioral elements: one that is similar to or at least in some way appropriate to the US and one that is similar to the response elicited by the CS prior to conditioning. In the case of conditioned taste aversions, a taste CS can elicit both ingestive and aversive responses prior to acquisition of a conditioned taste aversion. As indicated in Sec. II. B, the type of response is dependent on the bodystate. Before acquisition, a sweet taste elicits ingestive responses under most conditions. During acquisition, an association is formed between the sweet taste and illness (see Fig. 1). As a result of this association, the taste elicits a new response repertoire, weak illness responses (CRUS), and it changes the responses innately elicited by the CS from ingestive under most conditions to aversive under all conditions (CRCS). It should be noted that there is a critical difference between allesthesia or, for example, a food

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Figure 1 Neural connections during and after acquisition of conditioned taste aversions. Neural areas that store taste-illness association coordinate both responses associated with taste and responses associated with illness. The solid-line arrows represent the direction of the flow of information, the dotted-line arrows represent an existing unactivated connection, and the dotted lines represent the potential for a connection to be made. Abbreviations: URCS, unconditioned response to the conditioned stimulus; URUS, unconditioned response to the unconditioned stimulus; CRCS, conditioned response to the conditioned stimulus; CRUS, conditioned response to the unconditioned stimulus.

depletion-repletion palatability shift and a conditioned taste aversion based palatability shift. Food depletion-repletion shifts are tied to the internal state of the individual and as such are temporary. Sweet tastes become palatable again when the individual is no longer satiated. In conditioned

taste aversion, however, the palatability shifts are relatively permanent. A sweet taste can become palatable again only if the individual is exposed to the taste without negative consequences and unlearns the association between the taste and illness or relearns that the taste is safe.

Conditioned Taste Aversions

V. NEURAL MECHANISMS FOR CONDITIONED TASTE AVERSIONS The determination of the neural mechanisms for the acquisition of conditioned taste aversions involves the identification of at least seven types of pathways. One pathway type starts with the food stimulus (e.g., sweet taste) substrates and leads to the innate ingestive response repertoire substrates. The second pathway type starts with the illness stimulus substrates and leads to the illness response repertoire (e.g., lying-on-belly) substrates. The third pathway type starts with the food stimulus substrates and leads to the areas of integration. The fourth pathway type starts with the illness stimulus and leads to the areas of integration. The fifth pathway type starts from the areas of integration and proceeds to different areas for storage. The sixth pathway type starts with the food stimulus substrates, proceeds to areas of storage, and ends at the substrates for the aversive response repertoires. Finally, the seventh pathway type starts with the food stimulus substrates, proceeds to areas of storage, and ends at the substrates for the illness response repertoires. Most of the studies concerned with identifying the neural pathways mediating conditioned taste aversions have employed permanent lesioning as a technique and have concentrated on the ability of animals to acquire an aversion to a taste after lesioning. Several problems are associated with the interpretation of the results of these studies. Any deficit in conditioned taste aversion could be due to deficits in gustatory processing, illness processing, motor or response production deficits, maintenance of the gustatory trace so integration with illness can occur, integration, storage of the integrated information, or retrieval. In recognition of these limitations, more studies have begun to employ multiple physiological techniques. A.

Taste: Ingestive Responses

For most species studied, taste is the most salient or predominant stimulus associated with food. Aversions to other cues, such as odor, somatosensory stimulation to the tongue, visual cues from the food or the food source, and visual-tactile cues from the environment, can be conditioned. However, these aversions are weaker than aversions to taste (Best et al., 1977; Braveman, 1977; Hankins et al., 1973; Nachman et al., 1977; Taukulis, 1974). One exception is the quail, which develops stronger aversions to the color than to the taste of the food (Wilcoxin et al., 1971). This is not the case for all birds; taste is the predominant stimulus for broad-winged hawks and blue jays (Brett et al., 1976; Brower, 1969). When olfactory, visual, and auditory cues are presented

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in compound with taste and then followed by illness, stronger aversions to these cues can be obtained (Ellins et al., 1985; Galef and Osborne, 1978; Rusiniak et al., 1979). This effect has been termed taste potentiation (Rusiniak et al., 1979). Taste information is transmitted from taste receptor cells located primarily in the tongue to the chorda tympani and superficial petrosal branches of the facial nerve, the lingual branch of the glossopharyngeal nerve, and the vagus nerve (Hamilton and Norgren, 1984; Torvik, 1956) (see also Chapters 32, 33, 35 and 40). The gustatory afferent fibers from these nerves terminate in the ipsilateral rostral nucleus of the solitary tract (NST). Ascending axons from the NST terminate in the ipsilateral parabrachial nucleus (PBN) in rodents and lagomorphs and in the ventroposteromedial nucleus (VPMpc) of the thalamus in primates (Norgren, 1984; Travers et al., 1987). In rodents, neural areas rostral to the PBN are not critical for innate hedonic reactions to taste. In rats subjected to supracollicular decerebration that leaves functional only the first (NST) and second (PBN) central gustatory relay nuclei, intraoral taste stimulation elicits the same ingestive and aversive patterns of taste responsiveness at the same concentrations as it does in intact rats (Grill and Berridge, 1985). There are neurons in the PBN that project to oromotor nuclei (Travers and Norgren, 1983) and that both increase activity during orolingual movement and respond to the hedonic dimensions of taste (Schwartzbaum, 1983). This suggests that the PBN either generates or maintains the orolingual responses to taste (Travers et al., 1987). B.

Taste: Illness Responses

The stimuli that activate other sensory systems such as vision, audition, gustation, and olfaction are well known, and the sensations produced by activation of these systems are clearly definable. This is not the case for the illness sensory system. Within the context of the studies reviewed in this chapter, identification of stimuli that can activate the illness sensory system has been done almost entirely on the basis of whether a given stimulus can induce specific taste hypophagia, that is, a reduction in consumption of a specific food or drink after the stimulus has followed consumption of the food or drink. On the basis of this criterion, the list of stimuli that can activate the illness sensory system and thus are considered illness-inducing agents seems limitless. The organization of the brain is such that behaviors are endpoints of a lattice hierarchy, that is, a specific behavior can be triggered by different motivational systems. Food avoidance is no exception; reductions in food consumption

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can occur for a number of different reasons. Booth (1977) has suggested that rejection of a food could be a conditioned reaction based on states other than illness. He has provided evidence in both rats and humans that pairing a food with satiating properties can produce a subsequent reduction in the consumption of that food (Booth, 1985). In one study, human subjects were given high-calorie and low-calorie soups on two different occasions (Booth et al., 1982). They also were given a plate of small sandwiches to eat with the soup. The high- and low-calorie soups were given different and distinctive flavorings. The caloric value of the high-calorie soup was obtained by adding a starch that could not be detected by taste. On the first exposure, the subjects ate the same number of sandwiches after eating each of the soups. However, on the second exposure to each of the soups, the subjects ate fewer sandwiches with the high-calorie soup and more sandwiches with the low-calorie soup. Thus, in one trial, the subjects were able to learn to associate the taste of the soup with the satiating properties of the food and to adjust their consumption levels accordingly. This means that some reductions in food intake that occur after pairing a novel taste with administration of a drug may be the result of the satietyactivating properties of the stimulus, rather than its illnessactivating properties. Reductions in consumption also can occur under conditions of reward comparison or anticipatory contrast (Flaherty and Checke, 1982; Lucas et al., 1990). In this type of situation, animals are given access to a less preferred solution like saccharin prior to being given a more preferred solution like sucrose. They learn that the presence of saccharin predicts the future availability of sucrose, and as a result they reduce their consumption of the saccharin. In humans, this phenomenon is expressed in the voluntary reduction of intake of the main course to “save” room for dessert. Recently it has been suggested that certain drugs, such as morphine and cocaine, may serve a preferred reward function when they are paired with saccharin, rather than serving an illness-activating function (Grigson, 1997). Some investigators have used a comparison approach to identify illness stimuli (Lett, 1985; Parker, 1995). In this approach, they have identified LiCl as a putative illness stimulus and have compared the kinds of effects this stimulus produces with the kinds of effects other stimuli produce. If another stimulus does not produce similar changes, it is not regarded as an illness stimulus. As discussed in Sec. III. B, there is substantial evidence that LiCl does activate the illness sensory system and consequently illness behaviors. As a result of comparison with LiCl, many drugs that were thought to be illness-inducing agents have been reclassified. Drugs with apparent reinforcing

Chambers and Bernstein

properties that are self-administered in humans and animals (van Ree, 1979; Wise et al., 1976) or that produce conditioned place preferences (Katz and Gormezano, 1979; Reicher and Holman, 1977) can induce conditioned taste avoidance with equivalent doses. These drugs include amphetamine, cocaine, methamphetamine, methylphenidate, morphine, and phencyclidine (Parker, 1995). Their effects presented a paradox since they were believed to be both rewarding and apparently illness producing (Hunt and Amit, 1987). Parker (1995) argued that the learned food avoidance induced by these drugs is not the result of illness and that the association of these drugs with tastes is qualitatively different than that of LiCl. In a series of studies, she found that animals exhibited a substantially greater number of aversive orofacial responses when they were given a conditioned taste that had been paired with LiCl than one that had been paired with rewarding drugs (for a review, see Parker, 1995). This suggests that rewarding drugs produce taste avoidance without negative palatability shifts and thus, by definition, do not induce a conditioned taste aversion. As indicated above, a non-illness-based explanation for the paradoxical conditioned avoidance induced by rewarding drugs is anticipatory contrast (Grigson, 1997). It should be noted that adding the display of aversive orofacial response measures to taste avoidance is not sufficient for claiming that a drug is an illness-inducing agent. As indicated in Sec II. B, satiety also can produce aversive reactions to normally palatable tastes. Multiple measures of illness are needed when assessing the illness-inducing status of a drug. The sensation of illness is a conscious awareness of changes in the body. One sensation that is commonly reported during illness, and specifically reported after lithium administration, is nausea (Karniol et al., 1978; Schou, 1968). Garcia (1989) has held that nausea is the unifying symptom that ties together all the agents that can induce a conditioned food aversion, as defined by both reduced consumption and negative hedonic shift. Others have argued that nausea sensations play no special role in conditioned food aversions and that searches for common features of aversion-inducing agents in terms of symptoms are not likely to be fruitful (Gamzu, 1977; Gamzu et al., 1985; Goudie, 1979). However, most analyses of unifying features have looked at reduced consumption without more appropriate indices of the accompanying hedonic shift. In this vein, Pelchat et al. (1983) found that lactose, which does not produce nausea in humans, and LiCl, which does, induce comparable decreases in consumption, but only LiCl produces aversive orofacial responses. Investigations of nausea in animals have been hampered by a lack of knowledge of the specific body changes from which feel-

Conditioned Taste Aversions

ings of nausea arise. It is assumed that if nausea is reported in humans after administration of a drug, the body changes associated with this sensation (and perhaps the conscious awareness of this sensation as well) also will occur in animals. However, this assumption may not always be correct. It has been suggested that chin rubs specifically may reflect nausea in rats (Parker and MacLeod, 1991). This suggestion was based on finding that the antiemetic agent trimethobenzamide suppressed chin rubs in response to a sweet taste that had been paired with LiCl, but not to a bitter taste that had not been paired with illness. Even if chin rub is a nausea-triggered response, it seems unlikely that its expression is only at the service of neural areas mediating the sensation of nausea. Because LiCl is the putative illness-inducing agent, knowledge of the illness sensory system has been based primarily on studies identifying peripheral nerves and brain areas that respond when LiCl is administered. Niijima and Yamamoto (1994) have found that the c-fibers of the splanchnic nerve and the vagal nerve respond to intraperitoneal administration of LiCl and the magnitude of the response of the splanchnic nerve is greater than that of the vagal nerve. Also, there are neurons in the area postrema that respond electrophysiologically to LiCl (Adachi et al., 1991). The primary site of action of LiCl for conditioned taste aversions appears to be the area postrema. Bilateral subdiaphragmatic vagotomy does not prevent acquisition of LiCl-induced avoidance (Martin et al., 1978), but permanent lesions of the area postrema prevent many, although not all, of the effects of LiCl administration. Animals with permanent lesions of the area postrema do not display decreases in motor activity, lyingon-belly, delayed stomach emptying, and hypothermia after administration of LiCl (Bernstein et al., 1992; Ladowsky and Ossenkopp, 1986). Although both lesioned and sham animals show hypophagia in response to acute administration of LiCl, lesioned, but not sham, animals recover from hypophagia during chronic administration (Curtis et al., 1994; Eckel and Ossenkopp, 1993). Permanent lesions of the area postrema also prevent the learning of a taste avoidance and the shift in hedonic evaluation of a taste, as measured by display of aversive orofacial responses (Eckel and Ossenkopp, 1996; Ritter et al., 1980). This effect of lesioning on taste avoidance is specific to the area postrema and cannot be attributed to changes in gustatory processing or response production. In one study, we found that taste avoidance was blocked when reversible cooling lesions were confined to the area postrema and did not include the surrounding NST (Wang et al., 1997a,b). The lesions were administered only during acquisition, starting immediately after sucrose solution consumption and extending through the peak effec-

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tiveness of LiCl; the cooling lesions were not given during postacquisition testing. Rabin and colleagues have suggested that excitation of the area postrema constitutes a sufficient condition for the establishment of a conditioned taste aversion (Rabin et al., 1986). This hypothesis was based, in part, on the finding of a relationship between the sensitivity of the area postrema to angiotensin II in cats and rats and the ability of this substance to induce learned taste avoidance. The results of other studies also support this hypothesis. Electrical stimulation of the area postrema can serve as a US in a conditioned taste aversion paradigm (Gallo et al., 1988). In addition, a marker for neuronal activity, c-Fos-like immunoreactivity, is induced in the area postrema after LiCl administration during an acquisition trial and when LiCl is given unpaired with a taste solution (Chambers et al., 1999; Swank et al., 1995). All of these data, taken together with the lesion data, suggest that the area postrema is a necessary and sufficient part of the illness sensory system that mediates conditioned taste aversions induced by LiCl. The area postrema, however, is neither an essential nor a sufficient part of the illness sensory system for learned taste aversions induced by all agents. Nicotine induces illness responses, learned taste avoidance of sweet solutions, and shifts from ingestive to aversive orofacial responses (Ossenkopp and Giugno, 1990; Parker, 1995; Taylor, 1990). However, its site of action appears to be central rather than peripheral, and that this site is not the area, postrema (Iwamoto and Williamson, 1984; Ossenkopp and Giugno, 1990; Stolerman, 1988). The location of the site remains unknown; it could be distal to the area postrema or it could be part of another illness pathway. The vestibular system probably constitutes a second illness system. Vestibular stimulation through body rotation induces illness responses, conditioned taste avoidance, and conditioned taste aversion (Chinn and Smith, 1955; Cordick et al., 1999; Ossenkopp, 1983). Only the relationship between rotation-induced taste avoidance and the area postrema has been investigated, and this response is not mediated by the area postrema (Ossenkopp, 1983). Unilateral electrical stimulation or blockade of the vestibular nucleus induces peripheral vestibular symptoms and, as is true for the area postrema, it can be used as an unconditioned stimulus in a conditioned taste avoidance paradigm (Ballesteros and Gallo, 2000; Buresova et al., 1982). C.

Integration

The pathways carrying taste and illness information are anatomically close together in many different neural areas. This has led to the suggestion that perhaps association takes place at a number of levels. Three brain areas—the

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PBN, amygdala, and insular cortex—have received considerable attention as possible sites of integration. In this section we limit the discussion of this work to LiClinduced conditioned taste aversions. Neurons in the medial PBN transmit gustatory information and neurons in the lateral PBN transmit illness-related information that is important for taste aversion learning. Sucrose-best neurons have been located in the central lateral subnucleus and after saccharin consumption, c-Foslike immunoreactivity has been found in the dorsal lateral and central lateral subnuclei (Ogawa et al., 1987; Yamamoto et al., 1994a,b). Cells in the area postrema project to the PBN (Shapiro and Miselis, 1985; van der Kooy and Koda, 1983) and c-Fos-like immunoreactivity has been induced in the lateral PBN, specifically the external lateral subnucleus, and the medial PBN after administration of LiCl (Swank and Bernstein, 1994; Yamamoto et al., 1993). After acquisition of an aversion, exposure to a conditioned sweet taste induces c-fos-like immunoreactivity in the external lateral subnucleus, as well as the central lateral subnucleus of the PBN (Yamamoto, 1993, 1994). Bilateral reversible or permanent ibotenic lesions of the whole PBN attenuate or block acquisition of LiClinduced conditioned taste avoidance (Ivanova and Bures, 1990a; Scalera et al., 1995). When tetrodotoxin lesions are made 1, 2, or 4 days after the acquisition trial, learning also is disrupted (Ivanova and Bures, 1990b). Animals with permanent bilateral lesions of the medial PBN fail to show (1) reductions in consumption of a sweet solution that had been paired with LiCl or (2) increases in aversive orofacial responses (Grigson et al., 1998; Sakai and Yamamoto, 1998; Spector et al., 1992). Permanent and temporary cooling lesions of the lateral PBN also block the ability of animals to learn taste avoidance (Sakai and Yamamoto, 1998; Wang and Chambers, 1998, 2002). These results may be specific to gustatory and illness processing for conditioned taste aversions, since animals with PBN lesions are capable of learning a conditioned taste preference and a learned place avoidance that is based on LiCl administration (Grigson et al., 1998; Reilly et al., 1993). There is general consensus that the PBN is the initial area of taste-illness integration; however, there is disagreement as to whether it is located in the lateral or medial portion of the nucleus (see Reilly, 1999; Yamamoto et al., 1994b). It is unlikely that an intact PBN is sufficient for integration. Conditioned taste aversions cannot be learned when animals are placed under deep anesthesia before pairing the taste with LiCl (Bures and Buresova, 1979, 1989; Buresova and Bures, 1977). Also, rats decerebrated at the supracollicular level do not acquire a conditioned taste aversion (Grill and Norgren, 1978). Electrolytic lesions of the basolateral amygdala disrupt taste avoidance learning (Nachman and Ashe, 1974;

Chambers and Bernstein

Simbayi et al., 1986). This effect has been attributed to the damage of axons of passage since cutting the connections between the amygdala and insular cortex produces the same deficits as electrolytic lesions of amygdala and excitotoxic lesions have no effect on avoidance learning (Bermudez-Rattoni and McGaugh, 1991; Dunn and Everitt, 1988). However, in light of recent new evidence this conclusion needs to be reexamined. Careful behavioral and anatomical tract-tracing investigations have verified the role of the basolateral amygdala, but not the central amygdala, in acquisition of avoidance to novel gustatory stimuli. These studies are particularly persuasive because they used not only excitotoxic lesions but also tract tracing to establish that these lesions did not disrupt corticalamygdala connections (Morris et al., 1999). Another complication in interpreting the effects of amygdala damage on taste aversion learning involves the likelihood that different taste aversion conditioning methods may rely on different neural circuitry. For example, though Morris and colleagues found the basolateral amygdala to be critically involved in learning an avoidance of a novel taste, avoidance of tastes that were not novel was unaffected by basolateral amygdala lesions. Another procedural difference has been shown to critically affect the importance of the amygdala to taste aversion conditioning. This concerns the extent to which the conditioning protocol conforms to instrumental or Pavlovian procedures. When animals are conditioned and tested when drinking the CS taste from a bottle, they have direct control over their taste exposure. Therefore, the taste (CS)–illness (US) contingency may be characterized procedurally as classical conditioning, while the approach–illness (US) contingency may be characterized as instrumental learning. In contrast, when animals are exposed to the CS taste through intraoral infusions, they have little or no control over taste exposure (Schafe et al., 1998) and hence the classical components of the learning are isolated. The neural structures mediating different types of learning and memory have been shown to be dissociable (McDonald and White, 1993). The amygdala, for example, has been shown to be essential for classical conditioning tasks, particularly aversive tasks (Gallagher and Chiba, 1996; Lavond et al., 1993; McDonald and White, 1993), but not for tasks with a response requirement. Consistent with these findings, Schafe and colleagues (1998) found that the importance of amygdala to taste aversion learning depended heavily on whether the method of CS delivery was response contingent or not. When the conditioning procedure did not involve a response component (intraoral infusion), electrolytic and excitotoxic amygdala lesions eliminated conditioned taste aversion acquisition, that is, aversive orofacial responses were not exhibited. However, if the conditioning protocol did include a response requirement

Conditioned Taste Aversions

(bottle method), electrolytic lesions attenuated conditioned taste aversions, that is, some taste avoidance was still exhibited, and excitotoxic lesions had no effect on taste avoidance. These findings support the hypothesis that neural structures critical for conditioned taste aversion learning differ as a function of important, but subtle, procedural differences. Rats under anesthesia or bilateral cortical spreading depression who are force-fed with saccharin and then poisoned with LiCl do not acquire a conditioned taste avoidance, although they demonstrate unconditioned signs of illness (Bures and Buresova, 1989; Buresova and Bures, 1973). But when either the anesthesia or spreading depression is given after exposure to saccharin and before administration of LiCl, avoidance learning is not prevented. These results suggest that acquisition of taste avoidance requires cortical involvement in taste processing, but subcortical areas are sufficient for illness processing and integration. The dysgranular or gustatory insular cortex is involved in acquisition of conditioned taste avoidance induced by LiCl, but it is not essential. Rats with electrolytic lesions of the gustatory insular cortex show attenuated avoidance and no aversive orofacial responses (Braun et al., 1982; Kiefer and Orr, 1992). However, ibotenic lesions of this area have little effect on taste avoidance learning (Yamamoto and Fujimoto, 1991; Yamamoto et al., 1995). The results of the cortical spreading depression and electrolytic studies suggest there are axons of passage through the gustatory cortex that are important for learning taste aversion. Although rats with electrolytic lesions of the insular cortex can learn to reduce consumption of a taste paired with LiCl, more taste-LiCl pairings are required and they do not show aversive orofacial responses to the taste (Kiefer and Orr, 1992). Animals can learn to use body changes induced by LiCl as discriminative cues in learning tasks, which could account for the ability of these rats to learn taste avoidance after numerous trials (Mediavilla et al., 1998; Ossenkopp et al., 1997; Wilkin and Cunningham, 1982). Failure to exhibit aversive orofacial responses was not due to an inability to make aversive responses since the rats responded aversively to a quinine solution. It also was not due to a disruption of primary taste processing since rats with these lesions showed relatively normal ingestive-avoidance functions for basic tastes (Braun et al., 1982; Kiefer and Orr, 1992). On the basis of neuroanatomical tracing and lesion studies, Sakai and Yamamoto (1999) have suggested recently that the gustatory insular cortex is one route by which LiCl illness information travels from the lateral PBN to the basolateral amygdala. Another route is via the zona incerta, and midline and intralaminar thalamic complex. c-Fos-like immunoreactivity has been induced in

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the basolateral amygdala after administration of LiCl (Gu et al., 1993). Also, single excitotoxic lesions of the insular cortex, zona incerta, and midline and intralaminar thalamic complex have no effect on acquisition of a conditioned taste avoidance, but combined lesions of the zona incerta and gustatory insular cortex greatly attenuate acquisition (Sakai and Yamamoto, 1999). D.

Taste: Aversive and Illness Responses

When rats are poisoned after consuming a preferred sweet taste such as sucrose, their subsequent behavioral responses to sucrose resemble those exhibited after consumption of innately disliked bitter tastes such as quinine. They exhibit the following changes in their response to the taste CS: decreases in consumption levels, decreases in ingestive orofacial responses (a series of rhythmical mouth movements and alternations between tongue protrusions and tongue retractions that result in swallowing), increases in aversive orofacial responses (mouth gaping with tongue retraction followed by long-duration tongue protrusion and mouth closure, which results in a reduction in swallowing), spillage of food from eating containers, and defensive burying of drinking spouts (Berridge et al., 1981; Bowers et al., 1992; Carpenter, 1956; Garcia and Koelling, 1966; Parker, 1988; Rozin, 1967; Wilkie et al., 1979). Anecdotal reports in humans indicate that thoughts of conditioned taste aversions elicit facial expressions of loathing similar to those expressed in response to innately disliked foods. Furthermore, humans who avoid food followed by emesis report that the avoided food is distasteful (Garcia, 1989; Steiner, 1979). Conditioned tastes also evoke illness responses. Garcia (1989) has indicated that hearing or thinking about conditioned taste aversions elicits reports of nausea in humans, and as indicated in Sec. IV, conditioned tastes will elicit some of the unconditioned responses to LiCl, e.g., lying-on-belly and decreased heart rate (Kosten and Contreras, 1989; Meachum and Bernstein, 1990). When an animal encounters a conditioned taste stimulus after the taste has been paired with illness, expression of aversive and illness responses is dependent on successful association and the storage and retrieval of the association. A number of studies have used changes in electrophysiological responses and c-Fos induction in various neural areas after acquisition to assess involvement of these areas in the expression of a conditioned taste aversion. Also, neural areas have been lesioned after the first postacquisition trial in order to assess the importance of these areas in the retention of the conditioned taste aversion. It is not likely that the area postrema is involved in either aversive or illness reactions to a conditioned taste. Lesions of the area postrema after acquisition do not pre-

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vent avoidance of a conditioned sweet taste or the decrease in heart rate that occurs in response to a conditioned sweet solution (Kosten and Contreras, 1989; Rabin et al., 1984). Also, c-Fos-like immunoreactivity is not induced in the area postrema when rats are exposed to the conditioned taste after it has been paired with LiCl (Houpt et al., 1994; Swank and Bernstein, 1994). There is some evidence that the NST is involved in mediating aversive responses to conditioned tastes. Chang and Scott (1984) have demonstrated that the pattern of activity of NST neurons in response to a conditioned saccharin solution changes such that it more closely resembles the response to nonsweet stimuli such as quinine (see Chapters 33 and 35). Although sweet solutions do not induce c-Fos-like immunoreactivity in the intermediate zone of the NST before acquisition, they do after they are paired with LiCl (Houpt et al., 1994; Swank and Bernstein, 1994). Rats decerebrated at the supracollicular level do not retain an avoidance acquired prior to transection, and lesions of the gustatory rostral NST and medial PBN do not affect retention of avoidance acquired prior to lesioning (Grigson et al., 1997; Grill and Norgren, 1978). These data suggest that regions rostral to the PBN are essential for retention. Electrolytic and cytotoxic ibotenic lesions of the basolateral amygdala disrupt retention of avoidance learned prior to lesioning (Nachman and Ashe, 1974; Simbayi et al., 1986; Yamamoto, 1994). The insular cortex also is critically involved in retention of avoidance, since animals with these lesions do not retain a previously learned avoidance induced by LiCl (Braun et al., 1981; Ormsby et al., 1998; Yamamoto et al., 1980). This area probably is involved in retrieval rather than storage since fetal transplants can restore expression of an avoidance learned prior to lesioning (Ormsby et al., 1998). E.

Extinction

Extinction in associative learning involves presenting the CS (e.g., taste) without subsequent presentation of the US (e.g., illness). Extinction is a learning process; animals relearn the predictive value of the CS. In the case of taste aversion learning, they relearn that the taste stimulus no longer predicts illness if the food or drink is consumed. In the conditioned taste avoidance paradigm, the animal usually controls exposure to the taste CS. However, in traditional classical conditioning paradigms, the investigator controls exposure to the CS. This difference may account for the apparent resistance to extinction of conditioned taste aversions that is found in humans (Bernstein, 1991; Garb and Stunkard, 1974). In questionnaires, some humans have reported aversions persisting since childhood

or more than 50 years (Garb and Stunkard, 1974). Under conditions that force exposure to the CS in conditioned taste aversion situations, rates of extinction are comparable to those in more traditional classical conditioning studies (Spector et al., 1981, 1983). Once an animal has had one exposure to a conditioned taste unpaired with illness, it has experienced an extinction trial. This failure to experience illness after the exposure is processed in the brain, and each exposure that is not followed by illness weakens the taste-illness association and decreases the responses of neural areas mediating the aversion. This means that in the retention studies in which lesions were made after the first extinction trial, the results could be confounded by the initiation of an extinction process. It has been shown that c-Fos-like immunoreactivity is induced in the intermediate and caudal NST when animals are first exposed to a conditioned sweet solution, but after extinction there is little or no induction in these areas (Houpt et al., 1994). The neural areas involved in extinction most likely include those that mediate storage and retrieval, but other areas are probably also involved. The cortex does not appear to be necessary for extinction. Animals can extinguish a previously learned avoidance when they are anesthetized or under cortical spreading depression (Bures and Buresova, 1979). There are a number of factors that can modulate, that is, increase or decrease, the rate of extinction independently of acquisition. Adrenocorticotropin hormone and testosterone decrease and water deprivation increases extinction rate (Chambers and Sengstake, 1979; Kendler et al., 1976; Sengstake and Chambers, 1979). Excitotoxic lesions of the gustatory insular cortex and the whole hippocampus do not affect the ability of an animal to acquire an avoidance, but they do increase the rate at which an animal extinguishes the avoidance (Yamamoto et al., 1995). This suggests that these areas are involved in mediation or modulation of extinction.

VI. ROLE OF ESTROGEN IN CONDITIONED TASTE AVERSIONS Females and males differ in a number of behaviors associated with eating behavior. For example, there are striking sex differences in energy expenditure or activity levels, food intake, innate taste aversions and preferences, and learned taste aversions (see Beatty, 1979; van Haaren et al., 1990). In most cases the reason for these sex differences can be linked directly to differences in the reproductive and parental demands on females and males. For those behaviors that have been studied extensively, gonadal hormones

Conditioned Taste Aversions

have been found to play an essential role in the establishment and maintenance of these differences. There are primarily two periods in development during which gonadal hormones exert effects on the neural areas mediating sex differences. Gonadal hormones can act after puberty but they also can act during fetal life and/or the early part of postnatal life to alter neural development and subsequent responsiveness of neural areas to these hormones (see Arnold and Breedlove, 1985; Beatty, 1979; Goy and Goldfoot, 1973). The appropriate hormonal milieu is required during the fetal/neonatal period for expression of some sex differences (e.g., testosterone and its metabolites in males and no androgens in females), during the pubertal/postpubertal period for expression of other sex differences (e.g., testosterone in males and/or estradiol in females), and during both the fetal/neonatal and pubertal/postpubertal periods for expression of still other sex differences. For most behavioral sex differences, gonadal hormones play some role during both the fetal/neonatal and pubertal/postpubertal life span periods. In some of these cases, the hormonal milieu during the fetal/neonatal period simply alters the threshold level of hormone necessary to permit neural activation in adulthood. For example, there is a sex difference in the extinction of a conditioned taste avoidance in rats, mice, and chicks (Chambers and Sengstake, 1976; Clifton and Andrew, 1987; Ingram and Corfman, 1981). The expression of this sex difference is dependent on the presence of androgens in males during extinction (Chambers and Sengstake, 1979; Clifton and Andrew, 1987). Whereas gonadectomy of male rats reduces testosterone levels and accelerates extinction, exogenous treatment with physiological doses of testosterone or dihydrotestosterone restores extinction rates to those found in intact males (Chambers, 1976, 1980; Sengstake and Chambers, 1991). Females also exhibit slow extinction rates when they are administered testosterone. In two studies we found that for about half of the females, the amount of testosterone required to prolong extinction produced serum blood levels that fell at the high end of the physiological range found in intact males and for the other half it fell above (Sengstake and Chambers, 1991). In both cases, however, the amount of testosterone was higher than that required by males. When females were exposed to testosterone during fetal/neonatal development, the amount of testosterone that prolonged extinction in adulthood was similar to that of males. Taken together, these results suggest that both females and males possess the neural substrate upon which testosterone acts to modulate extinction. Further, the elevated levels of androgens in males during fetal/neonatal development do not direct the development

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and organization of this substrate; rather, they simply increase the sensitivity of this substrate to circulating levels of androgens in adulthood. A.

Food Intake

There is a systematic variation in consumption of food across the reproductive hormonal cycle of female members of several mammalian species. Food intake decreases during the follicular phase of the cycle and increases during the luteal phase in rats (Wade 1972), guinea pigs (Czaja and Goy, 1975), rhesus monkeys (Czaja, 1975), pigtail monkeys (Krohn and Zuckerman, 1938), baboons (Gilbert and Gillman, 1956), and humans (Dalvit, 1981). This hypophagia is due to elevated estradiol levels. Estradiol treatment in ovariectomized rats, guinea pigs, and rhesus monkeys lowers food intake (Czaja and Goy, 1975; Tarttelin and Gorski, 1973; Wade, 1972) and progesterone attenuates the inhibitory effects of estradiol on food intake (Kemnitz et al., 1989; Wade 1972). Elevations in the levels of estradiol in males also can effectively induce hypophagia. Several studies have shown that prepubertal and adult males treated with exogenous estradiol or implanted with estrogen-producing tumors [LTW(m)] exhibit decreases in food intake (Bernstein et al., 1985; Earley and Leonard, 1979b; Kuchar et al., 1982; Mordes et al., 1984). In addition to total food intake, the consumption of carbohydrates specifically also varies across the reproductive hormonal cycle of rats and humans; intake is lower prior to or during ovulation (Dalvit-McPhillips, 1983; Wurtman and Baum, 1980). Estradiol treatment in ovariectomized female rats decreases carbohydrate consumption as well as total food consumption. Estradiol most likely acts on a satiety mechanism. Several investigators have found that estradiol treatment reduces food intake by decreasing the size of the meals rather than the number of meals (Blaustein and Wade, 1976; Drewett, 1974). There is evidence to suggest that estradiol acts on a long-term satiety mechanism, that is, it regulates eating as a means of regulating body weight (Tarttelin and Gorski, 1973; Wade 1972). After ovariectomy, there is an increase in food intake and weight gain. In time, food consumption returns to control intact levels but body weight remains higher. Similarly, estradiol treatment in ovariectomized females causes only a transient decrease in eating but a lasting decrease in body weight. This conclusion is supported further by the failure of estradiol to induce decreases in food intake in females that do not gain weight after ovariectomy (Wade, 1972). Two primary mechanisms have been suggested to account for anorectic effects of estrogen, a peripheral metabolic mechanism and a neural mechanism. It has been

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proposed that estradiol acts on enzymatic mechanisms controlling fat storage and breakdown and that decreases in food intake are consequent to increases in fat metabolism (Wade and Gray, 1979; Wade and Schneider, 1992). Signals arising from adipose tissue would then augment short-term satiety signals through indirect or direct action on the brain. For example, estradiol could decrease lipoprotein lipase activity and thus increase the amount of long-chain fatty acids that reach liver or brain afferents that have connections to appetite or satiety neural areas. However, implantation of estradiol into the hypothalamus can induce hypophagia without affecting adipose lipoprotein lipase activity (Nunez et al., 1980). Also, decreases in food consumption occur when female rats are administered an estradiol-chemical delivery system, which slows the half-life of estradiol in the brain but accelerates its clearance from peripheral tissues (Simpkins et al., 1989). These results suggest that direct action of estradiol on neural receptors is sufficient to induce hypophagia. More recent evidence suggests that estradiol acts on receptors in the paraventricular nucleus of the hypothalamus. Intracranial application of estradiol into the paraventricular nucleus, but not the ventromedial hypothalamus, posterior hypothalamus, or medial preoptic hypothalamus, induces hypophagia in ovariectomized rats and guinea pigs and lesions of the paraventricular nucleus reduce the responsiveness of ovariectomized rats to estradiol (Butera and Beikirch, 1989; Butera and Czaja, 1984; Butera et al., 1990, 1992). Further, the hypophagic effects of estradiol are blocked by implanting of anisomycin, which inhibits protein synthesis, into the paraventricular hypothalamus (Butera et al., 1993). The corticomedial area of the amygdala also may be a site of action. Bilateral implants of estradiol benzoate into the corticomedial nuclei reduce food intake in ovariectomized females with and without ventromedial-arcuate hypothalamic lesions (Donohoe and Stevens, 1981). B.

Conditioned Taste Aversion

It is well known that high doses of estradiol can cause nausea and vomiting (Goodman and Gilman, 1975). In addition, a number of investigators have shown that estradiol can induce a conditioned taste avoidance when paired with a novel taste in rats (Gustavson et al., 1989a; Peeters et al., 1992) and mice (Miele et al., 1988). This effect is found in both females and males and it is not dependent on the form of estradiol or its route of administration (e.g., Bernstein et al., 1986; Ganesan and Simpkins, 1990; Merwin and Doty, 1994; Nicolaus et al., 1989; Yuan and Chambers 1999b). Estradiol also has been found to condition a shift in palatability. Decreases in ingestive orofacial responses

and increases in aversive responses were found in rats after 17-estradiol was paired with a novel sucrose solution, (Ossenkopp et al., 1996). In humans, a case study of a 15-year-old girl revealed incidences of nausea and repeated vomiting subsequent to being placed on birth control pills that contained ethinyl estradiol (Gustavson et al., 1988, 1989a). Concomitantly, the range of foods that she would eat became limited and for a few of the foods, she indicated a negative shift in palatability. She identified food as the source of her illness. When she was taken off the birth control pills, the vomiting subsided. One month after she stopped taking the pills, she participated in a conditioned taste aversion experiment. She showed suppression in consumption of the flavored water that had been paired with an ethinyl estradiol tablet but not to the flavored water that had been paired with a sucrose tablet. The estradiol tablet had triggered nausea and vomiting responses. It has been suggested that the aversive properties of estrogen play a role in the reduction of food intake observed after estrogen administration, particularly when the food is novel and hormone levels are high (Ganesan and Simpkins, 1990, 1991). The suppression of food intake is greater when animals are given a novel diet at the onset of estrogen treatment than when they are maintained on a familiar diet. Avoidance of the novel diet is evident in preference tests. This result, taken together with the finding of aversive orofacial responses after pairing estradiol with a novel sweet taste, supports the hypothesis that conditioned taste aversions can contribute to estrogen hypophagia (Ossenkopp et al., 1996). The aversive properties of estrogen also play a role in the acceleration of extinction of LiCl-induced conditioned taste avoidance that occurs when gonadectomized females and males are given continuous exposure to estradiol, starting before acquisition and continuing throughout extinction (Yuan and Chambers, 1999a). Recently, it was established that the same route of administration of the same dose of estradiol that was used to accelerate extinction can also induce a conditioned taste avoidance (Yuan and Chambers, 1999b). Further support for its aversive role comes from studies examining the effects of exposure to illness-inducing agents before acquisition or extinction of conditioned taste avoidance. Learning is attenuated when animals are exposed to an aversive agent before acquisition, and extinction is accelerated when they are exposed after acquisition but before extinction (Cannon et al., 1977; Domjan, 1978; Mikulka et al., 1977). These effects are found when the exposure agent is the same as the agent used to induce the conditioned taste avoidance and when the two agents are different. The former has been termed intra-agent disruption and the latter intera-

Conditioned Taste Aversions

gent disruption (Cannon et al., 1977). One of the explanations for these effects is as follows. Animals do not readily acquire aversions to familiar foods, especially foods eaten from infancy. Thus, when an animal is exposed to an aversive agent and only familiar foods are available, a dissociation between the illness induced by the aversive agent and food occurs. When a novel food is made available, this dissociation then interferes with the ability of an animal to associate the illness with the novel food or to maintain a previously acquired association. One important prediction can be made on the basis of this explanation. When the exposure agent is given before acquisition or before extinction, the aversion will be attenuated, but when it is given during acquisition or during extinction, the aversion will be strengthened. A number of studies have shown that avoidance learning is weakened or blocked and extinction is accelerated when estradiol is given before acquisition of a LiCl-induced avoidance and when it is given after acquisition, but before extinction (Chambers et al., 1998, 1999, 2002; De Beun et al., 1993; Merwin and Doty, 1994; Yuan and Chambers, 1999b). However, extinction is prolonged when estradiol exposure is initiated after the first extinction trial and continued throughout the rest of extinction (Yuan and Chambers, 1999b). It has been suggested that estrogen-induced nausea parallels the nausea induced by LiCl (Gustavson and Gustavson, 1987; Gustavson et al., 1989a). Consistent with this hypothesis is the finding that there is crossfamiliarization between LiCl and estradiol; exposure to LiCl before acquisition can attenuate an estradiol-induced avoidance as well as the reverse case discussed above (De Beun et al., 1993). This suggests that the aversive effects of estradiol and LiCl are mediated by the same neural pathway. As indicated in Sec. V.B, LiCl-induced avoidance is mediated by the area postrema (Ritter et al., 1980; Wang et al., 1997ab). Estrogen receptor–immunoreactive cells have been found in the area postrema, and lesions that include the area postrema and NST greatly attenuate the avoidance of a target diet available during estradiol infusions (Bernstein et al., 1986; Simonian and Herbison, 1997). These lesions also significantly attenuate the hypophagia and development of food aversions that accompany the growth of LTW(m) tumors, which are characterized by an elevation in circulating estrogens (Bernstein et al., 1986). However, we recently found that c-Fos-like immunoreactivity was induced in the area postrema during acquisition of an aversion produced by LiCl, but it was not induced by estradiol (Chambers et al., 2000). These data raise the possibility that LiCl-associated illness is qualitatively different than estradiol-associated illness.

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Given that estradiol is a satiety agent, perhaps the illness produced by estradiol is an extreme end of a satiety continuum. Excess consumption can produce nimiety and feelings of nausea, and administration of high doses of putative satiety agents can induce malaise. The short-term satiety agent cholecystokinin (CCK) is known to produce abdominal cramps, nausea, and emesis at high doses in humans (Miakiewicz et al., 1989; Stricker and Verbalis, 1991). In rats, CCK produces conditioned taste avoidance, passive aversive orofacial responses, a few active aversive responses, and defensive burying when it is paired with a novel taste (Bowers et al., 1992; Cross-Mellor et al., 1999; Eckel and Ossenkopp, 1995; Perez and Sclafani, 1991). C.

Estrogen and Anorexia Nervosa

Anorexia nervosa is an eating disorder with unknown physical or emotional causes. It occurs most often in young women (Crisp et al., 1976) and is characterized by a refusal to maintain body weight within the normal range for age and height, an intense fear of gaining weight, a disturbance of body image, and amenorrhea in postmenarchal females (DSM-IV-TR, 2000). A number of theories, ranging from psychosocial to physiological, have been proposed to account for anorexia nervosa, but most theories remain unsubstantiated or inadequate. There are several problems inherent in the study of anorexia nervosa. First, the originating cause for the reduction in food intake and body weight may not be what maintains the disorder once it is established (Casper, 1985). Second, it is possible that multiple causes interact with one another to produce the disorder. Third, what is known as anorexia nervosa may not be a unitary phenomenon. In recognition of this, two types of anorexia nervosa have been defined: restricting type and binge-eating/purging type (DSM-IV-TR, 2000). Finally, by the time anorexia nervosa is diagnosed, the individuals are suffering from symptoms of starvation. Thus, any differences found between anorexics and normal individuals may have more to do with starvation then they do with some originating cause. In 1986, Gustavson and Gustavson suggested that estrogen is an originating cause of anorexia nervosa in pubertal girls. They suggested that during fetal and early postnatal development, the central nervous system of some females is partially masculinized as a result of maternal pathology, medical treatments, or pollutants that increase androgen levels or mimic androgen (Gustavson et al., 1989a; Young, 1991). This results in a greater sensitivity to the aversive properties of estrogen. As a consequence, the increased production of estrogen at puberty can cause illness, which induces hypophagia and the development of conditioned food aversions. The conditioned food aversions in turn

922

contribute to the illness-induced hypophagia. The substantial weight loss that results leads to a reduction in estrogen production and, as a consequence, an elimination of illness. Any weight gains that occur lead to a restoration of estrogen production and a resumption of illness. This feedback loop ultimately evolves into a behavioral starvation system. In our reconceptualization of the model proposed by Gustavson and Gustavson (1989a), we suggest that estrogen produces decreases in food consumption by at least four different mechanisms, a simple satiety mechanism, a conditioned satiety mechanism, a simple malaise mechanism, and a conditioned food aversion mechanism (see Fig. 2). These four mechanisms could interact at puberty such that the lower elevations in estradiol occurring during the follicular and luteal phases of the hormonal cycle would induce a simple satiety-based anorexia and the higher rises in estradiol occurring midcycle would induce malaise-based anorexia. In Figure 2 we have suggested

Chambers and Bernstein

that high levels of estrogen cause an overstimulation of a satiety mechanism, which in turn activates a malaise mechanism. We have used the term malaise instead of illness to distinguish it from the type of illness induced by LiCl, which, as we suggested in the previous section, is qualitatively different than estrogen-induced illness. A loss in body weight would have two effects: it would decrease estrogen levels and, consequently, feelings of malaise, and it would stimulate hunger. The consequence of increases in eating and body weight would be elevations in estradiol levels and a return, first, of early satiety and then of malaise. Anorexia then becomes a learned means of reducing malaise. There are reduced blood levels of estrogen in patients suffering from anorexia nervosa (Halmi and Sherman, 1975; Russell et al., 1965) and neuropeptide Y, which has been shown to be a powerful stimulator of eating, is elevated in the cerebrospinal fluid of severely underweight anorexics (Clark et al., 1984; Kaye, 1996; Kaye et al., 1990). Once

Figure 2 Estrogen-based model for anorexia nervosa. It is suggested that at least four neural mechanisms contribute to anorexia: longterm satiety, food-satiety association, malaise, and food-malaise association. As the estrogen levels increase during the follicular phase of the menstrual cycle, the satiety-based mechanisms are activated. Further increases in estrogen activate the malaise-based mechanisms. The solid-line arrows represent the direction of the flow of information, the heavy lines represent excessive stimulation, solid circles represent inhibition, and the dotted-line arrows represent an existing unactivated connection.

Conditioned Taste Aversions

these individuals regain their normal weight, the levels of estrogen and neuropeptide Y return to normal. Kaye and colleagues have suggested that elevated levels of neuropeptide Y may contribute to the obsession with food that is evident in anorexics. Perhaps for individuals with anorexia nervosa, the aversive feeling of hunger is the lesser of two evils. Anorexia nervosa is more prevalent in women than it is in men, and, as indicated in the introduction to this section, every behavioral sex difference that has been studied in animals has been tied in some way to the gonadal hormones (Farrow, 1992; Halmi et al., 1987; Rolls et al., 1991). There is growing evidence of gonadal hormone involvement in human sex differences, and it is becoming apparent that the sex difference in anorexia nervosa is not an exception. There is a considerable amount of evidence to support an involvement of estrogen in anorexia nervosa. This evidence can be categorized in terms of the association of (1) estradiol with hypophagia, (2) fetal/neonatal androgenization with eating and body weight, and (3) fetal/neonatal androgenization with anorexia nervosa. First, as stated in the two preceding sections, exposure to estradiol induces hypophagia and aversions to novel foods, which can further contribute to the hypophagia. In addition, reduced palatability contributes to hypophagia, and reduced palatability is associated with satiety, illness, and learned aversions (see Secs. II. A, B and III. A). Early satiety and reduced palatability have been reported in patients with anorexia nervosa (Drewnowski, 1989; Holt et al., 1981). Also related to this issue is the evidence that antihistamines can attenuate estradiol-induced conditioned taste avoidance in rats (Rice, 1988; Rice et al., 1987), and they can speed recovery in some individuals suffering from anorexia nervosa (Halmi et al., 1986). Second, there is an association between neonatal masculinization and alterations in sensitivity to estrogen as well as body weight maintenance in rats. Conditioned taste avoidance can be induced with lower doses of estradiol in male rats than females (De Beun et al., 1991; Gustavson et al., 1989a; Rice et al., 1987). When female rats are masculinized with testosterone or diethylstilbestrol (DES) during neonatal development, the dose of estradiol that will induce conditioned taste avoidance is reduced (Gustavson et al., 1989b). In addition, fetal/neonatal masculinization can produce persistent changes in food intake and body weight maintenance in female rats. Exposure to testosterone propionate prenatally causes reductions in the body weight maintenance in adult females. Exposure to testosterone propionate or estradiol benzoate shortly after birth induces increases in food intake and body weight maintenance in adult females (Bell and Zucker, 1971; Slob and van der Werff ten Bosch, 1975).

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Finally, there is evidence of an association between fetal/neonatal androgenization and anorexia nervosa in humans. Women who were exposed to DES fetally are more likely to exhibit unexplained weight loss and a diagnosis of anorexia nervosa and/or bulimia than are unexposed women (Gustavson et al., 1989b, 1991). Further, the medical usage of DES began in 1947, peaked in 1964, and continued through 1971, and the agricultural usage of DES began around 1950 (Nash et al., 1983; Rico, 1983; Rumsey, 1983). This corresponds with the increase in the incidence of anorexia nervosa in 14- to 20-year-old females, but not other age groups, in the United States and Switzerland beginning about 1965 and continuing through 1976 (Jones et al., 1980; Willi and Grossmann, 1983). In addition, there is growing evidence for the existence of other environmental estrogens or ecoestrogens, which can have masculinizing effects in females during development (McLachlan and Arnold, 1996). That girls with anorexia nervosa were androgenized during fetal/neonatal development is supported further by the association between androgenization and abnormal gonadotropin regulation. Amenorrhea and subnormal levels of LH are found in girls with anorexia nervosa and are independent of weight loss in some anorectics (Halmi and Sherman, 1975; Newman and Halmi, 1988). Halmi (1974) found that amenorrhea preceded weight loss in 21% of the cases studied and was coincident with the onset of reduced eating in 52% of the cases. LH levels remain subnormal after a return to normal body weight (Isaacs et al., 1980). Abnormalities in gonadotropin regulation have been found in females of a number of different species that were androgenized during fetal/neonatal development (Barraclough and Leathem, 1954; Brown-Grant and Sherwood, 1971; Mennin and Gorski, 1975; Steiner et al., 1976; Tarttelin et al., 1975). There is enough support for an estrogen-based hypothesis to warrant serious consideration and further study, especially in light of the fact that many different kinds of chemical interventions have been employed without success (Mitchell, 1989). The role of estrogen in the triggering of anorexia nervosa after the beginning of menstruation is particularly promising. This hypothesis does not preclude the possibility that other factors contribute to anorexia nervosa. Certainly, social and cultural factors are powerful forces that could help to maintain and exacerbate the anorexia that is triggered by estrogen. However, it is unlikely that these factors are originating causes, particularly for girls who are in their teens. Psychological therapy generally has not been successful (Ratnasuriya et al., 1991). Follow-up studies 20 years after treatment have shown that 71% of patients had not recovered and, of these, 21% had died of suicide or complications of the disorder.

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VII.

Chambers and Bernstein

CONCLUSION

Conditioned taste aversions play a critical role in protecting us from eating harmful substances. Along with other feeding-associated mechanisms, they help establish our food and drink preferences and aversions. Since illness is the primary sensation that results in a taste aversion, it is highly likely that conditioned taste aversions will contribute to the anorexia prevalent in many physical illnesses. There is enough anecdotal evidence that indicates that when we are ill, we cognitively search for a food on which to place the blame, and the work on cancer anorexia and estrogen-based anorexia certainly substantiates this. Most of the emphasis in the study of conditioned taste aversions has focused on acquisition. This knowledge is important for preventing their development during illness. However, equally important is knowledge of how to eliminate them should they develop. More research is needed to identify the kinds of roles conditioned taste aversions play in different illnesses and the kinds of strategies that can be used to minimize their impact, in terms of both prevention and elimination, in the treatment of these illnesses. REFERENCES Ackroff, K., Vigorito, M., and Sclafani, A. (1990). Fat appetite in rats: the response of infant and adult rats to nutritive and nonnutritive oil emulsions. Appetite 15:171–188. Adachi, A., Kobashi, M., Miyoshi, N., and Tsukamoto, G. (1991). Chemosensitive neurons in the area postrema of the rat and their possible functions. Brain Res. Bull. 26:137–140. Arnold, A. P., and Breedlove, S. M. (1985). Organizational and activational effects of sex steroids on brain and behavior: a reanalysis. Horm. Behav. 19:469–498. Ballesteros, M. A., and Gallo, M. (2000). Bilateral tetrodotoxin blockade of the rat vestibular nuclei substitutes the natural unconditioned stimulus in taste aversion learning. Neurosci. Lett. 279:161–164. Barker, L. M., Best, M. R., and Domjan, M. (Eds.). (1977). Learning Mechanisms and Food Selection. Baylor University Press, Waco, TX. Barnett, S. A. (1958). Experiments on “neophobia” in wild and laboratory rats. Br. J. Psychol. 49:195–201. Barraclough, C. A., and Leathem, J. H. (1954). Infertility induced in mice by a single injection of testosterone propionate. Proc. Soc. Exp. Biol. Med. 85:673–677. Batson, J. D. (1983). Effects of repeated lithium injections on temperature, activity and flavor conditioning in rats. Anim. Behav. 11:199–204. Beatty, W. W. (1979). Gonadal hormones and sex differences in nonreproductive behaviors in rodents: organizational and activational influences. Horm. Behav. 12:112–163.

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933 Warren, R. P., and Pfaffman, C. (1959). Early experience and taste aversion. J. Comp. Physiol. Psychol. 52:263–266. Weiskrantz, L., and Cowey, A. (1963). The aetiology of food reward. Anim. Behav. 11:225–234. Wilcoxon, H. Dragoin, W., and Kral, P. (1971). Illness-induced aversions in rat and quail: relative salience of visual and gustatory cues. Science 171:826–828. Wilkie, D. M., Maclennan, A. J., and Pinel, J. P. (1979). Rat defensive behavior: burying noxious food. J. Exp. Anal. Behav. 31:299–306. Wilkin, L. D., and Cunningham, C. L. (1982). Pavlovian conditioning with ethanol and lithium: effects on heart rate and taste aversion in rats. J. Comp. Physiol. Psychol. 96:781–790. Willi, J., and Grossmann, S. (1983). Epidemiology of anorexia nervosa in a defined region of Switzerland. Am. J. Psychiatry 140:564–567. Wise, R., Yokel, P., and Dewitt, H. (1976). Both positive reinforcement and conditioned aversion from amphetamine and from apomorphine in rats. Science 191:1273–1274. Wurtman, J. J., and Baum, M. J. (1980). Estrogen reduces total food and carbohydrate intake, but not protein intake in female rats. Physiol. Behav. 24:823–827. Yamamoto, T. (1993). Neural mechanisms of taste aversion learning. Neurosci. Res. 16:181–185. Yamamoto, T. (1994). A neural model for taste aversion learning. In Olfaction and Taste XI, K. Kurihara, N. Suzuki, and H. Ogawa (Eds.). Springer, Tokyo, pp. 471–474. Yamamoto, T., and Fujimoto, Y. (1991). Brain mechanisms of taste aversion learning in the rat. Brain Res. Bull. 27:403–406. Yamamoto, T., Matsuo, R., and Kawamura, Y. (1980). Localization of cortical gustatory area in rats and its role in taste discrimination. J. Neurophysiol. 44:440–455. Yamamoto, T., Shimura, T., Sako, N., Sakai, N., Tanimizu, T., and Wakisaka, S. (1993). c-Fos expression in the parabrachial nucleus after ingestion of sodium chloride in the rat. NeuroReport 4:1223–1226. Yamamoto, T., Shimura, T., Sakai, N., and Ozaki, N. (1994a). Representation of hedonics and quality of taste stimuli in the parabrachial nucleus of the rat. Physiol. Behav. 56:1197–1202. Yamamoto, T., Shimura, T., Sako, N., Yasoshima, Y., and Sakai, N. (1994b). Neural substrates for conditioned taste aversion in the rat. Behav. Brain Res. 65:123–137. Yamamoto, T., Fujimoto, Y., Shimura, T., and Sakai, N. (1995). Conditioned taste aversion in rats with excitotoxic brain lesions. Neurosci. Res. 22:31–49. Young, J. K. (1991). Estrogen and the etiology of anorexia nervosa. Neurosci. Biobehav. Rev. 15:327–331. Yuan, D. L., and Chambers, K. C. (1999a). Estradiol accelerates extinction of a conditioned taste aversion in female and male rats. Horm. Behav. 36:1–16. Yuan, D. L., and Chambers, K. C. (1999b). Estradiol accelerates extinction of lithium chloride-induced conditioned taste aversions through its illness-associated properties. Horm. Behav. 36:287–298.

44 Clinical Disorders Affecting Taste: Evaluation and Management Steven M. Bromley University of Pennsylvania and Thomas Jefferson University, Philadelphia, Pennsylvania, U.S.A.

Richard L. Doty University of Pennsylvania, Philadelphia, Pennsylvania, U.S.A.

I.

INTRODUCTION

that can be associated with taste disturbance, (4) discuss clinically relevant anatomy and physiology associated with taste in the context of specific taste disorders, and (5) suggest options for patient management.

While disorders of taste are common in the general population, patients continue to be frustrated by an inability to find appropriate medical care (Deems et al, 1991; Hoffman et al., 1998). This frustration may stem, in part, from the fact that the spectrum of disorders influencing taste is broad, and the management of taste dysfunction requires a multidisciplinary mindset on the part of the physician or health care professional (Table 1) (Bromley, 2000). For example, which physician should manage an elderly patient who complains about a persistent “salty” taste and is known to have hypertension, thyroid disease, poor dentition, and gastroesophageal reflux? Should it be an internist, endocrinologist, dentist, or gastroenterologist? What about the depressed patient with throat or brain cancer who suffers from seizures or Bell’s palsy, continues to lose weight, has recurrent oral lesions, and complains that things “don’t taste right”? Such complexity helps to explain the difficulty that patients with chemosensory disturbance have in finding medical care, and why many physicians are uncertain how to manage such patients and where to refer them. The goals of this chapter are to (1) explore the numerous ways that patients present to their physician with taste complaints and what those complaints may imply, (2) provide an approach to obtaining a taste-focused medical history, physical exam, and sensory evaluation, (3) present the gamut of clinical disorders, across multiple specialties,

II. RANGE OF SYMPTOMS AND THEIR IMPLICATIONS There is tremendous variability in the ways patients present with taste problems. The range of disturbances include: (1) perceived loss or diminution of function, (2) Table 1 Medical Specialties Associated with the Management of Taste Dysfunction Cardiology Dentistry/dental surgery Dermatology Endocrinology Gastroenterology General and vascular surgery Gerontology Internal medicine/family medicine Neurology Neurosurgery Nutrition Oncology Otolaryngology–head and neck surgery Psychiatry/Psychology 935

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the distortion of everyday flavors, (3) persistent abnormal taste sensations in the absence of taste stimuli, and (4) syndromes of pain with or without taste triggers. One important distinction that should be made is whether the patient has difficulty with the perception of primary tastes versus the overall flavor of a substance. Primary taste sensations include sweet, sour, salty, bitter, and possibly metallic (iron salts), umami (monosodium glutamate, disodium gluanylate, disodium inosinate), and chalky (calium salts) (Kurihara and Kashiwayanagi, 2000; Schiffman, 1997). Chemical compounds can vary tremendously in their ability to stimulate these primary taste sensations. Somewhat like primary colors, these sensations blend to create variable “hues” of taste experience, and when they are combined with the sensory input from the olfactory and trigeminal systems, tasted substances acquire an aroma, texture, temperature, and spiciness—allowing for the overall perception of flavor (Woods, 1998). The primary flavor component of many “tastes” is, in fact, mediated by olfaction (e.g., chocolate, coffee, rose, strawberry, etc.). Stimulation of olfaction occurs during deglutition, when the molecules emanating from the food reach the olfactory mucosa via the so-called retronasal route. Therefore, when a patient complains of distorted flavor perception, it is essential to also consider coexisting or isolated impairment of the olfactory and trigeminal systems (see, e.g., Chapters 22 and 47). Impairment of taste can be devastating to a patient since it not only affects the ability to enjoy food products, but can ultimately alter food choices and patterns of consumption, thereby resulting in weight loss or weight gain, malnutrition, and possibly impaired immunity (Davidson et al., 1998; Mattes and Cowart, 1994; Schiffman and Wedral, 1996). People rely on taste function to gauge the quality of ingested nutrients; thus, in patients who are diabetic or hypertensive, the loss of taste may lead to dangerous overcompensation for the loss by adding too much sugar or salt to food (Bromley and Doty, 2002). Without proper gustatory function, people are at risk for ingesting potential toxins and spoiled foodstuffs (which usually taste “bad”) (McLaughlin and Margolskee, 1994). Importantly, there is evidence that taste is needed to trigger normal ingestive and digestive reflex systems such as the secretion of oral, gastric, pancreatic, and intestinal juices (Giduck et al., 1987; Schiffman, 1997) (see Chapter 42).

III. CLASSIFICATION OF GUSTATORY DISORDERS The classification of gustatory disorders follows a schemata similar to that of olfactory disorders:

Bromley and Doty

Ageusia: inability to detect qualitative gustatory sensations; i.e., absence of taste Hypogeusia: decreased sensitivity to tastants Dysgeusia: distortion in the perception of a normal taste (e.g., the presence of an unpleasant taste when a normally pleasant taste is perceived) Gustatory agnosia: inability to recognize a taste sensation, even though gustatory processing, language, and general intellectual functions are intact Additionally, patients often complain of oral sensations of burning or numbness that may or may not have its genesis in gustatory afferents. An extreme example is burning mouth syndrome (BMS), discussed in more detail later in this chapter, in which the patient experiences the sensation of “burning” within the mouth without obvious physical cause.

IV.

MEDICAL EVALUATION

A.

Medical History

A focused clinical history for a person complaining of chemosensory dysfunction, particularly taste, begins with an accurate description of the subjective problem, including: onset; frequency; timing; severity; exacerbating or relieving foods or products; relation to daily activities, bodily functions, and states of mind; prior treatments and their efficacy; and comorbid medical conditions. Particular note should be made in the history of problems with speech articulation, salivation, chewing, swallowing, oral pain or burning, dryness of the mouth, periodontal disease, foul breath odor, recent dental procedures or surgeries, recent radiation exposure, medications, and bruxism. Also, inquiry into the patient’s diet and oral habits may reveal exposure to oral irritants. Questions about hearing, tinnitus, and balance are important since the vestibulocochlear nerve (CN VIII) travels in close proximity to the facial nerve and can be susceptible to similar etiologies of disturbance (e.g., cerebellopontine angle tumor). Considering the occasional effect of gastroesophageal reflux on taste, asking about stomach problems may also be relevant. Asking about constitutional symptoms, such as fever, malaise, headache and body pains, and rashes, may also be important since such symptoms often accompany cancers or systemic inflammatory conditions (e.g., lupus). The possibility of seizure should be entertained, especially if the patient describes having “blackouts” or witnessed abnormal movements or actions. Lastly, a careful assessment of medication usage is critical and cannot be overemphasized.

Clinical Disorders Affecting Taste

B.

Physical Examination

In the evaluation of the oral cavity, an examiner should attend to the condition of the teeth and gums. Notably, a dysgeusia may result from exudates that commonly appear with gingivitis or pyorrhea. The nature and integrity of the fillings, bridges, and other dental work should be inspected. Visual inspection of the tongue surface may show signs of scarring, inflammation, or the absence, atrophy, or overgrowth of papillae. A whitish plaque on the tongue can be from candidiasis, lichen planus, leukoplakia, or food product. Inflammation of the tongue, or glossitis, may be present and can reflect local or systemic conditions. The physician should pay particular note to the color and degree of salivation. Palpation of the tongue should be performed to detect masses, neoplastic lesions, or collections deep in the tongue’s musculature. The neck should also be evaluated for the presence of masses, thyromegally, and carotid bruits. Neurologically, the integrity of CN VII, IX, and X can be evaluated by screening for deficits in nongustatory functions of these nerves, such as facial movements and expression, swallow function, gag, salivation, and voice production. To establish whether taste pores or papillae appear normal or are present in adequate numbers, the tongue can be stained with a dye and then photographed using a low power magnification (e.g., Doty et al., 2001; Miller and Reedy, 1990). Although not performed routinely, biopsy of circumvallate or fungiform papillae for detailed microscopic examination can establish whether pathological changes are present in taste bud tissue. C.

Diagnostic Testing

Because many complaints of taste loss really reflect smell loss, it is important to assess the patient’s olfactory function accurately, which is now easy to do given the commercial availability of quantitative smell tests (see Chapter 10). Unfortunately, taste testing is less practical, largely because of time considerations and the general lack of availability of commercially available and well-validated taste tests (see Chapter 37 for a review of taste tests). While a number of whole-mouth taste tests have been described in the literature, regional taste testing is required to establish the function of each of the nerves innervating specific taste bud fields, as whole-mouth tests are insensitive to even complete dysfunction of one or several of the nerves that innervate the tongue. Two approaches are available to regionally assess taste function psychophysically. In chemical testing, liquid stimuli are presented to selected target regions of the tongue or oral cavity via pipettes, special delivery devices, or filter paper disks. In electrical

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testing (also termed electrogustometry), low levels of electrical current are presented to taste bud fields. At low current ranges, gustatory—not somatosensory—afferents are activated. Equipment designed to present such electrical current to the tongue, termed electrogustometers, typically employ batteries to produce the current. One advantage of electrical testing is that there is no need to painstakingly mix and otherwise prepare chemical solutions prior to testing, unlike chemical testing where solutions must be made on a regular basis and can be used only for relatively short periods of time because of degradation and other factors. Another advantage of electrical over chemical testing is that no rinse trials are needed, saving considerable time. Moreover, electrogustometers are easily moved from place to place, unlike the movement of large volumes of liquids that is required in chemical testing. As noted in Chapter 37, however, regional test results can be misleading in some instances because the testing of small regions of the tongue may not provide an adequate sample of the whole taste bud field innervated by a given nerve. This is particularly true when a “patchy” lingual distribution of papillae is present. In general, since both chemical and electrical regional test results correlate highly with the number of fungiform papillae (Doty et al., 2001; Miller et al., 2002), it is essential for the examiner to be certain that the taste stimulus or electrode is placed on a tongue region containing papillae. Of course, too few papillae would be expected to be associated with overall poor taste test performance. Imaging studies can be helpful in the evaluation of gustatory disturbance (see Chapter 28). Plain radiographs of the teeth and jaw may reveal focal lesions of the teeth (e.g., fracture, cavity, impaction) or periodontal tissues (e.g., abscess extension) in proximity to gustatory fibers (e.g., chorda tympani–lingual nerve) and the tongue. Computed tomography (CT), however, is of greater utility in visualizing peripheral and central structures of the head and neck that are frequently affected by trauma, surgery, infection, cancer, and systemic disease. For example, fine axial “cuts” through the mastoid portion of the temporal bone can demonstrate subtle bony defects and mass lesions in and around the middle ear. Coronal CT scans are useful to evaluate paranasal anatomy. Noncontrast CT is used to show bony defects and acute blood accumulations, whereas contrast-enhanced CT highlights areas of inflammation or abnormal vascularity (as seen with many tumors). Magnetic resonance imaging (MRI) is the method of choice for soft tissue anatomy and central nervous system structures. Particularly when sequences such as diffusion-weighted imaging (DWI) and fluid-attenuated inversion recovery (FLAIR) are used, MRI can discern infarcts of variable ages and other changes within central

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gustatory pathways. Indeed, gustatory dysfunction has been related to MRI-established ischemia, hemorrhage, or demyelinating plaques in the brain stem and higher cortical areas of some persons with chronic dysgeusia (Onoda and Ikeda, 1999; Yabe et al., 1995). Gadolinium–diethylenetriamine pentaacetic acid (DTPA) enhancement is usually necessary to visualize lesions caused by demyelination, tumor, and infection. An electroencephalogram (EEG) should be obtained in all patients suspected of having seizure activity. Brain stem auditory evoked response (BAER) testing is useful to evaluate the continuity and symmetry of the brain stem auditory pathways and nuclei and, therefore, provides a screen for focal subcortical injury in this region. Although restricted in their resolution and general availability, functional imaging of the brain using positron emission tomography (PET), single proton emission computed tomography (SPECT), and functional MRI (fMRI) may be helpful in evaluating cortical and subcortical structures related to gustatory processing, particularly in the context of cerebrovascular disease, epilepsy, and dementia. Depending on the suspected underlying medical problem affecting taste, a number of laboratory studies are also available (Bromley, 2000).

V.

CAUSES OF TASTE DYSFUNCTION

A wide range of disorders and interventions has been associated with at least some disturbance of taste function. Mechanisms for the alteration of taste may include (1) the release of bad-tasting materials from oral medical conditions and appliances (e.g., gingivitis, purulent sialadenitis), (2) transport problems of tastants to the taste buds (e.g., excessive dryness of the oral cavity, damage to taste pores from a burn, candidiasis), (3) destruction or loss of taste buds themselves (e.g., local trauma, invasive tumor), (4) damage to one or more neural pathways innervating the taste buds (e.g., Bell’s palsy, trauma, dental or surgical procedures), (5) involvement of central neural structures (e.g., tumor, epilepsy, stroke), and (6) systemic disturbances of metabolism that presumably affect the taste system via any of the aforementioned mechanisms (e.g., medications, hypothyroidism, diabetes, liver or kidney disease). Although total loss or markedly diminished wholemouth gustation exists, such conditions are rarely produced by nonmetabolic diseases or disorders unassociated with central ischemic or other damage, since regeneration of taste buds can occur and peripheral damage alone would require the involvement of multiple pathways. Thus, while 433 of 585 patients (74%) with

verifiable olfactory loss complained to us of both smell and taste disturbance, less than 4% had verifiable wholemouth gustatory dysfunction, and even that dysfunction was limited (Deems et al., 1991). Regional deficits in taste dysfunction are much more common. For example, in one study, sensitivity to three relatively low concentrations of NaCl was measured on the tongue tip and 3 cm posterior to the tongue tip in 12 young (20–29 years) and 12 elderly (70–79 years) subjects. On average, the young subjects were more sensitive to NaCl on the tongue tip than on the more posterior stimulation site and exhibited, at both tongue loci, an increase in detection performance as the stimulus concentration increased. The elderly subjects, who would be expected to exhibit, at worse, moderate deficits on whole-mouth testing, performed at chance level (Matsuda and Doty, 1995). Patients with focal regions of taste impairment can, in some instances, perceive an alteration in overall gustatory perception. Thus, in one series, nearly 57% of patients presenting with Bell’s palsy complained of dysgeusia (Adour, 1982), and in a different study more than 9% of patients with surgically confirmed acoustic neuromas experienced loss of taste as a primary complaint (Harner and Laws, 1981). However, many people are unaware of such deficits. In fact, it is unusual for patients to specifically recognize their loss of taste sensation on half of the anterior tongue following unilateral sectioning of the chorda tympani in middle ear surgery. This lack of awareness stems, in part, from the redundancy of the multiple taste nerves, as well as compensatory mechanisms. Recently it has been found that anaesthetizing the chorda tympani nerve (CN VII) unilaterally enhances taste perception on the rear of the tongue (CN IX), particularly on the side contralateral to that of the anesthesia (Kveton and Bartoshuk, 1994; Yanagisawa et al., 1998). Such enhancement, which would be expected if significant regional lingual damage occurs, seems to be accentuated for unpleasant tastants and, moreover, seems to be associated with phantom tastes induced without stimuli being applied to the tongue. Yanagisawa et al. (1998) reported that this procedure increases the perceived intensity of bitter substances, such as quinine, applied to posterior taste fields, whereas the reverse occurs for the salty taste of NaCl. When both chorda tympani nerves were anesthetized, the effect was perceived bilaterally. In about 40% of their subjects, a phantom taste, usually localized to the posterior tongue contralateral to the anesthesia, appeared in the absence of stimulation and disappeared when the region of origin was anesthetized. Such phantoms may reflect central interactions among the taste nerves (e.g., release of inhibition), since the taste pathways do not cross until they reach the thalamus.

Clinical Disorders Affecting Taste

A. Disorders of the Oral Cavity and Related Structures That Affect Taste 1.

Oral Mucosa

The mucous membranes of the mouth, tongue, and oropharynx are directly exposed to the outside environment and, as such, are unique in their ability to protect, adapt, and regenerate in response to continuous chemical, mechanical, and thermal challenges. Digestion begins in the mouth with tastants being solubilized in saliva and exposed to a myriad of enzymes and resident microflora. A testimony to the durability of the oral mucosa is the fact that humans ingest many diverse foods over a broad range of temperatures and textures, often in the context of abrasive oral appliances and harmful habits such as tobacco use. Additionally, the oral epithelium tolerates large microbial concentrations of the order of a magnitude found elsewhere only in the colon. This durability may stem, in part, from antimicrobial properties of saliva and the high basal turnover rate of oral epithelial cells (Beidler and Smallman, 1965). Microorganisms that bind to surface cells of the mucosa are rapidly sloughed as the surface is renewed. Subsequently, disorders that alter the secretion and contents of saliva, the turnover rate of epithelial cells, and the balance of microorganisms within the epithelium, have the potential to alter proper mucosal function and to distort taste. By providing moisture and lubrication, saliva facilitates the chewing and swallowing of food, aids in the production of speech, acts as a buffer to acids and bases, and increases the delivery of solubilized chemicals to taste receptors (Mandel, 1989). Inadequate salivary flow via impaired production or secretion (e.g., numerous medications, sialolithiasis, irradiation of salivary glands) can result in (1) the tendency of oral mucosa to stick together, (2) impairment of eating and speech function, (3) aggravation of oral infections, (4) impaired ability to properly wear dental prostheses, and, in some instances, (5) abnormal flavor sensations. Clinically, this constellation of problems is considered xerostomia and may ultimately be the means by which many local and systemic diseases cause taste disturbance. Importantly, saliva likely plays a significant role in taste bud maintenance. Indeed, taste buds within the fungiform papillae of rats disappear following removal of the submandibular and sublingual salivary glands—disappearance that can be reversed by adding epidermal growth factor to their drinking water (Morris-Witman et al., 2000). There are a number of lesions involving the oral epithelium that can be associated with altered taste perception, tactile irritation, and possibly oral pain (Table 2), either by direct involvement with taste-related structures or through

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association with a taste-altering systemic illness (e.g., local or systemic cancer, autoimmune disorder, vitamin deficiency). Most healthy tissues of the oropharynx appear pink because the epithelium and connective tissues of the lamina propria are relatively translucent and allow red light to reflect from the underlying capillary bed. Disruption of the epithelial architecture, from such pathological processes as chemical necrosis of the surface layers, microbial colonies, exudate, and epithelial thickening from continued trauma, can scatter and reflect incident light creating a discolored whitish lesion. In regions of particular inflammation within the mucosa (e.g., glossitis), reddish lesions can predominate. For example, continued exposure to the heat and smoke from burning tobacco can lead to a rough, white, and fissured tongue surface, often with associated areas of inflammation, known as nicotinic stomatitis (Rossie and Guggenheimer, 1990). Leukoplakia is a clinical diagnosis reserved for whitish lesions that cannot be scraped off the tongue surface. It is important to recognize the presence of leukoplakia since approximately 20% of these lesions represent premalignant epithelial dysplasia, carcinoma in situ, or frank invasive squamous cell carcinoma (Allen and Blozis, 1998; Rodriguez-Perez and Banoczy, 1982). The apparent overgrowth or the exaggeration of the length and thickness of the filiform papillae (e.g., hairy tongue) can result from conditions that impair appropriate desquamation either by reducing salivary flow, limiting oral movements, or causing temporary imbalances in the growth of normal microbial flora (e.g., antibiotic therapy). Hairy tongue can sometimes be associated with a disagreeable taste or sensations of gagging when swallowing. Squamous cell carcinoma is particularly worrisome, since it is responsible for more than 90% of oral cancers, it has a considerable mortality rate (5-year survival rate – 52%), and it may have only subtle affects, if any, on one’s taste function (Salisbury, 1997). Arising from mucosal epithelium, squamous cell carcinoma is common on the ventral and lateral surfaces of the tongue and can be invasive, producing swelling and induration (Allen and Blozis, 1998). After several months of growth, the cancer can develop into an exophytic (sometimes endophytic), painful, ulcerated mass that bleeds easily and has a necrotic odor (Salisbury, 1997). The treatment of oral cancer, as well as other cancers of the head and neck, with agents like radiation and chemotherapy is more often associated with impairment of taste function than the cancers themselves. Radiation therapy alters taste bud cell turnover and can affect taste function, although return after radiation treatment has been observed (Conger, 1973). Additionally, radiation therapy fields often include salivary glands (e.g., parotid or submandibular) that are near the

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Table 2 Disorders Involving Structures of the Oral Cavity That Can Alter Taste Perception Epithelial Abnormalities Autoimmune Behcet’s syndrome Crohn’s disease Lupus erythematosus Pemphigus vulgaris Reiter’s syndrome Scleroderma Sjögren’s syndrome Wegener’s granulomatosis Discolored lesions Acanthosis nigricans Chronic ulcerative stomatits Diffuse or multiple papillary/verrucous lesions (e.g., hairy tongue) Erythema multiforme Leukoplakia Lichen planus Median rhomboid glossitis (MRG) Nicotinic stomatitis Reactive papillary hyperplasia Metabolic Primary amyloidosis (affecting tongue) Infection Candidiasis Ulcerative lesions coxsachievirus gonorrhea herpes simplex virus mycoses syphilis varicella/zoster Local injury Burns/chemical injury Radiation/chemotherapy Trauma

Neoplastic Kaposi’s sarcoma Oral melanoma Squamous cell carcinoma Tongue cancer (erosive) Verrucous carcinoma Odontogenic Diseases Acute necrotizing ulcerative gingivitis (ANUG, or “trench mouth”) AIDS-related peridontitis Dental cares/abscess Gingivitis (acute and chronic) Salivary Gland Abnormalities Acute suppurative sialoadenitis from: sialolithiasis radiation/chemotherapy Sjögren’s syndrome cystic fibrosis dehydration medications viral infections granulomatous disease (e.g., TB, sarcoid) benign lymphoepithelial lesion salivary gland tumor or cyst Chronic sialoadenitis from: recurrent sialolithiasis congenital or trauma-related stenosis foreign body salivary gland tumor or cyst Paranasal Sinus Abnormalities Postnasal drip Purulent sinusitis Gastroesophageal Reflux Disease

site of the primary tumor or lymph nodes that need treatment; this can result in irreversible damage to acinar cells, profound impairment of salivary flow, subsequent dessication of the mucosa, and xerostomia (Salisbury, 1997). Also, susceptibility to opportunistic infections (e.g., dental caries, candidiasis) increases after therapy. Necrosis of the tongue after arterial chemotherapy has been reported, in addition to the known direct affect on basal cell and taste receptor production (Duhra and Foulds, 1988; Shih et al., 1999). Local injury to the oral cavity from such insults as physical trauma, burns, and toxic chemical exposures can acutely alter flavor perception and create a dysgeusia associated with the region of mucosa involved. Applying a caustic substance to the oral mucosa can produce variable degrees of epithelial necrosis, resulting in whitish lesions, erosions, ulcerations, or blisters. For example, the inappro-

priate use of aspirin as a topical anesthetic causes a common and severe form of chemical burn to the mucosa (Allen and Blozis, 1998). Although most etiologies of local injury produce only transient lesions that last 1–2 weeks, repeated exposures (e.g., heavy use of O2-liberating mouthwashes) or injury (e.g., prolonged intubation with an oral endotracheal tube), especially in the context of underlying xerostomia, can produce more lasting and symptomatic lesions and can produce, in some patients, taste disturbances. Infections are common causes of distorted taste and oral pain. In fact, any local infection in and around the neural pathways or receptor sites associated with taste perception has the potential to disturb or impair taste. The mechanisms of taste disturbance can be direct inflammation and injury to a taste-related neural structure or the associated release of mucopurulent material that typically generates

Clinical Disorders Affecting Taste

a poor flavor. Important anatomical regions include (1) the surface of the tongue, mouth, and oropharynx, (2) the teeth and peridontal tissue, (3) the salivary glands, and (4) the middle ear. Candida albicans is a commensal yeast that normally exists, in small numbers, in the oral flora. However, when the oral immune system is not functioning optimally—as seen in the very young or old, in the HIVinfected, in patients with leukemia, or in those who have recently taken corticosteroids, chemotherapy, or broadspectum antibiotics—candidiasis of the tongue and oropharynx can ensue. The development of a pseudomembrane overlying an inflamed oral mucosa is called “thrush” and can be treated with antifungal therapy (Allen, 1992). There are other types of infectious diseases causing oral lesions, many of them viral, and most resuting in some form of pharyngitis, stomatitis, and an ulterative or blistered lesion of the oral mucosa. Typically they are treated with topical or systemic antibiotics. There are several autoimmune syndromes that affect the oral mucosa, usually by causing xerostomia and/or ulceration that, on rare occasion, can result in taste disturbance (for review, see Campbell et al., 1983). Sjögren’s syndrome (SS) is associated with salivary and lacrimal gland dysfunction—resulting in dry eyes and dry mouth (also called sicca syndrome)—and arthritis. If the symptoms of Sjögren’s syndrome occur in the context of another autoimmune disease (e.g., lupus, scleroderma, rheumatoid arthritis), it is said to be secondary SS. Systemic lupus erythematosus (SLE) is an multisystem inflammatory disease of small blood vessels that can produce a wide range of clinical symptoms including superficial ulcerative, vesicular, and white keratotic lesions of the tongue and oral mucosa. Behcet’s syndrome is also a multisystem disorder consisting of recurrent oral and genital ulcers, inflammatory disease of the eyes and gastrointestinal tract, arthritis, and, rarely, cranial neuropathies or meningoencephalitis. Pemphigus vulgaris is characterized by serum antibodies directed against components of the epidermis and mucosa, forming characteristic bulla that progress to superficial and friable eroded areas most often involving the palate, buccal mucosa, and tongue (Laskaris et al., 1982). 2.

Tongue

The tongue is a highly mobile muscular structure that helps to coordinate numerous functions within the mouth. These functions include (1) manipulating food during mastication (chewing), (2) squeezing food into the pharynx during deglutition (swallowing), (3) forming words during articulation (speech), (4) oral cleansing, and (5) taste. Embryologically, the anterior two thirds of the tongue (oral part) develops from the first brachial arch, while the

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posterior one third (pharyngeal part) develops from the second and third brachial arches, producing two distinct geographic regions identifiable by their nerve supply (CN VII and IX, respectively) and lymphatic drainage. The arterial supply to the tongue is chiefly through the lingual artery, which arises from the external carotid. Within the pharynx, the oral and nasal cavities communicate and allow the passage of volatile chemicals released during chewing into the nasal passages. Disorders of the tongue, such as neoplasms, infarction, and swelling, can not only affect overall lingual function, but can distort surface structures and impair taste. In primary amyloidosis, amyloid deposition within the lingual tissues can impair taste, which can be an early symptom of the disease (Ujike et al., 1987). 3.

Gingival/Dental/Periodontal Structures

Most diseases affecting the periodontal soft tissues (the gingiva) and the soft and hard palates (the periodontium) have an infectious component to their pathology. Dental infections are common among patients who neglect regular dental care. A dentoalveolar abscess, usually resulting from dental caries, can erode from the tooth to the adjacent gingival tissues to create a draining gingival fistula. These fistulas can drain bitter and foul-tasting purulent material. A peridontal cellulitis and/or abscess can involve the adjacent bone or perimandibular soft tissues and is usually associated with significant gingivitis, variable degrees of facial swelling, and fever. Acute necrotizing ulcerative gingivitis (ANUG, also known as trench mouth from the outbreaks among the troops in the trenches during World War I) is now not considered to be contagious but, rather, an opportunistic overgrowth of relatively common microbes in the mouth (Melnick, 1988). Predisposing factors include psychic and environmental stress, nutritional inadequacies, poor oral hygiene, and smoking. The clinical presentation is stereotyped and involves the sudden onset of gingival pain, bleeding, a bad taste in the mouth, and a fetid mouth odor that can be recognized by the patient (Melnick, 1988). A periodontal infection that is strategically located in the area of the chorda tympani-lingual nerve as it passes near the third molars can injure this nerve and affect taste on the ipsilateral side of the tongue. 4.

Salivary Gland Disorders

A primary function of the salivary glands in humans is to produce saliva, which, as previously noted, aids in the digestion and swallowing of food, provides immunological defense, releases hormones and hormone-like products, and mediates taste sensations (see Chapter 31). Besides function, the anatomical orientation of the glands is

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important to taste since cranial nerve VII, a major nerve responsible for gustatory function (see below), emerges from the stylomastoid foramen to enter the substance of the parotid gland. Therefore, lesions of the parotid (e.g., tumors, abscesses) can eventually involve this nerve and affect taste. Infections of the salivary glands (saladinitis) can be acute or chronic. Bacterial infections of the gland parenchyma are typically retrograde in nature and the result of a mechanical blockage or diminished production of saliva (Berndt et al., 1931; Gayner et al., 1998). However, there are viral infections, such as the mumps or HIV, which can produce localized gland disease (Gayner et al, 1998). Local symptoms of suppurative sialoadenitis include pain, swelling, induration, and suppurative discharge from the duct orifice into the oral cavity yielding a foul taste. Although infections can occur in any salivary gland, the parotid is the one most commonly involved (Gayner et al, 1998). High pH and viscosity of gland saliva likely contribute to calcium phosphate stone formation within the salivary duct, resulting in obstruction, stagnant salivary flow, dry mouth, and a suppurative infection. Contributing factors to infection include poor oral hygiene and compromised host resistence. Other than infection, there are some disorders that have a direct effect on the function of salivary glands, such as the aforementioned autoimmune disorder Sjögren’s syndrome. 5. Gastroesophageal Reflux Disease Gastroesophageal reflux disease (GERD) is defined as a recurrent regurgitation of acidic gastric contents up the esophagus as a result of transient or chronic relaxation of the lower esophageal sphincter (Isolauri et al., 1997). GERD symptoms can be obvious, such as recurrent midchest discomfort from esophageal irritation (heartburn), or they can be rather subtle, only manifesting as chronic cough, halitosis, hoarseness, or a recurrent acid or sour taste (Nelson et al., 2000; Richter, 1998). While it is not clear whether GERD can result in permanent injury to the gustatory mucosa, dysgeusias due to GERD do occur, but typically resolve with proper treatment of the reflux. B. Disorders of the Multiple Peripheral Neural Pathways That Affect Taste Depending on their location, taste receptors within the taste buds are innervated by branches of several cranial nerves, including CN VII, IX, and X, which, unlike CN I, are mixed motor and sensory nerves transmitting multiple forms of information. This becomes important when considering clinical syndromes that involve taste dysfunction since variable degrees of either motor or sensory

Bromley and Doty

disturbance may be present, depending upon where and to what extent the taste-mediating cranial nerve is injured (see Table 3). All the peripheral gustatory fibers enter the brain stem and project to the nucleus of the tractus solitarius (NTS), which begins in the rosterolateral medulla and extends caudally along the ventral border of the vestibular nuclei. 1.

The Facial Nerve (CN VII)

The facial nerve (CN VII) supplies the muscles of facial expression, the stapedius muscle of the middle ear (which helps to dampen the movement of the stapes in response to loud auditory stimuli), the sublingual and submandibular salivary glands, the lacrimal glands, and the membranes of the oral and nasal cavities. In addition, this nerve conveys taste sensation from the taste buds found in the fungiform and filiform papillae on the ispilateral anterior two thirds of the tongue (via the lingual nerve to the chorda tympani) and on the soft palate (via the lesser palatine nerve branch of the greater petrosal nerve). Because of the relative long and winding course of the facial nerve, it is susceptible to peripheral injury in a number of locations. Thus, from the tongue, gustatory fibers travel through the lingual nerve, passing within the soft tissue of the jaw along the medial surface of the mandible in close proximity to the mandibular third molar. At this point they become the chorda tympani nerve, which penetrates the skull via the stylomastoid foramen and turns within the tympanic cavity of the middle ear along the medial surface of the tympanic membrane between the malleus and the incus. The fibers then synapse with the geniculate ganglion at the bend of the facial nerve within the facial canal, where the projection becomes the nervus intermedius, which travels next to the eighth nerve through the internal auditory meatus into the cerebellopontine angle. This nerve subsequently penetrates the caudal border of the pons near the medulla and enters the brainstem (Fig. 1). Bell’s palsy is among the most common causes of facial nerve injury resulting in characteristic unilateral facial weakness and gustatory loss (Fig. 2). Clinical criteria for Bell’s palsy include the sudden weakness of all muscles of one side of the face in the absence of CNS, ear, or cerebellopontine disease (Taverner, 1973). The etiology is still considered idiopathic, although the viral hypothesis is the most widely accepted (Adour et al., 1975; Takahashi et al., 2001). In fact, the herpes simplex virus 1 genome has been isolated from facial nerve endoneural fluid in affected patients (Murakami et al., 1996). It affects males and females of all ages. Although Bell’s palsy is frequently associated with ipsilateral loss of taste over the anterior two thirds of the tongue, the patient may or may not

Clinical Disorders Affecting Taste

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Table 3 Disorders of Peripheral Nerves That May Alter Taste Perception Cranial Nerve VII (facial nerve) Infection/polyneruitis Bell’s palsy Botulism Guillain-Barré syndrome Herpes zoster Human immunodeficiency virus Kawasaki disease Leprosy Lyme disease Otitis media Acute bacterial Chronic bacterial Cholesteatoma Syphilis Tuberculosis Neoplastic Glomus tumor Primary parotid tumor Schwannoma/acoustic neuroma Temporal bone cancer/metatases Trauma Barotrauma Birth trauma Injection of local anesthesia (e.g., dental procedure) Middle ear surgery Temporal or mandibular bone fracture Third molar extraction Neurovascular Brain stem stroke (pontine) Inflammatory Multiple sclerosis Sarcoidosis Melkersson syndrome Cranial Nerve IX (Glossopharyngeal nerve) Trauma Bronchoscopy Laryngoscopy Penetrating neck trauma Skull base fracture Tonsillectomy

Neoplastic Glomus jugulare tumor Neurofibroma Skull-based metastases Neurovascular Carotid artery aneurysm or dissection Brain stem stroke (pontine, medullary) Infection Botulism Guillain-Barré syndrome Herpes zoster of petrosal ganglion (rare) Leprosy Meningitis Otitis media Pharyngeal/retroparotid abscess Inflammatory Multiple sclerosis Sarcoidosis Cranial Nerve X (vagus nerve) Trauma Neck surgery Penetrating trauma Skull base fracture Infection Cytomegalovirus Guillain-Barré syndrome Leprosy Meningitis Pharyngeal/retroparotid abscess Poliomyelitis Neoplastic Glomus jugulare tumor Neurofibroma Skull-based metastases Neurovascular Brain stem stroke (medullary) Carotid artery aneurysm or dissection Degenerative Multisystem atrophy Progressive bulbar palsy Inflammatory Multiple sclerosis Sarcoidosis

recognize this loss. Involvement of taste in Bell’s palsy localizes the lesion to the neural pathways from the pons, along the nervus intermedius portion of the facial nerve, through the geniculate ganglion, to the point where the chorda tympani joins the facial nerve in the facial canal. A more severe form of Bell’s palsy, Ramsay Hunt syndrome, results from active infection of the geniculate ganglion with herpes and involves pain and vesicles in the external auditory canal or soft palate. Bartoshuk and Miller (1991) describe a patient with this syndrome whose neural

involvement included both CN V and CN IX on the left side. Ratings of the intensity of four major taste qualities were obtained at regular intervals across a nearly 2-year period for the front, back, and palate regions. The entire left side of the patient was devoid of taste function about three months after the herpes zoster oticus attack. Although all taste qualities were perceived on the left rear and palate regions after about a year, only sweetness was perceived on the left front. Even by the end of study, full taste recovery of the anterior tongue was not evident.

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A

B Figure 1 (A) Pathways of the taste fibers involving the facial nerve (CN VII). From the anterior two thirds of the tongue, taste fibers follow the lingual nerve near the third molar, along the medial aspect of the mandible, and then leave the lingual nerve to become the chorda tympani nerve proper. The chorda tympani nerve then passes through the middle ear (see Fig. 1B) to join the facial nerve in route to the brain stem. The winding course of these taste fibers leaves them susceptible to trauma, both accidental and iatrogenic, at a number of locations (see text). (B) A depiction of the middle ear demonstrating the close proximity of the facial nerve/chorda tympani fibers to the tympanic membrane, ossicles, and the tensor tympani and stapedius muscles. (Artist: Paul Schiffmacher, University of Pennsylvania. Copyright © 2001, Richard L. Doty.)

Clinical Disorders Affecting Taste

Figure 2 Unilateral facial paralysis as a result of Bell’s palsy shown in resting (A), smiling (B), and tongue protrusion (C) states. Clinical evaluation revealed an 83-year-old woman with hypertension, hypercholesterolemia, and paroxysmal atrial fibrillation who woke up with a right facial droop association with slurred speech, drooling from the right side of the mouth, and decreased taste on the right anterior two thirds of the tongue. There was no evidence of stroke. (Copyright © 2001, Steven M. Bromley.)

Typical Bell’s palsy begins with pain in or behind the ipsilateral ear with subsequent unilateral facial weakness within several days (maximum paralysis within 48–72 hours). In addition to ipsilateral taste loss, hyperacusis (due to weakness of the stapedius muscle) is present in many cases. The taste-salivary reflex also can be compromised in Bell’s palsy. This reflex results in salivation when a gustatory stimulus, such as a weak acid, stimulates the taste receptors on the anterior tongue, and reflects a relay from the nucleus of the solitary tract to the parasympathetic fibers innervating the salivary glands. Regeneration of the facial nerve after Bell’s palsy may be abnormal, such that novel innervation to nearby structures can produce synkinesis of the facial musculature when chewing or tearing of the ipsilateral eye while eating (“crocodile tears”). Melkersson syndrome, a familial disorder of recurrent Bell’s palsy (sometimes alternating), can result in permanent facial plegia and is associated with lingua plicata (furrowed or fissured tongue) and recurrent facial edema. In general, taste dysfunction resulting from trauma is much less common than posttraumatic smell dysfunction,

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with solitary ageusia of one or more primary taste modalities occurring in less than 1% of persons with major head injury (Deems et al., 1991; Sumner, 1967) (see Chapter 45). Only rarely are trauma-related taste disturbances unaccompanied by smell impairment. The prognosis for posttraumatic ageusia is far better than for posttraumatic anosmia, and recovery from ageusia usually occurs in most patients over a period of a few weeks to months. The etiology of posttraumatic taste dysfunction varies. Head trauma that results in injury to the middle ear, a finding often encountered with temporal bone fractures, can potentially result in both ipsilateral impairment of taste function and injury to the preganglionic parasympathetic fibers, thus preventing salivary secretion. It has been estimated that approximately 7–10% of temporal bone fractures result in facial nerve dysfunction (see Chang and Cass, 1999, for review). Injury to the lingual nerve from trauma in and around the mouth and tongue is another cause of taste impairment. The lingual nerve is a branch of the mandibular division of the trigeminal nerve CN V3, and it is the most proximal pathway to the tongue for general somatic sensation (touch) and special visceral sensation of taste (due to coexisting chorda tympani fibers). Thus, injury to the jaw resulting in mandibular fracture (e.g., punch with a fist) or to the inside of the mouth (e.g., difficult orotracheal intubation, aggressive dental procedure) can result in numbness and taste loss over the anterior two thirds of the ispilateral side of the tongue affected. Compression injury to the lingual nerve may have a greater effect on taste than on fine-touch sensory components, since the chorda tympani fibers of the conjoined nerve are more superficial and posterolateral and therefore more susceptible to partial transection or pressure on the side of the nerve (Wantanabe et al., 1995). A number of surgical procedures have been associated with taste dysfunction. Ear surgery, including tympanoplasty, mastoidectomy, and stapedectomy, can damage CN VII (Bull, 1965; Kveton and Bartoshuk, 1994; Moon and Pullen, 1963). The chorda tympani nerve fibers are at particular risk from surgical procedures that involve the middle ear. This nerve is typically stretched or sectioned during surgery for otosclerosis, often resulting in taste loss or dygeusia. Such aberrations can last long after the operation. Thus, Bull (1965) found that 78% of patients with bilateral section and 32% of patients with unilateral section of the chorda tympani had persistent adverse symptoms. Laryngoscopy and endotracheal intubation can have rare complications that result in lingual nerve injury, often with coexisting hypoglossal nerve damage (Evers et al., 1999; James, 1975; Silva et al., 1992). However, the specific incidence of taste disturbance with and without somatosensory loss following these procedures has not yet

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been well defined. The common dental procedure, third molar (wisdom tooth) extraction, is clearly associated with risk of damage to the lingual nerve. The reported incidence of this problem varies widely (0.2–22%), possibly reflecting variability in the surgical techniques that are employed (Robinson et al., 2000; Scrivani et al., 2000). The lingual nerve lies in close proximity to the mandibular third molars, making it susceptible to dental manipulation and injury. This nerve may also be injured by the injection of local anesthesia, either by direct contact with the needle or as a result of neurotoxic effects of the anesthetic compound (typically lidocaine and epinephrine) (Nickel, 1993; Shafer et al., 1999). Recently, Shafer et al. (1999) quantitatively evaluated taste function in 17 patients before third molar extraction and at 1 month and 6 months thereafter. These investigators found that taste deficits can persist for at least 6 months after surgery, even though they do not typically result in patient complaints of taste loss, and patients with the most deeply impacted teeth exhibit the most severe loss. In a retrospective study of patients’ perception of taste after lingual nerve injury and repair, Scrivani et al. (2000) found that among 22 patients who demonstated a postinjury, prerepair sensory deficit to the lingual nerve, 20 patients perceived a subjective disturbance of whole-mouth taste function. While repair of this nerve provided significant improvement in trigeminal somatosensory function (82%), improvement in wholemouth taste was moderate at best (35%). Infections can also result in taste disturbance. Otitis media is a suppurative infection of the middle ear caused by such organisms as Streptococcus pnenoniae, Haemophila influenzae, Moraxella catarrhalis, and group A streptococci, and Pseudomonas aeroginosa (chronic infection). Recurrent aggressive cases of otitis media can result in unilateral taste loss or dysgeusia due to chorda tympani nerve injury as it passes through the middle ear. One series reports that otitis media accounts for approximately 3.1% of acute facial palsies (Takahashi et al., 1985). CNS infections such as lyme, tuberculosis, and occasionally HIV are routine suspects when a patient presents with a possible “Bell’s palsy,” often warranting a lumbar puncture for cerebrospinal fluid analysis. Other inflammatory disease states, such as sarcoidoisis or Wegener’s granulomatisis, can also cause isolated facial nerve injury and possibly taste disturbance (Bibas et al., 2001). Tumors growing anywhere along the path of the facial nerve—from the oral cavity (see previous section), through the skull base, temporal bone and middle ear, to the cerebellopontine angle and the brainstem—can cause a neuropathy of the nerve. Loss of hearing in conjunction with a unilateral facial paresis suggests the presence of an acoustic neuroma within the cerebellopontine angle,

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involving both the seventh and eighth cranial nerves (Harner and Laws, 1981). In a study of 81 patients with pathologically confirmed temporal bone and lateral skull base malignancies, 14 (17.3%) presented with a facial nerve paralysis; this was felt to be an adversve prognostic sign since increasing levels of facial nerve dysfunction (based on the House-Brackmann standardized scoring criteria) related to increasing levels of treatment failure (Manolidis et al., 1998). 2.

The Glossopharyngeal Nerve (CN IX)

The glossopharyngeal nerve (CN IX) provides innervation to the stylopharyngeus muscle (motor), the parotid gland (autonomic), the tympanic cavity and plexus (sensory), the eustachian tube (sensory), the baroreceptors of the carotid sinus (sensory), and the pharyngeal mucosa (sensory). Regarding taste (special visceral sensory), the lingualtonsillar branch of the glossopharyngeal nerve supplies circumvallate and most foliate taste buds within the posterior third of the tongue. The pharyngeal branch of the glossopharyngeal nerve innervates taste buds in the nasopharynx. The nerve cell bodies of these gustatory afferent fibers are located immediately outside the jugular foramen—through which they eventually pass to enter the skull—in the petrosal ganglion. Clinically important functions of this nerve include (1) the salivary reflex (a flow of saliva from the parotid gland through Stensen’s duct in response to highly seasoned foods on the tongue), (2) the gag reflex (an elevation and constriction of the pharyngeal musculature retraction of the tongue in response to stimulating the base of the tongue and posterior pharyngeal mucosa), and (3) the carotid sinus reflex (vagally mediated reflexive parasympathetic slowing of heart rate with mechanical stimulation of the carotid sinus). From a practical point of view, the nerve is purely sensory, only supplying motor innervation to the stylophrngeus muscle, which cannot be tested clinically. The glossopharyngeal nerve is small and, unlike the facial nerve, relatively well protected, making isolated lesions of this nerve extraordinarily rare. In fact, disorders that involve this nerve almost invariably affect the nearby vagus, accessory, or hypoglossal nerves. Nevertheless, trauma-related damage to the glossopharyngeal nerve can occur as a result of tonsillectomy, bronchoscopy, or laryngoscopy (Arnhold-Schneider and Bernemann, 1987; Donati et al., 1991; Ohtsuka et al., 1994a, b; Vories, 1999), reflecting the proximity of the lingual branch of this nerve to the muscle layer of the palatine tonsillar bed (Ohtsuka et al., 1994). Neoplasms that occur along the course of glossopharyngeal nerve only rarely present with isolated impairment of the nerve. Vernet’s syndrome,

Clinical Disorders Affecting Taste

consisting of loss of taste in the posterior third of the tongue, paralysis of the vocal cord, palate, pharynx, trapezius, and sternocleidomastoid muscles, is caused by lesions of the jugular foramen inside the skull that affect cranial nerves IX, X, and XI. Usually it is of traumatic origin (e.g., skull base fracture); however, it can occur as a result of internal carotid artery aneurysms, tumors, granulomas, or thrombosis of the jugular bulb (Haerer, 1992). Glossopharyngeal neuralgia is an important syndrome involving this nerve in which eating, swallowing, or talking may bring on a lancinating pain from one side of the throat, along the course of the eustachian tube to the tympanic membrane and ear. Like trigeminal neuralgia, there can be trigger zones for pain, and with the glossopharyngeal nerve they are usually the base of the tongue, pharyngeal wall, or tonsillar regions. While most cases are considered idiopathic, glosspharyngeal neuralgia has been associated with tongue carcinoma (particularly the posterior third), laryngeal carcinoid, a cerebellopontine angle tumor, and neck trauma (Webb et al., 2000). Rarely, there can be a CNIX-mediated disturbance of taste with increased salivation that accompanies diseases of the middle ear (Haerer, 1992). This results from the fact that the tympanic plexus is an element of glossopharyngeal nerve (Carpenter, 1985). 3. The Vagus Nerve (CN X) The vagus nerve (CN X) mediates sensation from the vocal cords, external ear, external auditory canal, and external surface of the tympanic membrane, as well as visceral sensation from the larynx to the gut. CN X innervates the taste buds on the epiglottis, aryepiglottal folds, and esophagus (via the internal portion of the superior laryngeal branch). Afferent fibers from the superior laryngeal nerve project centrally via their cell bodies in the inferior nodose ganglion. This important nerve provides motor function to the smooth muscle of the pharynx, larynx, and viscera, as well as to all striated muscles of the pharynx, larynx, and palate, except the stylopharyngeus (CN IX) and tensor veli palatini (CN V3) muscles. Therefore, proper functioning of this nerve is essential to the swallowing mechanism. Like CN IX, the vagus penetrates the skull through the jugular foramen. Clinically important reflexes of this nerve include the vomiting reflex (a more severe form of the gag reflex involving the vagus nerve), the swallowing reflex (swallowing in response to pharyngeal wall and back of tongue stimulation by a food bolus), the cough reflex (cough in response to stimulation of the pharynx, larynx, trachea, bronchial tree, tympanic membrane or external auditory canal), the sucking reflex (sucking by an infant in response

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to tactile stimulation of the lips near the buccal angle), and the carotid sinus reflex (mentioned above, the efferent limb). In terms of localization, it is important to note that pharyngeal branches separate from the vagus nerve high in the neck; thus, the absence of any sensory changes in the pharynx suggests that the neural lesion is below this level. While a number of disorders can affect the vagus nerve, such as tumors, vascular lesions, and infections, it is unclear whether isolated vagal injury can actually result in a subjective impairment or distortion of taste. 4.

Other Nerves Relevant to Taste

The trigeminal nerve (CN V), largely responsible for somatosensory function of orofacial stuctures, is important to consider with other gustatory-mediating cranial nerves since tactile, thermal, and spicy sensations are essential to the overall flavor of a tasted substance and, anatomically, the gustatory fibers of the facial nerve (chorda tympani) travel in conjunction with the mandibular-trigeminal fibers of the lingual nerve for a short distance near the tongue— making both of these nerves susceptible to orofacial causes of injury. Isolated damage to the auriculotemporal nerve (ATN) can produce a rare clinical syndrome of posttraumatic gustatory neuralgia. This syndrome is recognized as tasteinitiated, episodic, paroxysmal lancinating facial pain (often electric shock–like) in the cutaneous distribution of the auriculotemporal nerve (De, 1990; Scrivani et al., 1998; Sharav et al., 1991; Soria et al., 1990; Truax, 1989). Posttraumatic abnormalities involving this nerve were originally described by Lucie Frey (Frey, 1923), such as sweating and flushing in this same region of the ATN following a taste stimulus (Frey’s syndrome). Although typically provoked by tastants, one report suggests that Frey’s syndrome can be elicited by the smell of food or even emotional excitement (Scrivani et al., 1998). Reported causes include head trauma, parotid gland surgery, temporomandibular joint (TMJ) surgery, carotid endarterectomy, orthognathic surgery, oncological surgery, viral illness, and local infection (Scrivani et al., 1998; Sharav et al., 1991; Truax, 1989). C. Disorders Involving Parts of the Central Nervous System That Affect Taste After the facial, glossopharyngeal, and vagus nerves enter the brain stem and synapse in descending (and overlapping) order within the NTS of the medulla, forming the gustatory nucleus, the taste pathways progress to primary and secondary cortical taste regions. Within the brain stem, the cells of the NTS also make viscerovisceral and

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viscerosomatic reflexive connections, via the interneurons of the reticular formation, with cranial nuclei that control (1) the muscles of facial expression, (2) taste-mediated behaviors, including chewing, licking, salivation, and swallowing, and (3) preabsorptive insulin release (Smith and Shipley, 1992). In primates, the projections from the NTS to higher centers ascend with the central tegmental tract within the brain stem and synapse within the parvicellular division of the ventroposteromedial thalamic nucleus (termed the thalamic taste nucleus or TTN) (Beckstead et al., 1980; Onoda and Ikeda, 1999). From the TTN, fibers then project to the primary taste cortex, which is located deep in the parietal operculum and adjacent parainsular cortex (Norgren, 1990; Pritchard et al., 1986) near somatosensory centers for oral sensation and motor centers for the control of jaw and tongue movement. PET and fMRI studies have noted that tastants seem to activate insular and perisylvian regions, including the frontal operculum, superior temporal gyrus (opercular part), and inferior parts of the pre- and postcentral gyrus (Faurion et al., 1998; Gloor, 1997). A secondary cortical taste region is located in the caudomedial/caudolateral orbitofrontal cortex, just rostral to the primary taste cortex (Rolls and Baylis, 1994). The role of each gustatory cortical region in the processing of taste information remains unclear. None appear to be purely gustatory, often containing neurons responsive to touch and temperature, as well as taste (Pritchard, 1991). The orbitofrontal cortex processes information from several sensory pathways (including olfactory, gustatory, and visual) and has been linked to emotion, feeding, and social behaviors in both monkeys and humans (Rolls and Baylis, 1994; Rolls et al., 1998). The degree to which taste function is represented on each side of the brain is also poorly understood. In an fMRI study, Cerf et al. (1998) found evidence that the superior part of the insula is likely bilaterally innervated; however, significant lateralization of taste function was found in the inferior insula, such that in righthanded subjects there was relatively more left inferior insula activation, whereas in left-handed subjects the reverse was true. Small et al. (1997) observed that patients who had a right anterior temporal lobectomy had elevated citric acid recognition thresholds, but normal detection thresholds, relative to both controls and patients who had undergone left temporal lobectomy. Employing PET, these researchers noted that orally presented citric acid increased regional cerebral blood flow (rCBF) bilaterally in the caudolateral orbitofrontal cortex, but relatively more in the right anteromedial temporal lobe and in the right caudomedial orbitofrontal cortex. They speculated that (1) damage to the area corresponding to the rCBF increase in right anterior medial temporal lobe in normal subjects may account for elevation in taste recognition thresholds in

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lobectomized patients, and (2) taste recognition requires further processing by structures located in the anteromedial temporal lobe. Salivary gland secretion is controlled centrally by the autonomic nervous system and involves multiple structures. While CN IX provides secretomotor function to the lingual glands, as well as to the parotid gland, the fibers of CN VII, in connection with the superior salivatory nucleus, innervate the sublingual and submandibular glands, as well as the buccal glands. Salivation can be produced by electrical stimulation of the brain stem between or adjacent to CN VII and IX (Grovum et al., 2000), the lower Rolandic cortex, the lower precentral gyrus, the postcentral gyrus, the insula, and regions within the frontal cortex (Penfield and Jasper, 1954). Seizures involving the lower Rolandic region are commonly associated with hypersalivation (Penfield and Jasper, 1954). Damage to brain stem structures that subserve taste may produce dysgeusia, although usually not without significant impairment of other cranial nerves or long tracts (see Table 4). Notable brain stem areas involved in taste that are susceptible to injury include the tractus solitarius and its nucleus (where injury produces ipsilateral ageusia) and the pontine tegmentum, which involves both gustatory lemnisci (where injury produces bilateral ageusia). Bilateral injury to the thalamus can result in ageusia, although unilateral lesions above the brain stem do not usually cause complete loss of function (likely due to the multiple areas

Table 4 Disorders of Central Structures That Can Alter Taste Perception Neurovascular (stroke, arteriovenous malformation) Brain stem Nucleus of the tractus solitarius Pontine tegmentum Insula (parietal lobe/frontal lobe) Thalamus Seizures Amygdala Hippocampus Parietal operculum Rolandic operculum Medial aspect of the right T1 gyrus Anterior part of the right T2 gyrus Neoplastic Brain stem tumor Parietal lobe tumor Temporal lobe tumor CNS infection (e.g., herpes encephalitis) Inflammatory Multiple sclerosis Rasmussen’s encephalitis

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involved in processing taste information). An isolated unilateral lesion to the thalamus or the parietal lobe may cause contralateral taste disturbance. Taste function is clearly at risk by strategically placed CNS lesions, as seen in cases of stroke (either ischemic or hemorrhagic) or demyelinating disorders such as multiple sclerosis (Catalanotto et al., 1984; Onoda and Ikeda, 1999; Yabe et al., 1995). In a review of 15 reported cases, Onoda and Ikeda (1999) found that unilateral impairment of taste function occurs with injury to any one of several central structures, including (1) the pons, involved in 8 cases (53.3%), (2) the thalamus, involved in five cases (33.3%), (3) the midbrain, involved in one case (6.7%), and (4) the internal capsule, involved in one case (6.7%). Interestingly, based on the side of the respective central lesion and its effect on either unilateral or contralateral gustatory function, they surmised that the gustatory pathways ascend ipsilaterally from the medulla, cross over in their course from the pons to the midbrain, and synapse within the contralateral thalamus. It should be noted, however, that while this interpretation supports a primarily contralateral projection of taste information to the cerebral cortex in humans, evidence exists using gustatory-evoked potentials that unilateral taste stimulation can evoke contralateral and bilateral cortical responses (Kida, 1974; Plattig, 1968). Pritchard et al. (1999) performed formal gustatory testing in six

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stroke patients with unilateral lesions of the insular cortex and three patients with brain damage outside the insula and found that, compared to controls, damage to the right insula produced ipsilateral taste deficits in recognition and intensity, whereas damage to the left insula caused an ipsilateral deficit in taste intensity but a bilateral deficit in taste recognition. Based on this information, the authors hypothesized that taste information from both sides of the tongue must pass through the left insula for proper recognition to occur. In a case presenting with taste dysfunction to our center (Fig. 3), the MRI showed increased signal intensity suggestive of ischemia in a region of the upper medulla near the right NTS (note that, in this image, the patient’s right side is on the left of the picture, and the left is on the right of the picture). Taste dysfunction, as indicated by our taste-identification test and a localized complaint of dysgeusia, was greater on the right than on the left side of the tongue. Both CN VII and CN IX seem to be involved. Gustatory perception may also be affected by cortical injury to areas of association cortex outside of direct gustatory processing. The concept of left unilateral neglect, typically resulting from contralateral injury to the right hemisphere (e.g., parietal lobe, thalamus, basal ganglia), appears to exist for taste in a manner similar to the visual, auditory, tactile, and olfactory sensory systems

Figure 3 (Left) Axial T2 (2500/90) MR scan through the upper brain stem reveals a hyperintensive infarct lesion (4  3 mm) in the right medulla (arrow A). Note also large infarct (15  8 mm) inside the white matter of the left cerebellum (arrow B). (Right) Scores on a taste identification test showing a relative decrement on right side of tongue from this 65-year-old-woman with a history of transient ischemic attacks who developed a persistent salty/metallic dysgeusia and soreness on the right side of the tongue following a severe 2day bout of emesis accompanied by marked dehydration and increased blood pressure but unaccompanied by fever. (Copyright © 2000, Richard L. Doty.)

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(Bellas et al., 1988). In fact, a specific syndrome of buccal hemineglect may exist in which patients neglect mouth and food products in the left half of the mouth and are unable to initiate chewing and swallowing when food is in this location (Andre et al., 2000). This syndrome can result in choking or a socially embarrassing tendency to drool and regurgitate unnoticed food. With migraine headache, it is not clear whether the prodrome or acute headache phase involves definable gustatory phenomena as seen with olfaction. However, certain tastes may, in fact, induce a migraine (Blau and Solomon, 1985). More commonly, taste function is altered by seizure activity. Following in the footsteps of Penfield and Jasper (1954), who explored the function of the brain by observing the effects spontaneous and induced epileptic seizures, Hausser-Hauw and Bancaud (1987) found six specific areas of the brain where gustatory sensations result from electrical stimulation. These areas included the right rolandic operculum, right parietal operculum, right amygdala, right hippocampus, medial aspect of the right T1 gyrus, and the anterior part of the right T2 gyrus. Like olfactory auras, gustatory auras represent seizure activity predominately within the temporal lobes, but they can also be linked on rare occasion to epileptiform activity in the orbitofrontal lobes (Acharya et al., 1998; Hausser-Hauw and Bancaud, 1987; Roper and Gilmore, 1995). Etiologies of the seizures can vary, and identifiable causes include mesial temporal sclerosis, tumors, vascular malformations, infections (e.g., tuberculosis, herpes simplex), and Rasmussen’s encephalitis. Examples of taste sensations that have been reported in such cases include “peculiar,” “rotten,” “sweet,” “like a cigarette,” “like rotten apples,” and “like vomitus” (for review, see West and Doty, 1995). However, some of these “tastes” likely represent smell sensations that are miscategorized as tastes by both the patients and their physicians. D.

Systemic Disorders and Medications

Excessive dryness of the oral cavity and hyperviscosity of saliva accompany several diseases (e.g., Sjogren’s syndrome, xerostomia, pandysautonomia, diabetes mellitus) and are common side effects of a number of medications (e.g., anticholinergics, antidepressants, antihistamines). Although such conditions likely alter taste when chronic, acute effects are less well understood. In general, hyposalivation, per se, appears to have little influence on taste sensitivity in the short run (Christensen et al., 1984). A number of systemic disorders have been associated with taste dysfunction. Both chronic renal failure (Middleton and Allman-Farinelli, 1999) and end-stage liver disease (Bloomfield et al., 1999; Davidson et al., 1998) can

produce altered taste function, which, in some cases, seems to improve following transplantation (Bloomfield et al., 1999). Apparently the taste dysfunction associated with liver failure is not necessarily associated with a vitamin deficiency and may be centrally mediated (Bergasa, 1998), although it is generally believed that poor nutritional states can be related to taste impairment (Davidson et al., 1998). Avitaminosis, particularly of the B group and C, can result in pellagra, sprue, pernicious anemia, scurvy, or glossitis. Additionally, zinc and copper deficiencies may produce taste disturbances (Mattes and Cowart, 1994). In patients younger than 55 years with uremia, a significant impairment of taste recognition, independent of zinc deficiency, was found by Ciechanover et al. (1980) for salty, sweet, bitter, and sour compared to controls. The impairment for sour and bitter, however, showed improvement immediately after a dialysis session. Interestingly, idiopathic dysgeusia has been reported with blood transfusions (Erick, 1996), and we are familiar with one leukemia patient who could ascertain, by taste, whether transfused blood came from a smoker or a nonsmoker. There is considerable evidence that diabetes is associated with loss of taste function. Thus, there appears to be a progressive loss of taste beginning with glucose and extending to other sweeteners, salty stimuli, and then all stimuli (Hardy et al., 1981). In newly diagnosed non–insulin dependent diabetics (based on plasma glucose concentration and glycosolated hemoglobin percentage), quantifiable taste impairment not only exists, but may be somewhat reversible with correction of the hyperglycemia (Perros et al., 1996). Interestingly, about 10% of first-order relatives of late-onset diabetics exhibit decreased taste suprathreshold taste function that is rather specific to glucose (Settle, 1981). Whether these individuals are more prone to subsequent development of diabetes is not known, but is an intriguing possibility since about 10% of such relatives eventually develop this disease. Hypothyroidism has been reported to result in significant changes in taste-detection thresholds (McConnell et al, 1976); however, the degree of taste dysfunction does not appear to correlate with the severity of the disease (Pittman and Beschi, 1967). Importantly, there is suggestion that replacement of thyroid hormone may normalize threshold sensitivity and suprathreshold performance, although dysgeusias may still be present (Deems et al., 1991). Pharmacological agents appear to result in taste disturbances much more frequently than olfactory disturbances. Over 250 medications reportedly alter the sense of taste, including antiproliferative drugs, antirheumatic drugs, antibiotic drugs, psychotrophic drugs, and drugs with sulfhydryl groups, such as penicillamine and captopril

Clinical Disorders Affecting Taste

(Ackerman and Kasbekar, 1997; Bromley, 2000; Deems et al., 1991; Doty et al., 1991; Schiffman, 1997; Schiffman et al., 1998) (see Table 5). Angiotensin-converting enzyme (ACE) inhibitors are considered by many authors to be most commonly associated with taste disturbance, such as hypogeusia, or excessive metallic, bitter, or sour dysgeusic tastes (Ackerman and Kasbekar, 1997). Terbinafine, a commonly used antifungal medication, has been linked to Table 5 Systemic Disorders and Agents That May Affect Taste Medications Antibiotics/antifungals Anticonvulsants Antidepressants Antihistamines and decongestants Antihypertensives and cardiac medications Antiinflammatory medications Antimanic medications Antineoplastics Antiparkinson medications Antipsychotics Antithyroid medications Lipid-lowering medications Muscle relaxants Nutritional Blood transfusions Cancer-related wasting syndrome Chronic renal failure/uremia HIV wasting syndrome Liver disease (cirrhosis) Malnutrition Trace metal deficiency (zinc, copper) Vitamin deficiency (B3, B12, C) Endocrine Adrenocortical insufficiency Congential adrenal hyperplasia Cushing’s syndrome Cretinism Diabetes mellitus Hypothyroidism Panhypopituitarism Pseudohypoparathyroidism Psychiatric Bulimia Conversion disorder Depression Malingering Schizophrenia Genetic Familial dysautonomia (Riley-Day) Late-onset hereditary spinocerebellar degeneration Machado-Joseph disease variant Turner’s syndrome/gonadal dysgenesis

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taste disturbance that reportedly can last from 2 days to 3 years (Duxbury et al., 2000). In a study of six psychotropic medications, including amytriptyline, clomipramine, desimpramine, imipramine, doxepin, and trifluoperazine, Schiffman et al. (1998) found that these drugs not only have a taste of their own, but they can significantly alter the intensity of other tastants (such as salt and sugar). She also noted that among the medications used in the treatment of HIV and AIDS, the protease inhibitors have a taste themselves and appear to adversely modify the taste perception of other taste compounds (Schiffman et al., 1999). Repeated oral use of some topical agents, such as hydrogen peroxide or steroids, may affect taste.

E.

Psychiatric Disorders

There are multiple psychiatric conditions known to affect taste function. Patients who are clinically depressed may complain of an unpleasant or diminished taste that is not readily explainable by medications or other causes (Miller and Naylor, 1989); however, the problem with taste can be reversible (Gangdev, 1995). When formally tested, depressed patients demonstrate a higher recognition threshold for sucrose compared to nondepressed controls, a finding that is possible related to a subtle carbohydrate intolerance observed in many depressed patients (Amsterdam et al., 1987; Steiner et al., 1969). Gustatory hallucinations are associated, on rare occasion, with schizophrenia, cocaine use, and a number of other organic conditions (Carter, 1992; Siegel, 1978). Patients with bulimia nervosa appear to have impaired satiety responses based on the fact that postingestion of a glucose load, bulimics, unlike controls, do not find sweet tastes less pleasant (Rodin et al., 1990). Bulimic patients also appear to have a selective loss of taste on the palate, presumably from the local effect of acidic vomitus during a purging episode (Rodin et al., 1990). In the context of litigation or identifiable secondary gain, a physician may need to consider malingering on the part of the patient who is complaining of taste loss or distortion. Conversion disorder involving taste is also a possibility in some psychiatric cases. F.

Genetic Disorders

Familial dysautomonia (Riley-Day syndrome), an autosomal recessively inherited disease, is a sensory neuropathy that mostly affects Ashkenazi Jewish patients and results in defective autonomic control, insensitivity to pain, hyporeflexia, impaired lacrimation, the absence of fungiform

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papillae and taste buds on the tongue, and impairment of taste (Gadoth et al., 1997). Fukutake et al. (1996) described another inherited disease entity in two Japanese siblings that consists of late-onset progressive ataxia, global thermoanalgesia, sensorineural deafness, mild autonomic disturbances, and ageusia in the context of absent fungiform papillae and taste buds. This particular disease entity may represent a variant of late-onset hereditary spinocerebellar degeneration. A few of the same authors reported a case of a family with a possible new variant of Machado-Joseph disease (confirmed with genetic testing) associated with a sensory dominant axonal neuropathy, autonomic disturbances, and the absence of fungiform papillae (Uchiyama et al., 2001). Gonadal dysgenesis, particularly Turner’s syndrome, has been reportedly linked to a number of sensory abnormalities, including gustatory dysfunction (Henkin, 1967; Valkov et al. 1975). G. Special Circumstances 1. Aging Regional tests of taste function demonstrate marked losses in older persons (Matsuda and Doty, 1995). However, such regional deficits need not translate into marked overall whole mouth loss, given the redundancy of the system. Nonetheless, whole-mouth taste acuity does decline with age, and the elderly tend to perceive tastes as being less intense than do younger persons. Fortunately, the amount of decline is not as marked as that seen for olfaction (Cowart, 1989; Schiffman, 1997; Weiffenbach et al., 1986). Nonetheless, in conjunction with the loss seen with the sense of smell, decrements in taste function—and most significantly age-related dysgeusias—can be very detrimental to some of the elderly, leading to anorexia, weight loss, malnutrition, impaired immunity, and worsening of medical illnesses (Mattes and Cowart, 1994; Schiffman and Wedral, 1996). 2. Pregnancy Pregnancy proves to be a unique condition with regard to taste function. In one study of 46 pregnant and 41 nonpregnant women controls, Duffy et al. (1998) noted that quinine hydrochloride (bitter), while perceived as unpleasant became less disliked during the course of pregnancy, and that the sensitivity to bitter taste was highest in the first trimester. The authors suggest that this increase in dislike and intensity of bitter tastes during the first trimester may help to ensure that pregnant women avoid poisons during a critical phase of fetal development. Similarly, a relative increase in the preference for salt in the second and third trimesters may support the ingestion of much-needed elec-

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trolytes to expand fluid volume and support a varied diet (Duffy et al., 1998). 3.

Burning Mouth Syndrome

Burning mouth syndrome (BMS), often referred to as glossodynia or glossalgia, is the subjective sensation of intense “burning” pain within the mouth without obvious physical cause. Often accompanied by dry mouth, thirst, and altered or dysgeusic taste, BMS usually begins by late morning and is continuous once it has started for the day (Gorsky et al., 1987; Grushka and Sessle, 1991). Interestingly, most of the BMS patients who present for treatment are postmenopausal; however, studies vary in terms of the effectiveness of hormone-replacement therapy in reducing oral symptoms. Numerous etiologies suggested by researchers, many of them amenable to treatment, include nutritional deficiencies (e.g., iron, folic acid, B vitamins, zinc), diabetes mellitus (possibly predisposing to oral candidiasis), denture allergy, mechanical irritation from dentures or oral devices, parafunctional habits of the mouth (e.g., tongue thrusting, teeth grinding, jaw clenching), tongue ischemia as a result of temporal arteritis, oral candidiasis, periodontal disease, reflux esophagitis, and geographic tongue (see Tourne and Fricton, 1992, for review). Psychological factors are clearly important in the diagnosis and management of BMS, since psychological dysfunction (e.g., anxiety, depression) is common in this population (Gorsky et al., 1991). Both neuropathic and psychiatric factors linked to patients with BMS may help to explain why tricyclic antidepressants (e.g., amitriptyline, desipramine, nortriptyline) and benzodiazepines are reportedly useful as therapy in some cases. It is not clear whether the oral burning pain is a result of central or peripheral mechanisms. In a study of 9 men and 24 women, with an average age of 60 years, Formaker et al. (1998) found that the application of a topical anesthetic (dyclonine HCl) reduced the perceptual intensity of salt and sugar, but had variable affects on the burning sensation, such that 12 patients demonstrated increased burning, 14 patients had burning that did not change, and 7 patients experienced decreased burning. Topical capsaicin may also be helpful, although clinical trials using this substance have not been reported. VI.

TREATMENT

As with other sensory systems, prognosis for functional recovery of taste appears to be related to the kind of neural damage. For example, permanent sensory abnormalities are more likely to result from nerve section of a gustatorymediating cranial nerve than a crush injury (Robinson et

Clinical Disorders Affecting Taste

al., 2000). In general, however, the taste nerves and buds are relatively resilient, and spontaneous resolution of dysfunction is not uncommon (Deems et al., 1996). The factors responsible for such resolution, however, are not clear, and depending on the underlying disorder affecting taste function, some therapeutic strategies can be efficacious. In the case of neural damage from viruses, presumably the damaged taste afferents regenerate. In cases of taste loss secondary to hypothyroidism, thyroxin-replacement therapy may be beneficial (Mattes and Kare, 1986). Similarly, taste impairment due to nutritional deficiencies may improve with supplementation and a regular diet. Conditions such as Bell’s palsy and radiation-induced xerostomia generally improve over time. When identified, infection and inflammation of the mouth surfaces and perioral anatomical structures should be treated with the appropriate antibiotic or anti-inflammatory medication. Medication-related changes in taste can, in some instances, be reversed by discontinuance of the offending drug, by employing alternative medications, or by changing drug dosage. The health care professional should be aware of the fact, however, that many pharmacological agents appear to induce long-term alterations in taste that may take months to disappear even after discontinuance of the drug. Although the Physician’s Desk Reference (PDR) can be helpful in identifying medications that may result in drug-related taste dysfunction, there is lack of uniformity and control in the acquisition of this information. Many agents are listed with the potential side effects of “altered taste,” “ageusia,” “dysgeusia,” “bad taste,” “metallic taste,” or “bitter taste” without the benefit of a controlled trial. Therefore, caution should be used in simply attributing a patient’s taste disturbance to his or her medication regimen based on PDR classification alone. Most cases of idiopathic dysgeusias spontaneously resolve within a year or two (Deems et al., 1996). In some cases, antifungal and antibiotic treatments have been reported to be useful in resolving dysgeusias, although double-blind studies of the efficacy of such treatments are lacking. Chlorhexidine employed in a mouthwash has been suggested as having possible efficacy for some salty or bitter dysgeusias, possible as a result of its strong positive charge (Helms et al., 1995). Again, however, definitive clinical trials have not been performed. Trauma- and surgery-related injury to peripheral nerves supplying taste (e.g., lingual nerve, facial nerve) may be amenable to surgical repair, particularly in the early months immediately following injury (Robinson et al., 2000; Zuniga et al., 1994). In a series of 53 patients with prior lingual nerve injury who underwent direct reapposition of the nerve with epineurial sutures, Robinson and colleagues found that 41% more patients could detect

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some taste solutions postoperatively (Robinson et al., 2000). Recovery of sensory function after repair can vary, however, with patients finding more improvement in light touch and pain functions (Robinson et al., 2000). Postrepair, patients do not appear to regain completely normal sensation or get relief from spontaneous paresthesias or pain syndromes, but many patients do recover some sensory function and fewer bite their tongues by accident (Robinson et al., 2000). Proper oral hygiene and routine dental care are extremely important ways for patients to protect themselves from disorders of the oral cavity that can ultimately result in taste disturbance. Patients should be warned not to overcompensate for their taste loss by adding excessive amounts of sugar or salt. In situations where xerostomia and excessive dryness exists, a physician can offer artificial saliva (e.g., Xerolube) to improve comfort and mucosal function. Accentuating the other sensory experiences of a meal, such as food texture, aroma, temperature, and color, can optimize the overall eating experience for a patient. In some cases, a flavor enhancer like monosodium glutamate (MSG) can be added to foods to increase palatability and encourage intake (Bellisle, 1998; Schiffman, 1997). VII.

CONCLUSIONS

Clinical disorders that influence taste are numerous and may involve multiple organ systems. Because of this, the appropriate diagnosis and management of a patient with gustatory complaints requires a multidisiplinary mindset on the part of the evaluating physician. A disturbance of taste may represent a spectrum of possible problems from local (e.g., oral cavity) to more generalized (e.g., systemic disorders or agents). The ability to taste reflects the proper function of many systems within the body and, as such, may be considered a marker for good health. Conversely, altered taste function, particularly dygeusias that make food distasteful, can influence nutrition and lead to unhealthy states of being, not to mention adversely affecting a person’s ability to enjoy food products and overall quality of life. In this chapter we have presented multiple disorders across medical specialties that can influence taste, described anatomy and physiology relating these disorders to the taste system, and provided basic information for the evaluation and management of patients who complain of taste disturbance. ACKNOWLEDGMENTS The development of this chapter was supported, in part, by Grants PO1 DC 00161, RO1 DC 04278, RO1 DC 02974,

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and RO1 AG 27496 from the National Institutes of Health, Bethesda, MD (R. L. Doty, Principal Investigator).

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45 Head Injury and Taste Richard M. Costanzo, Laurence J. DiNardo, and Evan R. Reiter Virginia Commonwealth University, Richmond, Virginia, U.S.A.

I.

patient reportedly regained his ability to discriminate between sweet and bitter and even between the “flavors of beef and mutton.” Since his sense of smell remained “absolutely annihilated,” Ferrier concluded that in this patient the return of taste was not due to a coincident improvement in the sense of smell. Ferrier further suggested that reversible taste loss may be attributed to lesions of the lower part of the temporosphenoidal lobe. Modern studies suggest that ageusia occurs in approximately 0.5% of all head injuries (Costanzo and Becker, 1986; Costanzo and Zasler, 1991; Deems et al., 1991; Leigh, 1943). However, it may be more common when there is a concurrent loss of olfactory function (5–7%) (Sumner, 1967). In Deems and colleagues’ study of 750 patients evaluated for chemosensory disturbances (Deems et al., 1991), there were 132 cases involving head injury. Of these, 46 complained of dysgeusia. However, only 7 had a measured taste loss, while 113 had a measured smell loss. These findings are typical of patients with head trauma. Such individuals will often report a taste loss, although most will actually have a smell loss rendering them unable to detect aromas and flavors of foods. Patients who present with posttraumatic ageusia should thus be carefully evaluated for concurrent loss of olfactory function. The patient and/or the physician may not be aware of a smell loss. This chapter presents a review of gustatory dysfunction following head injury. A discussion of the mechanisms involved in such dysfunction is presented, along with information on diagnostic, management, and rehabilitation issues for patients with impaired gustatory function.

INTRODUCTION

The gustatory system is responsible for four basic taste experiences: salty, sweet, sour, and bitter. A fifth quality, called umami, has been postulated to describe the taste of monosodium glutamate and related compounds (Rolls et al., 1998). However, these taste bud–mediated sensations should not be confused with flavor sensations like coffee, chocolate, strawberry, lemon, steak sauce, barbeque sauce, etc., which are mediated via the olfactory system. During deglutition, volatiles from foodstuffs or drinks stimulate the olfactory receptors retronasally (i.e., through the nasopharynx), imparting such sensations. Additional components of flavor include texture, temperature, oral irritation, pungentness, and other somatosensory sensations. Although many of these interrelated components of flavor can be impaired or distorted following traumatic cranial nerve or brain injury, nasal or oral trauma, and surgical or medical therapies, such impairment is extremely rare. In general, most “taste” losses reflect damage to the olfactory system (see Chapters 22, 30, and 44). The first medical reports of head trauma–related taste loss appeared in the latter half of the nineteenth century. Ferrier (1876) was one of the first to report a case of posttraumatic “taste” loss that supposively could not be explained by the loss of olfactory function. A previous report by Ogle (1870) had described ageusia in patients with head injury, but attributed the taste loss to a loss of smell and inability to discriminate aromas and flavors. Ferrier’s patient, however, was said to have recovered his ability to taste without any improvement in smell. This 959

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MECHANISMS OF INJURY

Several mechanisms have been postulated to explain posttraumatic taste dysfunction, including (1) direct injury to the tongue and its epithelium, (2) damage to cranial nerve VII (facial), IX (glossopharngeal), or X (vagus), and (3) focal cortical contusion or intraparenchymal hemorrhage. A.

Injury to the Tongue

Tongue injuries may occur during head trauma as a result of mandibular or maxillary fractures, foreign body penetration, or self-inflicted bite wounds. This can result in localized or diffuse edema of the tongue musculature and epithelium. Rarely significant laceration or ischemic injury due to vascular compromise in the neck or within the tongue can lead to scarring or atrophy of large segments of the tongue. Taste disturbances may improve as tissue edema or hematoma resolve and epithelial repair occurs. However, permanent paresthesia and taste dysfunction can result from particularly extensive lesions. B.

Cranial Nerve Damage

Cranial nerves VII, IX, and X carry afferent gustatory information. Damage to these nerves may occur at any point in their course from the brainstem to the periphery and result in taste disturbances. CN VII is the most vulnerable of these structures owing to its circuitous extracranial path. The gustatory portion of the facial nerve, which supplies taste to the anterior two thirds of the ipsilateral tongue, initially leaves the brainstem with the facial nerve. These fibers synapse at the geniculate ganglion contained within the temporal bone and then course through the middle ear and mastoid bone. Taste fibers leave the motor component of the facial nerve at its mastoid segment as the chorda tympani nerve. The chorda tympani then traverses back across the middle ear space to join the lingual branch of CNV and then projects to the tongue. Head trauma may result in temporal bone fracture and facial nerve injury. Temporal bone fractures are classified as either transverse or longitudinal based on relationship to the long axis of the temporal bone. Longitudinal fractures comprise 70–90% of all temporal bone fractures, with only 10–20% resulting in facial nerve injury, while transverse fractures comprise 10–30% of all temporal bone fractures, with 50% resulting in facial nerve disruption (Sofferman, 1993). The facial nerve is particularly vulnerable to injury in its labyrinthine segment near the geniculate ganglion, given the narrow confines of the bony canal through which it passes (Eby et al., 1988). Consequently, this area is frequently the site of facial nerve injury, as the “classic”

transverse and longitudinal temporal bone fractures pass through or near this area. The chorda tympani nerve, which derives its name from its appearance as it transverses the medial surface of the tympanic membrane, is particularly susceptible to injury from trauma involving the middle ear and tympanic membrane. CN IX and X supply taste to the posterior one third of the tongue and to the pharynx. Injury to these nerves is relatively uncommon since they travel deep within the neck and exit the skull base at the well-protected jugular foramen. Traumatic disruption of these structures may result in decreased gag reflex, vocal cord paralysis, and aspiration on swallowing since these nerves also supply motor and sensory function to the larynx and pharynx. Despite the well-established relationship between skull base fractures and unilateral taste disturbances, Obreboski and colleagues (1976) found only a 0.9% incidence of measurable unilateral gustatory loss with maxillofacial fractures. Although the etiology of the disturbances was not addressed, the results suggest that injury to the most peripheral portions of the gustatory cranial nerves can produce dysgeusia and ageusia. Therefore, patients with facial fractures alone may have taste disturbances. C.

Brain Contusion and Hemorrhage

A central etiology for posttraumatic taste disorders has been postulated by several authors (Combarros et al., 1994; Costanzo and Zasler, 1991; Fujikane et al., 1999; Rousseaux et al., 1996), although the site of lesions causing ageusia has been the subject of much speculation (Ferrier, 1876; Helsmoortel, 1929; Semira, 1957; Sumner, 1967). Traumatic brain injury (TBI) involving the frontal and the temporal lobes may offer a plausible explanation for bilateral gustatory losses (Sumner, 1975). These structures are well known to be predisposed to contusional injury from coup-contra-coup forces generated by acceleration or deceleration forces (Auerbach, 1989) and have been implicated in cases of posttraumatic anosmia (Costanzo and Zasler, 1991; Zilstorff, 1955). Herberhold and Westhofen (1980) hypothesized that the ventroposteromedial thalamic nucleus is the site of the posttraumatic anosmia-ageusia syndrome. Focal contusions in gustatory cortical regions caused by a closed head injury are also likely to contribute to impaired taste function (Helsmoortel, 1929; Rousseaux, 1956; Semira, 1957). Recently magnetic resonance imaging (MRI) studies have shown that thalamic and ventral forebrain structures play an important role in gustatory function (Cerf et al., 1998; Fujikane et al., 1999; Rousseaux et al., 1996). The use of MRI and functional imaging may be of diagnostic value in localizing cortical lesions. In addition to cortical foci,

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hemorrhagic lesions in the midbrain (Johnson, 1996; Rousseaux et al., 1996) and in the pons (Kojima and Hirano, 1999; Rousseaux et al., 1996; Sunada et al., 1995) have been shown to cause hemiageusia. Disturbances of taste following head injury may result from bleeding, contusions, and infarcts in a variety of regions of the CNS. The identification and localization of specific brain structures may be helpful in the assessment of patients with posttraumatic dysgeusia. III. A.

CLINICAL ASSESSMENT History

A complete patient history is fundamental in the initial evaluation of posttraumatic ageusia. Many of the same issues that complicate the workup of posttraumatic anosmia also apply to taste disturbance (see Chapter 30). Preexisting gustatory dysfunction must first be excluded. Patients should be interviewed to determine the level of gustatory function prior to head trauma. For example, a history of previous head and neck injury, surgical procedures, or medications can be contributing factors in gustatory dysfunction. In addition, it is known that about one quarter of the U.S. population are “nontasters” to some bitter tastants (Bartoshuk et al., 1998). These individuals have few, if any, receptors to phenylthiocarbamide and chemically related compounds and perceive some bitter substances less intensely. Any history of pre- or posttraumatic olfactory dysfunction should also be elicited, given the interrelationship between these senses and patients’ difficulty in discerning between olfactory and gustatory loss (Deems et al., 1991). Occasionally, changes in eating habits or appetite and sometimes altered behavior or mood changes can signal taste dysfunction. Neuropsychiatric conditions directly related to the traumatic brain injury (TBI) may on occasion present with altered taste perception. Posttraumatic epilepsy may present with gustatory auras. On rare occasion the ictal phenomena itself, particularly with temporolimbic seizure foci, may manifest with dysguesia as the sole symptom or one of several sensory symptoms (Doty et al., 1991). The clinician must also consider organic affective disorders, particularly depression, and their possible impact on chemosensory function, including taste (Settle and Amsterdam, 1991). Depressive illness is a well-recognized sequela of TBI (Gualtieri and Cox, 1991; Martzke et al., 1997). A few studies have objectively evaluated this neuropsychiatric sequela utilizing DSM-III diagnostic criteria. For example, Varney and colleagues (1987) interviewed 120 patients with TBI who were referred for neuropsychological evaluation. They found that 77% of this patient

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group fulfilled the DSM-III diagnostic criteria for major depressive disorder. Of those patients with depression, 46% believed that their depression did not begin until more than 6 months after the injury. Some authors have theorized that rates of affective disease in persons with TBI are 5–10 times those seen in the general population (Gualtieri and Cox, 1991). Appropriate treatment of the depressive condition via pharmacological and counseling interventions should in theory help remedy taste dysfunction if the etiology relates at least in part to the organic affective disorder (McAllister, 1985). A careful review of all current medications should be included to identify any that are known to alter taste. Some antirheumatic and antiproliferative medications have been reported to alter taste. Schiffman notes that the presence of sulfhydryl groups in these compounds might be responsible for dysgeusia (Schiffman, 1983) (see also Chapter 44). Use of medications is a major factor contributing to taste loss among the elderly (Schiffman et al., 1999). Table 1 lists drugs commonly used in patients recovering from head injury that can interfere with taste function. Normal saliva production is necessary for the appropriate interaction of taste stimuli and receptors. In severe brain injury inadequate swallowing and overproduction or stasis of saliva in the oral cavity may result in abnormal taste function. Similarly, previous head and neck irradiation or connective tissue disorders may result in decreased saliva production and, consequently, dysgeusia. Posttraumatic changes in salivary production and composition should be considered. The physician should seek history of prior head and neck surgery. Submandibular gland excision, for example, may result in lingual nerve injury. Other common procedures such as tonsillectomy, uvulopalatopharyngoplasty, or tongue base reduction or advancement performed for obstructive sleep apnea have been reported to produce dysguesia (see Chapter 44). Skull base surgery or high carotid endarterectomy can result in injury to cranial nerves

Table 1 Drugs Used in Head-Injured Patients That May Interfere with or Alter the Quality of Taste Sensations Amitriptyline Amphetamines Baclofen Carbamazepine Dantrolene sodium Levodopa Nortriptyline Phenytoin Trazodone

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IX or X. Additionally, stretching or transection of the chorda tympani nerve can occur during ear surgery. Patient motivation and medicolegal concerns should be considered when evaluating posttraumatic taste disturbances. Complete obliteration of taste is unlikely to result from head trauma given the redundant innervation, although this can be seen with central lesions. A functional component or even malingering should, therefore, also be considered in cases of complete loss. Gustatory function testing may be of help in providing objective assessment of the presence and degree of gustatory loss (Bartoshuk, 1989; Frank and Smith, 1991; Lawless and Zwillenberg, 1983; Obrebowski et al., 1977) (see Chapter 37). In addition to taste, CN VII, IX, and X serve other functions. Inquires into deficiencies of these functions may be useful in determining the mechanism of gustatory loss. CN VII is responsible for a major portion of taste. Weaknesses of the facial musculature, impaired stapedial muscle reflex with altered sound perception, decreased lacrimation with ocular dryness, and periauricular parathesia may also accompany injury to this nerve. CN IX function is difficult to assess independently. Dysphagia may be reported, but often no deficits are noted. CN X has a more significant projection to the pharyngeal musculature than does the glossopharyngeal nerve. In addition, ipsilateral vocal cord function and laryngeal sensation are provided by branches of the vagus nerve. Consequently, dysphagia and hoarseness often accompany vagal nerve injuries, producing obvious functional deficits and patient complaints. These deficits may be worsened by a concomitant CN IX lesion. CN V and VIII are not directly responsible for taste but are in close proximity to the chorda tympani and petrous portion of the facial nerve, respectively. Parathesia of the ipsilateral anterior tongue occurs with disruption of the lingual branch of the trigeminal nerve, which travels with the chorda tympani. Audiolory and vestibular complaints are common after head trauma (Abd al-Hady et al., 1990; Griffiths, 1979; Sakai and Mateer, 1984). Occasionally, these may be the rest of intrapetrous disruption of CN VIII or its end organs, which lie in close proximity to the facial nerve. This tends to be seen more commonly in transverse fractures of the temporal bone. B.

Head and Neck Exam

A complete head and neck examination is important in the evaluation of patients with posttraumatic gustatory complaints. The endoscopic nasal examination and olfactory testing must not be overlooked since olfactory losses (see Chapters 1, 10, 22, 28, and 30) may result in altered taste perception.

The physical examination should focus particular attention on the tongue and the quantity and quality of saliva. Both dorsal and ventral surfaces of the tongue should be inspected for atrophy, edema, or, in the acute setting, hematoma or laceration. Tongue sensation (CN V) and mobility (CN XII) should also be evaluated. The oral mucosa should be moist throughout, and saliva should be clear and fluid, rather than tenacious. Saliva should be freely expressible from Wharton’s and Stenson’s ducts with massage of the appropriate gland. In addition, poor dentition or dental appliances that might mask or interfere with taste should be considered. Other components of the exam seek to rule out associated cranial nerve injuries. The palate is examined for lesions, gag reflex, and mobility (CN IX and X). The otologic examination may reveal evidence of temporal bone trauma such as hemotympanum, ear canal laceration or bony step-off, cerebrospinal fluid otorrhea, or Battle’s sign (ecchymosis overlying the mastoid region). Audiologic testing and, perhaps, electronystagmography should be considered so as to fully evaluate the function of CN VIII. Facial nerve evaluation includes not only motor function testing, but also studies of lacrimation, stapedial reflex response, and, if possible, salivary flow rate and pH monitoring. These studies assist in determining the site of facial nerve lesion and are possibly of prognostic significance in facial nerve dysfunction (May, 1986). The laryngeal exam performed with mirror or fiberoptic scope may sugggest CN X injury if vocal cord mobility or laryngeal sensation are impaired. C.

Radiography

As with posttraumatic anosmia, plain films are of little value in evaluating traumatic taste disturbances. Axial and coronal computerized tomography (CT) scanning with thin (5 mm) cuts through the temporal bone may identify bony fractures in proximity to CN VII or the chorda tympani nerve, or rarely fractures involving the jugular foramen potentially compromising CN IX and X. More recently, MRI has been used to trace the course of the facial nerve itself (Kemink, 1989). Unfortunately, little success has been reported in imaging traumatic facial nerve injuries. MRI studies are also useful in delineating brain injury either acute or chronic, such as cerebral hemorrhage, infarct, or contusion (Cerf et al., 1998; Fujikane et al., 1999; Rousseaux et al., 1996). Rousseaux and colleagues reported a case of a 68-year-old patient with an abrupt reduction in both gustatory and olfactory perception that resulted in a drop in appetite and a weight loss of 10 kg (Rousseaux et al., 1996). MRI revealed that the lesion involved the dorsomedial thalamic nuclei and the adjacent part of the intralamina nuclei. The patient

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reported that the pleasant character of foods had disappeared and they had became unpleasant, suggesting that the dosomedial thalamus may play a role in the hedonic perception of foods. D.

Gustatory Function Testing

A number of gustatory function tests have been described (Bartoshuk, 1989; Frank and Smith, 1991; Lawless and Zwillenberg, 1983; Yamauchi et al., 1995). (Specific gustatory function tests are discussed in detail in Chapter 37.) Gustatory function testing is warranted in any patient with complaints of posttraumatic gustatory dysfunction, as a high percentage will actually have only olfactory loss as the etiology (Deems et al., 1991). In this setting olfactory function testing is also indicated. An intriguing approach to determining the source of posttraumatic taste loss has been described by Miller and Reedy (1990). Human tongues were stained with methylene blue and examined with video microscopy. When the taste buds remained innervated, the taste pores stained blue. With the interruption of innervation, the pores did not stain. This may help differentiate central from peripheral posttraumatic ageusia. Local anesthetics applied to the tongue may also be useful in differentiating between a peripheral and central origin for distorted and phantom taste sensations. E.

Treatment, Prognosis, and Recovery

In most instances, no specific treatment exists for posttraumatic ageusia. Theoretically, timely decompression of the facial nerve compromised by temporal bone fracture offers possible improvement in gustatory function; however, mechanisms of recovery are not well known. Improved function in gustatory brain centers recovering from hemorrhagic cortical contusions offers another possibility for recovery of gustatory function. Spontaneous recovery from posttraumatic ageusia occurs more frequently than from posttraumatic anosmia (Ferrier, 1876; Rotch, 1878; Sumner, 1967; Szmeja et al., 1974). Improvement or resolution of taste loss usually occurs over several months after the injury. Additionally, different taste qualities (salty, sweet, bitter, sour) may be affected differently. Several reports have noted that bitter sense is particularly susceptible to traumatic insults (Henkin et al., 1971). Semira (1957) found that the different taste sensations recover at different rates. Specifically, sweetness, thought to be disproportionately mediated by the facial nerve, appears to recover before bitterness, which is thought to be disproportionately mediated by the glossopharyngeal nerve. Specific mechanisms of recovery remain unclear.

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IV.

SPECIAL CONSIDERATIONS

A.

Disability Evaluation and Activities of Daily Living

The American Medical Association impairment rating system, “Guides to the Evaluation of Permanent Impairment,” rates total bilateral loss of taste as 3% impairment of the whole individual, whereas complete unilateral or partial bilateral loss precludes any impairment rating from being assigned (AMA, 1993). This may be insensitive in assessing the level of functional disability resulting from total or partial loss of taste function. The impairment rating may not represent the true functional disability associated with taste disorders, depending on the importance of taste in that person’s daily life, vocationally and otherwise. To evaluate the degree of functional disability and resultant handicap for an individual patient, the professional must evaluate that patient’s activities of daily living, vocational status, and avocational pursuits. From a functional standpoint the main areas that should be considered relative to potential adverse effects of taste loss are appetite and diet, vocational and homemaking pursuits, and avocational interests. Since the patient may experience an array of gustatory dysfunctions ranging from complete ageusia to dysgeusias and parageusias, it is critical for the clinician to delve into the functional areas of gustatory performance once objective evaluation has been completed and a deficit detected. Individuals with posttraumatic ageusia may never again appreciate the gustatory sensations we all enjoy on a regular basis and the associated hedonistic pleasures that accompany them. There are no well-controlled studies of the effect of ageusia on posttraumatic vocational reentry. Certain vocational pursuits such as culinary work would obviously become more difficult without a sense of taste. Appropriate job analysis is critical prior to making final employment reintegration recommendations. Additionally, employee and employer education and on-site job modifications may further assure smooth work reentry. The ageusic homemaker will generally have problems with cooking owing to the inability to taste what is being prepared for consumption by the family. Appropriate education regarding labeling of cooking materials and spices is critical. Additionally, patients should be instructed that following recipes verbatim is probably safer than trusting one’s own taste if it is aberrant. Avocational activities may also be adversely affected by loss of taste following head trauma. Activities such as entertaining and cooking may lose at least a component of their inherent hedonic value secondary to the inability to perceive tastes. From a simple quality-of-life standpoint,

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we seldom think what it would be like not to be able to taste. What would the texture of cotton candy be like without the sweetness, the crunch of a dill pickle without the sourness, or the aroma and texture of bacon without the saltiness? B.

Medicolegal Considerations

Objective quantification of gustatory function is critical with regard to any medicolegal matter for which a clinician is consulted. Subjective reports or crude bedside testing are unreliable in differentiating true gustatory loss from olfactory loss and determining the precise degree of each. Elaborate medicolegal protocols that assess reproducibility of results may be utilized, particularly if there is a question regarding the veracity of patient reports. Congruence between expected objective results and bedside testing is also critical in the medicolegal context. In any legal case, a thorough history including both medical and psychiatric details is indicated to rule out other disorders that may affect taste. C.

Counseling and Information Resources

Although relatively uncommon following traumatic head injury, gustatory deficits do occur and should be a topic of inquiry during the standard review of systems for headinjured patients. Typically, patients with gustatory alterations report changes in the taste of their food. Taste sensations allow us to perceive foods as salty, sweet, sour, or bitter, or combinations thereof. Taste is one of several components that make up the flavor of food along with the food’s tactile qualities, temperature, and retronasally induced smell and aroma. When taste is lost or significantly altered, the flavor of food is obviously compromised. Individuals with parageusias may actually avoid foods that stimulate “distasteful” sensory input. In the extreme, the ageusic patient may have a significant loss of appetite secondary to the inability to perceive taste at any level. As with olfactory dysfunction, nutrition, as well as medical well-being, can be compromised by ageusia. Patients may seek out “compensatory” food items that may be excessively salty or sweet in an effort to make foods more palatable. The hypertensive patient with loss of “salt” sensation may inadvertently “oversalt” his food, resulting in increased serum sodium with subsequent fluid retention and worsening of blood pressure control. The diabetic patient may oversweeten foods and beverages, leading to poor blood glucose control. Patients with ageusia compensate by choosing foods for other aspects of hedonic stimulation other than taste, such as texture, temperature, or smell. The dysgeusic patient may try to

heighten stimulation in other preserved taste modalities or via stimulating CN V by the use of spices or “hot” food items. Rehabilitative interventions for the patient with gustatory compromise may include menu planning, instruction regarding compensatory dietary alterations to increase mealtime pleasure, and dietary consultation. As with olfactory deficits, enhancement of meal enjoyment can be accomplished by incorporating more “visual cues” in meal preparation in the form of colors or textures, varying food temperatures, as well as use of various condiments and seasonings depending on the specific taste deficit present. As with any sensory loss, the affected person may experience psychiatric sequelae such as grief. Typically, the psychoemotional responses associated with loss of taste are underestimated by clinicians. Counseling can assist the person with ageusia in dealing with the loss and accepting that he or she is not alone. As with any type of counseling, empathy, support, and education are critical factors. Discussion of sensory loss with ageusic persons who have already adjusted to their disability can provide additional insights for the newly ageusic patient. The earlier intervention begins the sooner the patient can adapt to the loss. Table 2 lists some of the practical issues for the patient with ageusia. V.

SUMMARY

Alteration in gustatory function occurs in fewer than 0.5% of all head injuries. Often both patient and physician are unaware of this loss. A detailed history and thorough head and neck exam including cranial nerve testing are primary components in the assessment of gustatory dysfunction. The clinician should assure that proper consideration has been given to nontraumatic etiologies of gustatory dysfunction, including drug side effects, affective disorders such as depression, and other preexisting conditions. Special gustatory function tests play an important role in

Table 2 Issues for Patients with Altered Taste Perception Prevent overindulgence as compensation for bland taste of food Avoid failure to eat due to bland taste of food Monitor for excessive intake of salt or sugar Assess job requirements relative to the functional need for taste perception Monitor freshness of perishable foods Adhere to recipes to avoid overseasoning food Label similar-appearing condiments Compensate for taste loss by optimizing food texture, aroma, and color Consider counseling to help cope with sensory loss

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verifying and quantifying loss. This may be of particular significance in medicolegal cases. Although there are no specific treatments for posttraumatic ageusia, careful follow up may reveal spontaneous recovery. In addition, the functional consequences of gustatory loss should be evaluated and compensatory strategies recommended when feasible. REFERENCES Abd al-Hady, M. R., Shehata, O., el Mously, M., and Sallam, F. S. (1990) Audiological findings following head trauma. J., Laryngol. Otol., 104:927–936. American Medical Association (AMA). (1993). Guides to the Evaluation of Permanent Impairment. American Medical Association, Chicago. Auerbach, S. H. (1989). The pathophysiology of traumatic brain injury. In Traumatic Brain Injury: State of the Art Reviews in Physical Medicine and Rehabilitation, L. J. Horn and D. N. Cope (Eds.). Hanley and Belfus, Philadelphia. Bartoshuk, L. (1989) Clinical evaluation of the sense of taste. Ear Nose Throat J. 68:331–337. Bartoshuk, L. M., Duffy, V. B., Lucchina, L. A., Prutkin, J., and Fast, K. (1998). PROP (6-n-propylthiouracil) supertasters and the saltiness of NaCl. Ann. NY Acad. Sci. 855:793–796. Cerf, B., Lebihan, D., Van de Moortele, P. F., Mac, L. P., and Faurion, A. (1998). Functional lateralization of human gustatory cortex related to handedness disclosed by fMRI study. Ann. NY Acad. Sci. 855:575–578. Combarros, O., Miro, J., and Berciano, J. (1994). Ageusia associated with thalamic plaque in multiple sclerosis. Eur. Neurol. 34:344–346. Costanzo, R. M., and Becker, D. P. (1986). Smell and taste disorders in head injury and neurosurgery patients. In Clinical Measurements of Taste and Smell, H. L. Meiselman and R. S. Rivlin (Eds.). MacMillian Publishing Company, New York, pp. 565–578. Costanzo, R. M., and Zasler, N. D. (1991). Head trauma. In Smell and Taste in Health and Disease, T. V. Getchell, R. L. Doty, L. M. Bartoshuk, and J. B. Snow, Jr. (Eds.). Raven Press, New York, pp. 711–730. Deems, D. A., Doty, R. L., Settle, R. G., Moore Gillon, V., Shaman, P., Mester, A. F., Kimmelman, C. P., Brightman, V. J., and Snow, J. B., Jr. (1991). Smell and taste disorders, a study of 750 patients from the University of Pennsylvania Smell and Taste Center. Arch.Otolaryngol.Head.Neck Surg. 117:519–528. Doty, R. L., Bartoshuk, L. M., and Snow, J. B. Jr. (1991). Causes of olfactory and gustatory disorders. In Smell and Taste in Health and Disease, T. V. Getchell (Ed.). Raven Press, New York, pp. 449–462. Eby, T. L., Pollak, A., and Fisch, U. (1988). Histopathology of the facial nerve after longitudinal temporal bone fracture. Laryngoscope 98:717–720.

965 Ferrier, D. (1876). The hemispheres considered physiologically. In The Functions of the Brain. G. P. Putnam’s Sons, New York, pp. 181–211. Frank, M. E., and Smith, D. V. (1991). Electrogustometry: a simple way to test taste. In Smell and Taste in Health and Disease, T. V. Getchell, R. L. Doty, L. M. Bartoshuk, and J. B. II Snow (Eds.). Raven Press, New York, pp. 503–514. Fujikane, M., Itoh, M., Nakazawa, M., Yamaguchi, Y., Hirata, K., and Tsudo, N. (1999). [Cerebral infarction accompanied by dysgeusia—a clinical study on the gustatory pathway in the CNS.] Rinsho Shinkeigaku 39:771–774. Griffiths, M. V. (1979). The incidence of auditory and vestibular concussion following minor head injury. J. Laryngol. Otol. 93:253–265. Gualtieri, T., and Cox, D. R. (1991). The delayed neurobehavioural sequelae of traumatic brain injury. Brain Inj. 5:219–232. Helsmoortel, J. (1929). Les troubles d’olfaction et du gout. J. Belge. Neurol. Psychiatry 29:298. Helsmoortel, J., Nyssen, R., and Thienpont, R. (1936). Six cas d’anosmie-aguesie d’orgine traumatique. Acta Psychiatr.Neurol.Scand. XI:251. Henkin, R. I., Schechter, P. J., Hoye, R., and Mattern, C. F. T. (1971). Idiopathic hypogeusia with dysgeusia, hyposmia, and dysosmia: a new syndrome. JAMA 217:434–440. Herberhold, C., and Westhofen, M. (1980). [On the central-nervous localization of the anosmia-ageusia-syndrome (author’s transl).] Laryngol.Rhinol.Otol.(Stuttg.) 59:570–574. Johnson, T. M. (1996). Ataxic hemiparesis and hemiageusia from an isolated post-traumatic midbrain lesion. Neurology 47:1348–1349. Kemink, J. L. (1989) Gradolinium diethylenetriaminepentoacetic acid contrast–enhanced magnetic resonance imaging in facial nerve lesions. Arch. Otolaryngol. Head Neck Surg. 115:661. Kojima, Y., and Hirano, T. (1999). [A case of gustatory disturbance caused by ipsilateral pontine hemorrhage.] Rinsho Shinkeigaku 39:979–981. Lawless, H., and Zwillenberg, D. (1983). Clinical testing of taste and olfaction. Trans.Pa.Acad. Ophthalmol. Otolaryngol. 36:190–196. Leigh, A. D. (1943). Defects in smell after head injury. Lancet 1:38–40. Martzke, J. S., Kopala, L. C., and Good, K. P. (1997). Olfactory dysfunction in neuropsychiatric disorders: review and methodological considerations. Biol. Psychiatry 42:721–732. May, M. (1986). Differential diagnosis by history, physical findings, and laboratory results. In The Facial Nerve, M. May (ed.). Theime, Inc., New York, pp. 181–216. McAllister, T. W. (1985). Carbamazepine in mixed frontal lobe and psychiatric disorders. Clin. Psychiatry 46:393–394. Miller, I. J., Jr. and Reedy, F. E., Jr. (1990). Variations in human taste bud density and taste intensity perception. Physiol. Behav. 47:1213–1219. Obrebowski, A., Pruszewicz, A., Baranczakowa, Z., and Flieger, S. (1976). [Early and delayed changes in the olfactory and gustatory function following maxillofacial fractures.] Otolaryngol. Pol. 30:391–398.

966 Obrebowski, A., Pruszewicz, A., Flieger, S., and Baranczakowa, Z. (1977). [Usefulness of smell and taste examination in the assessments of late results of craniofacial trauma.] Czas. Stomatol. 30:343. Ogle, W. (1870). Anosmia or cases illustrating the physiology and pathology of the sense of smell. Med. Chir. Trans. 53:263. Rolls, E. T., Critchley, H. D., Browning, A., and Hernadi, I. (1998). The neurophysiology of taste and olfaction in primates, and umami flavor. Ann. NY Acad.Sci. 855:426–437. Rotch, T. M. (1878). A case of traumatic anosmia and ageusia. Bost. Med. Surg. J. 91:130–132. Rousseaux, M. (1956). A propos d’un cas de fracture du craine avec anosmie et ageusie. Rev. Otoneuroophtalmol. 28:168. Rousseaux, M., Muller, P., Gahide, I., Mottin, Y., and Romon, M. (1996). Disorders of smell, taste, and food intake in a patient with a dorsomedial thalamic infarct. Stroke 27:2328–2330. Sakai, S. C., and Mateer, C. A. (1984). Otological and audiological sequelae of closed head injury. Semin. Hearing 5:157–174. Schiffman, S. S. (1983). Taste and smell in disease. N. Engl. J. Med. 308:1275–1280. Schiffman, S. S., Zervakis, J., Suggs, M. S., Shaio, E., and Sattely-Miller, E. A. (1999). Effect of medications on taste: example of amitriptyline HCL. Physiol Behav. 66:183–191. Semira, C. (1957). Le alterazioni della sensibilita olfactiva e gustative consecutive a traumi cranici. Minerva Otorhinolaring. 7:3.

Costanzo et al. Settle, R. G., and Amsterdam, J. D. (1991). Depression and the chemical senses. In Smell and Taste in Health and Disease, T. V. Getchell (Ed.). Raven Press, New York, pp. 851–862. Sofferman, P. A. (1993). Facial nerve injury and decompression. In: Surgery of the Ear and Temporal Bone, J. B. Nadol and H. F. Schuknecht (Eds.). Raven Press, New York, pp. 329–344. Sumner, D. (1967). Post-traumatic ageusia. Brain 90:187–202. Sumner, D. (1975). Disturbance of the senses of smell and taste after head injuries. In: Handbook of Clinical Neurology, P. J. Vinken and G. W. Bruyn (Eds.). North-Holland Publishing Co., Amsterdam, pp. 1–25. Sunada, I., Akano, Y., Yamamoto, S., and Tashiro, T. (1995). Pontine haemorrhage causing disturbance of taste. Neuroradiology 37:659. Szmeja, Z., Pruszewicz, A., Tokarz, F., Zwozdziak, W., and Obrebowski, A. (1974). [Value of otoneurologic examinations in the certification of late results of craniocerebral injuries.] Patol. Pol. 25:469–473. Varney, N. R., Martzke, J. S., and Roberts, R. J. (1987). Major depression in patients with closed head injury. Neuropsychology 1:7–9. Yamauchi, Y., Endo, S., Sakai, F., and Yoshimura, I. (1995). [Whole mouth gustatory test (Part 1)—basic considerations and principal component analysis.] Nippon Jibiinkoka Gakkai Kaiho 98:119–129. Zilstorff, K. (1955). Anosmia and ageusia, presumably resulting from anoxia. Acta Otolaryngol. 45:371.

46 The Vomeronasal Organ Peter A. Brennan and Eric B. Keverne University of Cambridge, Cambridge, United Kingdom

similar appearance of bipolar receptor neurons in the VNO to those in the main olfactory epithelium in Golgi-stained preparations. The specific nerves that Jacobson had described were actually vomeronasal nerves conveying chemosensory information from the VNO to the olfactory bulb. Most of the early work on the VNO involved comparative studies on the occurrence and structure of the VNO in a number of different species. However, the role of the VNO remained elusive until relatively recently, when techniques were developed to selectively ablate the vomeronasal organ without affecting other chemosensory systems. Mating behavior in male hamsters was the first of many examples of species-specific behavioral and physiological responses that have been found to be mediated by the vomeronasal system (Powers and Winans, 1975). For a long time it was unclear how stimuli could gain access to the narrow lumen of the VNO. The anatomy of the VNO suggested that a vascular pumping mechanism could suck stimuli into the VNO. This was finally confirmed by Meredith and O’Connell (1979), who observed mucus being sucked into the opening of the VNO in anaesthetized hamsters, following stimulation of the superior cervical sympathetic ganglion. This also established the important role of the mucus secretions as a transport medium for vomeronasal stimuli. The last decade has seen a revolution in our understanding of VNO function. Molecular biological techniques have identified genes for vomeronasal receptors. Furthermore, the projections of sensory neurons expressing a specific receptor can now be traced from the VNO to the accessory olfactory bulb (AOB). Biochemical

The vomeronasal organ (VNO), otherwise known as Jacobson’s organ, is a bilateral tubular structure located at the base of the nasal cavity on each side of the nasal septum. It is present in a wide variety of vertebrates and is typically enclosed along all or most of its length in a prominent bony or cartilaginous capsule. The VNO is closed at its posterior end and opens at the anterior end into the nasal cavity, via the vomeronasal duct. However, in some species, especially carnivores and ungulates, there is also a connection with the oral cavity, via the nasopalatine duct. The VNO was first described by Ludvig Jacobson in 1813 (Trotier and Døving, 1998). Jacobson provided detailed descriptions of the rich blood supply to the VNO and its innervation from the trigeminal nerve. In addition, he noted a completely separate nerve, consisting of large fascicles that innervated the organ along its length, which he was able to follow back to the main olfactory bulbs (Fig. 1). Jacobson could only guess at the function of this new organ based on its anatomical appearance. He considered the possibility that the VNO might have a sensory role. However, it was not obvious why a separate chemosensory system would be required in addition to the well-established sensory organs of smell and taste. Furthermore, Jacobson was impressed by the extensive glandular structure of the VNO and came to the reasonable conclusion that it was most likely to be a secretory organ that was nonetheless important for chemosensory function. According to a recent review by Døving and Trotier (1998), it was not until the improved histological techniques of the late nineteenth century that it became evident that the VNO was a sensory organ. This was due to the 967

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Figure 1 Cross section through the head of a typical rodent showing the vomeronasal organ and the vomeronasal nerves, which project to the accessory olfactory bulb at the rear of the main olfactory bulb.

techniques have identified a range of species-specific vomeronasal stimuli, which are being used in single cell electrophysiology to determine the response properties of individual, vomeronasal receptor neurons. As a result of the application of this wide variety of techniques, the importance of the vomeronasal system in the life of most vertebrates is now more fully appreciated.

I.

STRUCTURE AND FUNCTION OF THE VNO

In cross section the VNO has a characteristic crescent shape with a thick, pseudostratified sensory epithelium on the medial, concave side of the lumen, and a thinner, ciliated nonsensory epithelium laterally (Fig. 2). The sensory epithelium consists of layers of bipolar vomeronasal receptor neurons (VRNs), which project a single dendrite to the epithelial surface. The terminal knobs are covered with microvilli, unlike the cilia found on olfactory receptor neurons in the main olfactory epithelium. In addition, the sensory epithelium also contains supporting cells and prominent mucus glands. As in the main olfactory epithelium, receptor neurons in the vomeronasal organ have a limited life span, being continually replaced from basal stem cells. New VRNs appear to be concentrated particularly at the margins of the sensory epithelium where it borders the respiratory epithelium (Weiler et al., 1999). Because the VNO is a blind-ended, mucus-filled tube, the VRNs are isolated from the airstream that passes through the nasal cavity during normal respiration. Consequently, stimulus access to the VNO is entirely dependent on a vascular pumping mechanism (Salazar and Sánchez Quinteiro, 1998). Large blood vessels and sinuses

Figure 2 Schematic representation of a coronal section through the vomeronasal organ. Changes in the volume of the highly vascularized cavernous tissue cause pressure changes that pump substances in and out of the lumen of the organ. This is aided by the rigid cartilaginous capsule.

are found laterally to the VNO lumen and are innervated by the autonomic nervous system. Electrical stimulation of the nasopalatine nerve, or the superior cervical sympathetic ganglion, causes a vasoconstriction of the blood vessels within the VNO capsule, reducing the volume of blood in the cavernous tissue. This lowers intraluminal pressure, aided by the resilient bony casing and cartilaginous surrounding tissue, and draws fluid in from the vicinity of the vomeronasal duct opening (Meredith and O’Connell, 1979). Along with the vasodilatation caused by activity in vasoactive intestinal polypeptide (VIP) nerve fibers (Matsuda et al., 1996), it comprises a pumping mechanism for moving stimuli in and out of the lumen of the organ. The vomeronasal pump is not activated continuously, or linked to respiratory cycles, but is activated in situations of arousal, especially associated with the investigation of novel stimuli (Meredith, 1994). The large influx of mucus into the VNO during pump activation carries with it vomeronasal stimuli, which are often proteins, or are associated with proteins. In general, direct physical contact with these nonvolatile stimuli appears to be necessary for their effects, and specific behaviors are often used to facilitate the delivery of stimuli to the VNO. Snakes and lizards have a highly specialized tongue that flicks out to sample prey stimuli and delivers them to the vomeronasal ducts in the roof of the mouth (Burghardt and Pruitt, 1975). In many mammals, such as dogs and rodents, the licking and nuzzling of urine marks introduces vomeronasal stimuli into the nasal cav-

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ity, from where they can be taken up into the VNO in the mucus. Other mammals, especially ungulates, depend on flehmen behavior in which the head is lifted and the upper lip curled back, drawing stimuli from the oral cavity by way of the nasopalantine duct (Müller-Schwarze, 1979). II.

SPECIES DISTRIBUTION OF THE VOMERONASAL ORGAN

Jacobson studied the VNO in a variety of domestic mammals. Subsequently it has been found in many other vertebrates, although there are species differences in its occurrence and elaboration (Dawley, 1998). A VNO is present in most amphibians, reptiles, and mammals. However, it is absent in fish, crocodilians, marine turtles, and exclusively marine mammals, including porpoises, dolphins, whales, and manatees. This suggests that a VNO may be of little use in an exclusively aqueous environment. However, the olfactory epithelia of fish contain a mixed population of receptor neurons, some of which have apical cilia like mammalian olfactory receptor neurons, and some of which have apical microvilli similar to VRNs. Moreover, chemosensory receptor genes have been found in fish olfactory epithelia, which are homologous to the V2R class of genes for mammalian vomeronasal receptors (Speca et al., 1999). Therefore, vomeronasal receptor neurons may have become partitioned into a separate organ during evolution, as an adaptation to cope with the delivery of relatively nonvolatile stimuli in the terrestrial environment. The vomeronasal system also appears to be of limited use for airborne species, as it is absent in birds and many bats, although there are a few bat species in which it is particularly well developed (Bhatnagar and Meisami, 1998). In addition to physical features of the environment that may impose restrictions on the importance of the vomeronasal system, the lack of flexibility of vomeronasal responses may limit its role in species with complex social systems. Thus, although there are well-developed VNOs in platyrrhine New World monkeys, VNOs appear to be absent from adult catarrhine monkeys, such as macaques, baboons, and gibbons, as well as the great apes, i.e., gorillas, chimpanzees, and orangutans (Bhatnagar and Meisami, 1998). III. DO HUMANS POSSESS A FUNCTIONAL VOMERONASAL ORGAN? Human societies and relationships are extremely complex, requiring flexible behavior that does not come under the obligatory regulation of any one sense (Keverne, 1983a). Chemosensory cues undoubtedly influence human sexual

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behavior via the main olfactory system. However, it is unlikely that the relatively inflexible responses elicited by vomeronasal stimulation play an important role in humans. Anatomical studies support this view. Although a VNO does develop prenatally in humans, it is generally thought to degenerate around the time of birth. Nevertheless, there have been recent claims for a functional human VNO (Monti-Bloch et al., 1998). A bilateral, tubular, epithelial structure can often be found in biopsy and autopsy investigations of the adult human nasal septum. However, different investigators report markedly differing frequencies for its occurrence in adults. Moreover, there are numerous histological differences from the typical VNO found in other mammalian species. The epithelium is thin and the lateral and medial surfaces appear similar, covered in cilia typical of nonsensory respiratory epithelium (Smith et al., 1998). Bipolar cells with microvillar processes have been described, but they share immunohistochemical characteristics with neuroendocrine cells scattered through human nonsensory, respiratory epithelium (Johnson, 1998). Moreover, the epithelium does not stain for olfactory marker protein (OMP), which is a characteristic feature of mature chemosensory neurons (Takami et al., 1993). There is also no evidence in adults of a neural projection from the VNO and no sign of an AOB, although both are present in the fetus (Meisami and Bhatnagar, 1998). Finally, molecular genetic studies suggest that human olfactory sensibilities are in decline, and all the vomeronasal receptor genes that have been detected so far in humans have turned out to be pseudogenes. Therefore, although a vestigial VNO-like structure may be present in some adult humans, the evidence suggests that it is unlikely to be functional in any meaningful way.

IV. THE CHEMOSENSORY ROLE OF THE VOMERONASAL SYSTEM The vomeronasal system is often regarded as being specialized for mediating pheromonal communication. However, this can be misleading, as the term pheromone is often used rather loosely (Johnston, 1998). Although there is some justification for using the term in the context of stereotyped responses, it is often misapplied to substances that exert more subtle biases on behavior. Therefore, the term pheromone, which was originally devised for insects, may best be avoided when considering vertebrates (see Chapter 17). Furthermore, in some species the vomeronasal system also responds to “nonpheromonal” stimuli. For instance, although the vomeronasal system is important for reproductive behavior in garter snakes, it is also vital for prey tracking and striking behavior and for predator avoidance

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(Millar and Gutzke, 1999). These do not involve conspecific cues and are certainly not pheromones. There is no straightforward answer to Jacobson’s puzzle as to how the function of the vomeronasal system differs from that of the main olfactory system. In general, the main olfactory system responds to an enormous variety of volatile odorants and can link them with great flexibility to different responses. In contrast, the vomeronasal system is a more specialized chemosensory system that responds to a narrower range of mainly nonvolatile stimuli that are unlikely to gain access to the main olfactory epithelium. Furthermore, the vomeronasal system elicits more stereotyped endocrinological and behavioral responses than the main olfactory system via a relatively direct neural pathway. Of the species-specific responses mediated by the vomeronasal system, those that influence reproduction in rodents have been studied most extensively. One of the first effects to be attributed to the vomeronasal system was that elicited by vaginal fluid produced by sexually active female hamsters (Powers and Winans, 1975). This hamster vaginal fluid (HVF) contains components that attract males to lick it and thereby introduce it to their VNO. Exposure of male hamsters to HVF elicits mounting behavior and increases testosterone levels. Ablation of the VNO abolishes the HVF-induced testosterone rise and seriously disrupts mounting behavior in sexually inexperienced males (Meredith, 1986). The vomeronasal system is also very important in mice. Lesions of the VNO abolish the aggressive behavior in male mice that is elicited by exposure to urinary stimuli from sexually mature males (Maruniak et al., 1986). It is also likely to be important in their territorial behavior. Volatile stimuli released from urine marks attract the attention of other males. However, it is the nonvolatile proteins in the urine that stimulate countermarking and which are likely to be conveyed by the vomeronasal system (Humphries et al., 1999). In addition to mediating immediate behavioral responses, activation of the vomeronasal system can also result in endocrinological changes that have longer-term effects. Studies using ablation of the VNO have demonstrated the important role played by conspecific vomeronasal stimuli in regulating the reproductive state of female mice (Keverne, 1983b). Urinary stimuli from females delay the onset of puberty and suppress estrus cyclicity in grouped females (Lee and Boot, 1955). In contrast, male urinary stimuli generally have estrus-inducing effects on females. They accelerate the onset of puberty (Vandenbergh, 1969), induce estrus in anestrous females (Whitten, 1956), and prevent pregnancy in recently mated females (Brennan, 1999; Bruce, 1960). Although exposure to male urinary stimuli will block the pregnancy of a newly

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mated female mouse, this does not occur when she is reexposed to stimuli from the male with whom she has mated. This is because the female learns about the urinary stimuli to which she is exposed at mating (Brennan et al., 1990). This learning and subsequent recognition of the mating male’s individual urinary cues depends on the vomeronasal system and is vital to prevent the abortion of his own offspring (Lloyd-Thomas and Keverne, 1982). Therefore, in addition to conveying information about gender identity, the vomeronasal system can also convey information about individual identity. This is also apparent in the ability of male hamsters to respond differentially to HVF from individual females. Preexposure to HVF from a certain female increases a male’s subsequent investigation of that female compared to a novel female (Steel, 1984). Male hamsters with VNO ablations are unable to distinguish between HVF of different females (Johnston, 1998).

V.

THE NATURE OF VOMERONASAL STIMULI

One of the first vomeronasal stimuli to be identified was termed aphrodisin, a 17 kDa protein found in HVF that elicits mounting behavior in sexually naïve male hamsters (Singer and Macrides, 1990). More recently, a 20 kDa chemoattractant protein isolated from earthworm electric shock secretion has been found to act as a potent vomeronasal stimulus in the completely different context of prey striking behavior in garter snakes (Wang et al., 1997). Nineteen kDa proteins, known as major urinary proteins (MUPs), are found at high levels in the urine of sexually active male mice. Moreover, effects such as puberty acceleration and pregnancy block are elicited by the protein fraction of male mouse urine (Marchlewska-Koj, 1981; Vandenbergh et al., 1975). However, these findings do not necessarily imply that the proteins themselves are functioning as the stimuli. All these proteins belong to the lipocalin family of ligand-binding proteins, having a characteristic barrel structure enclosing a hydrophobic ligand-binding site (Bocskei et al., 1992). Therefore, ligands released from the binding proteins, rather than the proteins themselves, could act as vomeronasal stimuli. The MUPs in male mouse urine bind a variety of small, volatile ligands (Fig. 3). The main ligands present in the urine of sexually mature male mice are (R,R)-3,4-dehydroexo-brevicomin, (S)-2-sec-butyl-4, 5-dihydrothiazole, E,E-farnesene, E--farnesene, and 6-hydroxy-6-methyl-3heptanone. In spite of their different chemical structures, these compounds have been reported to accelerate puberty in female mice when administered individually in urine from castrated males (Novotny et al., 1999). Furthermore, a mixture of brevicomin and thiazole and a mixture of the

The Vomeronasal Organ

Figure 3 Volatile constituents of male mouse urine that are bound to the major urinary proteins and are likely to signal male gender identity. They have been reported to accelerate puberty and induce estrus in female mice.

farnesenes have both been reported to be effective in inducing estrous in anestrous female mice when administered in water (Jemiolo et al., 1986; Ma et al., 1999). These male stimuli may therefore share a common neuroendocrine mechanism for estrous induction. The importance of the MUPs may be to act as a reservoir for the ligands, allowing their prolonged release from urine marks over a period of many days. However, although it is clear that the ligands convey information about gender identity, it is less certain that they could account for the recognition of individual male mice that is observed in the context of pregnancy block. This is more likely to involve the MUPs, which are highly polymorphic (Robertson et al., 1996), and likely to differ among individuals in the wild (Robertson et al., 1997). Different variants of MUP may bind different cocktails of ligands, the MUPs themselves may be involved in presenting the ligands to the receptors, or the MUPs themselves may interact directly with receptors. It is interesting that glands in the VNO produce a protein named vomeromodulin, which is another member of the lipocalin family of ligand-binding proteins. Thus, the vomeronasal system may also be responsive to volatile stimuli that are transported into the VNO by ligand-binding proteins in the mucus (Miyawaki et al., 1994). VI. TRANSDUCTION MECHANISMS OF VOMERONASAL RECEPTOR NEURONS Transduction mechanisms of VRNs differ from that of main olfactory receptor neurons. VRNs do not possess cyclic nucleotide gated ion channels and do not respond to

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cyclic nucleotide injection (Liman and Corey, 1996). Instead, work on a number of different species suggests that an IP3 signaling pathway is activated by vomeronasal stimuli. Purified aphrodisin, isolated from HVF, increases IP3 production in hamster VNO membranes without altering c-AMP production and does not affect second messenger production in the main olfactory epithelium (Kroner et al., 1996). The IP3 pathway is also important in vomeronasal transduction in garter snakes. Chemoattractive proteins from their earthworm prey act via a G-protein coupled receptor to increase IP3 levels, and dialysis of IP3 into snake VRNs elicits an inward current (Taniguchi et al., 2000). The IP3 pathway appears to activate a cation channel, leading to an inward current, which is not dependent on Ca2 release from intracellular stores. The membrane channel that is opened by IP3 is likely to be a transient receptor potential channel (TRP2), which has been localized to the apical microvilli of VRNs (Liman et al., 1999). The electrophysiological responses of VRNs are abolished in knockout mice that lack the TRP2 channel leading to specific behavioral deficits (Stowers et al., 2002; Leypold et al., 2002). Interestingly, the human homologue of this channel contains multiple stop codons and therefore is likely to be a pseudogene, reinforcing the evidence that humans lack a functional vomeronasal system. Specific expression of the TRP2 channel in VNO sensory microvilli is consistent with IP3 activation opening TRP2 channels leading to an inward current and a depolarization of membrane potential. Patch clamp recordings from single receptor cells in vitro have highlighted the sensitive nature of VRNs. Although they typically exhibit a low spontaneous activity, a current injection of only 1 or 2 pA is sufficient to fire a series of action potentials (Trotier et al., 1998). This sensitivity is due to a high input resistance and a membrane potential close to threshold, which is maintained by the electrogenic Na/K pump. The action potential firing rate increases linearly with increasing current injection up to 10 pA in rat VRNs, which suggests that it has the potential to encode stimulus strength. However, unlike the rapid adaptation seen in olfactory receptor neurons, VRNs respond to maintained current injection with steady trains of action potentials, with little sign of adaptation (Liman and Corey, 1996). This is consistent with the prolonged periods of exposure required to induce the endocrinological effects in rodents (Rosser et al., 1989). Interestingly, some inhibitory reponses to putative vomeronasal stimuli have also been reported (Moss et al., 1997). Therefore, a maintained level of activity in VRNs during sustained exposure might be subject to inhibition from other vomeronasal stimuli at the level of the receptor neuron.

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VOMERONASAL RECEPTOR GENES

Molecular biological techniques have recently yielded valuable information about putative vomeronasal receptor proteins. Two main classes of receptors termed V1R and V2R have been cloned from rodent VRNs (Dulac and Axel, 1995; Herrada and Dulac, 1997; Matsunami and Buck, 1997; Ryba and Tirindelli, 1997). They are both seven transmembrane domain, G-protein–linked receptors, but share little homology with either each other or with main olfactory receptors. The V1Rs comprise a family of around 137 genes (Rodriguez et al., 2002). They exhibit significant variability in their transmembrane domains, which presumably constitute the ligand-binding site (Dulac and Axel, 1995). In contrast, the ligand-binding site in V2Rs is likely to be in the large and highly variable extracellular N-terminal domain, similar to that of metabotropic glutamate receptors and Ca2-sensing receptors. The V2Rs comprise a family of up to 140 genes, although some of these are pseudogenes, which lack one or more exons, or have mutations that prevent transcription of functionally normal receptors. In situ hybridization has revealed that the two classes of vomeronasal receptors are segregated in the vomeronasal epithelium of mammals. The cell bodies of VRNs that express V1Rs are located in the superficial third of the epithelium, nearest the lumen. Cell bodies of VRNs expressing V2Rs are located in the basal two thirds of the sensory epithelium (Herrada and Dulac, 1997). These two zones are also characterized by the expression of different G-proteins (Berghard and Buck, 1996). Thus, V1Rs are likely to be coupled to Gi2, whereas V2Rs are probably linked to Go. VIII. SEGREGATED PATHWAYS TO THE ACCESSORY OLFACTORY BULB As the two classes of vomeronasal receptor are only distantly related, they may respond to very different classes of stimuli. Moreover, there is good evidence that VRNs expressing each class of vomeronasal receptor project separately to distinct anterior and posterior subdivisions of the AOB (Halpern et al., 1998). These subdivisions can be visualized using antibodies to cell surface proteins such as the cell adhesion molecule OCAM (Campenhausen von et al., 1997). The have also been identified using monoclonal antibodies and lectin-binding patterns (Mori et al., 1987; Schwarting et al., 1994; Takami et al., 1992). The more superficial VRNs expressing V1Rs project to the anterior part of the AOB, whereas VRNs in the basal region of the epithelium, in which V2Rs are found, project to the posterior part of the AOB (Halpern

et al., 1995). Voltage-sensitive dyes and population potential recordings have demonstrated that these subdivisions of the AOB are functionally distinct (Sugai et al., 1997). Activity evoked in either region by selective stimulation of their vomeronasal nerve input does not propagate across the anatomical boundary. Furthermore, stimulation of the vomeronasal nerves produces an oscillating local field potential, which is more heavily damped in the anterior AOB than the strong oscillations produced in the posterior AOB. Therefore, differences in synaptic connectivity or pharmacology may contribute to functional differences between the two subregions. The anterior/posterior subdivision of the AOB has been found in a range of species including mice, rats, rabbits, guinea pigs, and opossums. This suggests that it is a general feature of the vomeronasal system, constituting separate pathways for information from the two classes of VRNs about distinct types of vomeronasal stimuli. A major question concerns the nature of the information conveyed by these parallel vomeronasal pathways. A number of possibilities exist: protein stimuli, as opposed to small volatile stimuli; male stimuli, as opposed to female stimuli; and stimuli that elicit neuroendocrine effects as opposed to stimuli that elicit behavioral responses. Support for the first of these possibilities comes from in vitro studies of rat VRNs (Krieger et al., 1999). Volatile components of rat urine activated the Gi2 pathway, corresponding to the V1R class of receptor, which projects to the anterior AOB. In contrast, the rat urinary protein alpha(2u)-globulin activated Go, corresponding to V2R expressing VRNs that project to the posterior AOB. An alternative approach has been to use the expression of immediate-early genes such as c-fos as markers of neuronal activity induced by vomeronasal stimuli. In mice, exposing females to urinary stimuli in soiled male bedding increases the number of Fos positive neurons in the VNO and in the AOB. This effect is enhanced in estrous females, especially following mating (Brennan et al., 1992). Cues in male urine mainly activate the anterior part of the AOB, suggesting preferential activity of V1R receptors (Brennan et al., 1999; Halem et al., 1998).

IX. INFORMATION CODING IN THE VOMERONASAL SYSTEM An understanding of sensory coding in the vomeronasal system requires information about the stimulus specificity of individual VRNs. Calculations based on probes that both recognize genes and hybridize with Go suggest that only one receptor is expressed in each sensory neuron (Belluscio et al., 1999; Rodriguez et al., 1999). This is supported by electrophysiological recordings from rat

The Vomeronasal Organ

vomeronasal epithelium, which have found a high degree of specificity in the responses of individual VRNs (Leinders-Zufall et al., 2000). The first steps have been taken towards an understanding of how the input from the VRNs is organized at the level of the AOB. Groups led by Peter Mombaerts and by Catharine Dulac and Richard Axel have genetically manipulated mice to express the taulacZ construct linked to the expression of a single, vomeronasal receptor gene (Belluscio et al., 1999; Rodriguez et al., 1999). This has allowed the visualization of the axonal projections of VRNs that express a specific receptor of the V1R class. These VRNs have cell bodies located in the superficial region of the vomeronasal sensory epithelium and project exclusively to the anterior region of the AOB. At first sight, the projections appear considerably less specific than the high degree of convergence seen in the main olfactory bulb. In the AOB, VRNs typically project to between 10 and 20 glomeruli out of a few hundred. There is some degree of consistency in the overall pattern of projection between the right and left AOBs and among different individuals. However, the relatively poorly defined glomeruli in the AOB complicate the interpretation of these findings. Mitral cells in the main olfactory bulb (MOB) project a single primary dendrite to a single glomerulus, and therefore collect information from a single receptor type. In contrast, mitral/tufted (M/T) cells in the AOB have a branched primary dendritic tree that projects to around 5–10 glomeruli (Mori, 1987). Although a M/T cell may collect information from several glomeruli, they may all be innervated by VRNs expressing the same receptor. If this is the case, each M/T cell would process information from a given receptor type, leading to highly specific M/T-cell responses that is similar to the situation in the MOB. Alternatively, each primary dendrite may project to glomeruli receiving input from a different receptor type. Information about different vomeronasal stimuli could then be integrated at the level of the M/T cells (Fig. 4). Considering that there are relatively few receptor types responding to a restricted range of vomeronasal stimuli, this may be important in the detection of specific blends of individual vomeronasal stimuli. Alternatively, it could provide a means of increasing the complexity of the vomeronasal signal to facilitate the fine discrimination among individuals.

X. NEURAL PROJECTIONS OF THE VOMERONASAL SYSTEM The distinction between the vomeronasal system and the main olfactory system is emphasized when their central

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projections are considered. VRNs project via the vomeronasal nerves to the AOB, which is located in the dorsocaudal part of the MOB, although entirely separate functionally (MacLeod and Reinhardt, 1983). Olfactory information from the MOB has access to neocortical processing through widespread projections, including those to the orbitofrontal and insular cortex, via the thalamus. In contrast, the AOB only projects sub-neocortically, to the medial amygdala (MeA), the posterior medial part of the cortical amygdala (PMCoA), the bed nucleus of the stria terminalis (BNST), and the bed nucleus of the accessory olfactory tract (BAOT) (Scalia and Winans, 1975). These amygdala regions then project to the medial hypothalamus both directly and via the BNST (Swanson and Petrovich, 1998). Thus, the vomeronasal system has relatively direct projections to hypothalamic centers controlling behavioral and neuroendocrine output (Fig. 5) (Li et al., 1990). The anatomical differences between the two olfactory systems reflect the different nature of the information that they convey (Meredith, 1991). The main olfactory system has the complex role of discriminating among the enormous variety of odors present in the environment and must be able to associate them flexibly with different behavioral responses. In contrast, the vomeronasal system has more straightforward task of detecting a limited range of stimuli and initiating relatively stereotyped behavioral and endocrine responses. The separate streams of information, from V1Rs to the anterior AOB and from V2Rs to the posterior AOB, are not maintained in their central projections. Halpern and Mori have injected small amounts of neural tracers into either the anterior AOB or posterior AOB in mice and opossums (Martínez-Marcos and Halpern, 1999b; von Campenhausen and Mori, 2000). In both cases the tracer was anterogradely transported to axon terminals distributed evenly throughout the MeA, PMCoA, BNST, and BAOT. There are no obvious differences in extent or density in the patterns of projection from either subregion of the AOB. There are reciprocal projections from these regions back to the AOB, and most of these innervate both the anterior and posterior regions equally. However, there is a population of neurons in the PMCoA that provide a reciprocal innervation of only the posterior AOB (Martínez-Marcos and Halpern, 1999a). Thus, although the PMCoA receives information form the whole of the AOB, it may send feedback only to the posterior region. The highly distributed pattern of projections from the AOB was still observed when only a few M/T cells had taken up any tracer (von Campenhausen and Mori, 2000). Individual M/T cells in both the anterior and posterior AOB are, therefore, likely to send a divergent projection to all of these targets. In this respect, the projection of M/T

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Figure 4 Possible projection patterns of vomeronasal receptor neurons (VRNs) to mitral/tufted (M/T) cells in the accessory olfactory bulb (AOB). (a) M/T cells may receive input from multiple glomeruli, which in turn receive input from a single receptor type. This form of connectivity is essentially similar to that seen in the main olfactory bulb. (b) The specificity of input to individual glomeruli may be maintained, but the M/T cells may send their primary dendrites to different types of glomeruli. This arrangement would give greater opportunity for integration of information from different receptor types at the level of the AOB.

Figure 5 The vomeronasal system projects exclusively subneocortically and has a relatively direct influence on hypothalamic centers controlling behavioral and endocrine responses. Information from basal and apical vomeronasal receptors is kept separate at the level of the accessory olfactory bulb, but is widely distributed at the level of the amygdala. Input from the main olfactory system can influence behaviors controlled by the vomeronasal system by way of projections to the medial amygdala. Abbreviations: AOB, accessory olfactory bulb; BAOT, bed nucleus of the accessory olfactory tract; BNST, bed nucleus of the stria terminalis; MeA, medial amygdala; PA, posterior amygdala; PMCoA, posterior medial cortical amygdala.

cells in the AOB resembles the highly distributed projection of MOB mitral cells to the piriform cortex (Haberley, 1985). This type of neural architecture is characteristic of highly parallel processing, probably involving a sparse representation of vomeronasal information at the level of the amygdala. Therefore, although there is a relatively direct projection from the VRNs to hypothalamic areas and the range of vomeronasal stimuli is likely to be small, the neural architecture is not consistent with a simple labeled line coding of information. The representation of information in the vomeronasal system may be more similar to the pattern recognition system that is thought to operate in the main olfactory system. Although both the MOB and AOB project to the amygdala, they terminate in adjacent, nonoverlapping areas (Scalia and Winans, 1975). The MOB projects to the anterior and posterior lateral parts of the cortical amygdala, and although there are both direct and indirect projections from these areas to the vomeronasal parts of the amygdala, there are no reciprocal connections. Therefore, the main olfactory system can influence the neuroendocrine output of the vomeronasal system. This may provide an explanation of why VNO ablation disrupts mounting behavior in inexperienced male hamsters but has a less serious effect in experienced males (Meredith, 1986). The first mating experience seems to involve a learning process by which odor cues received via the main olfactory system become capable of eliciting full mounting behavior. This occurs via links with the vomeronasal amygdala, which controls mounting behavior via an output to the medial hypothalamus (Meredith, 1998). Interestingly, exogenous, intraventricular administration of LHRH is able to compensate for VNO lesions

The Vomeronasal Organ

and to restore sexual behavior in inexperienced males (Fernandez-Fewell and Meredith, 1995). Thus, the vomeronasal system may mediate the effects of HVF to elicit mounting behavior through increased LHRH release in the medial preoptic area (Meredith, 1998). The medial hypothalamic projections of the accessory olfactory amygdala are especially prominent and are involved in the control of reproductive, defensive, and ingestive behaviors. Mating induces the expression of immediate early gene markers in the central projections of the vomeronasal system (MeA, BNST, medial preoptic area, ventromedial hypothalamus, and arcuate nucleus) in both males and females (Bressler and Baum, 1996; Halem et al., 1998). There is a sexual dimorphism in the VNO and its central projections (Segovia and Guillamón, 1993), although the extent to which these relate to anterior or posterior AOB, and hence different classes of VNO receptor, is not known. However, these sexual dimorphisms in the morphology of the VNO projection circuit may not be reflected in the functional responsiveness of the neurons in this circuit as assessed by c-Fos expression (Bressler and Baum, 1996).

XI. SYNAPTIC PLASTICITY IN THE AOB AND ITS IMPLICATIONS FOR INFORMATION PROCESSING Learning plays a major role in the responses to odor stimuli conveyed by the main olfactory system. Likewise, the effect of experience cannot be ignored when considering the responses mediated by the vomeronasal system. This has been studied most extensively in female mice, which learn the characteristics of urinary stimuli from the individual male with which they mate (Brennan and Keverne, 1997). Learning occurs during a sensitive period of around 4 hours following mating. This enables a female mouse to recognize the pregnancy-blocking urinary stimuli from her mate and prevents them from aborting his offspring. Surprisingly, infusions of local anesthetic into the amygdala during the sensitive period do not impair memory formation. This is because memory formation depends solely on neural changes occurring in the AOB (Kaba et al., 1989). The vaginocervical stimulation at mating increases noradrenaline release in the AOB from centrifugal, locus coeruleus projections. Depletion of noradrenaline in the AOB or infusions of noradrenergic antagonists into the AOB during the sensitive period disrupt memory formation (Kaba and Keverne, 1988; Rosser and Keverne, 1985). This suggests that the noradrenergic projection from the locus coeruleus plays a vital role in memory formation by signaling that mating has occurred. The AOB has a relatively simple structure, which is similar to the olfactory bulb of lower vertebrates and

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consists of three main cell types (Mori, 1987). M/T cells collect the input from the vomeronasal nerves and project centrally, forming the excitatory pathway by which the pregnancy-blocking signal is conveyed, via the amygdala, to the hypothalamus. There are two main classes of inhibitory interneurons in the AOB. Periglomerular cells provide feedback inhibition at the glomerular level and may regulate input from the vomeronasal nerves. Granule cells are the major class of interneuron. They form reciprocal dendrodendritic synapses with M/T cells in the external plexiform layer and provide feedback control of M/T cell activity. Despite its simple structure, memory formation can be explained by changes in the strength of the glutamatergic synapses from M/T cells to granule cells (Brennan et al., 1990). Increased noradrenaline levels at mating are hypothesized to cause a long-term enhancement at the glutamatergic synapses of the sub-population of M/T cells activated by the male urinary stimuli. As the M/T cells form reciprocal synapses with inhibitory granule cells, the increased excitation of the granule cells results in an increased inhibitory feedback onto the M/T cells. During subsequent exposure to the urinary stimuli of the mating male, the M/T cells that convey the pregnancyblocking signal are subject to an increased inhibitory control. This disrupts the pregnancy-blocking signal at the level of the AOB (Fig. 6). However, when the female is exposed to urinary stimuli from a different male, they activate a different subpopulation of M/T cells that are not subject to enhanced inhibitory control. The male urinary signal is thus transmitted to hypothalamic levels resulting in pregnancy block. There is a substantial amount of evidence to support this hypothesis. A glutamate challenge causes greater release of the inhibitory transmitter GABA in the AOB of mated compared to nonmated females (Brennan et al., 1995). Moreover, the ratio of the inhibitory transmitter GABA to the excitatory transmitters glutamate and aspartate is increased in mated females when they are exposed to the urinary stimuli of the mating male. These findings are consistent with an increase in the inhibitory control of M/T cells in the AOB following mating. The underlying synaptic changes have also been observed more directly. The postsynaptic densities on glutamatergic synapses from M/T cells to granule cells are longer in mated compared to nonmated females (Matsuoka et al., 1997). This occurs without changes of the granule cell to M/T cell inhibitory synapses or to synapses in the glomerular layer. Similar increases in the inhibitory control of mitral cells are associated with odor learning in the MOB and appear to be a general feature of chemosensory processing (Brennan and Keverne, 1997). Mitral cells in the MOB have extensive secondary dendritic trees that extend over large areas of the bulb. Thus, changes in lateral inhibition between

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When female mice are exposed to male urinary stimuli without mating, memory formation does not occur. On the contrary, exposure without mating appears to cause a longlasting depression of glutamatergic transmission at M/T to granule cell synapses. This is evident in an increased release of the excitatory transmitters aspartate and glutamate in response to male urinary stimuli 2 days following exposure without mating (Brennan et al., 1995). The decreased inhibitory control of M/T cells is consistent with the priming effect of preexposure to male urine, which enhances the effect of male urinary stimuli to accelerate puberty in female mice (Mucignat Caretta et al., 1995). It is also consistent with the finding that the exposure of male hamsters to urine from female hamsters decreases the length of the postsynaptic densities of the glutamatergic synapses from M/T to granule cells (Matsuoka et al., 1998). Such a “wind-up” of M/T cell transmission may be important in maintaining a response to the vomeronasal stimuli during the long periods of exposure that are necessary to elicit their neuroendocrine effects (Rosser et al., 1989). Taken together, these various lines of evidence suggest that synaptic plasticity of the reciprocal synapses between M/T and granule cells may play a vital role in controlling the transmission of information through the vomeronasal system. Figure 6 Changes in the strength of the reciprocal synapses between mitral/tufted (M/T) and granule cells regulate the transmission of vomeronasal information by the accessory olfactory bulb (AOB). (a) Feedback inhibition from granule cells regulates the output of M/T cells that is transmitted centrally, resulting in the estrus-inducing effects of male chemosignals. (b) Following mating, the M/T cells respond to the urinary stimuli from the mating male are subject to greater inhibitory feedback from granule cells. This disrupts the transmission of the pregnancyblocking signal at the level of the AOB. (c) Preexposure to urinary stimuli appears to decrease the strength of excitatory synapses from mitral to granule cells. The reduction in feedback inhibition of M/T cells enhances the effectiveness of subsequent exposure. Periglomerular (PG) cells are likely to control the glomerular input to M/T cells from the vomeronasal nerve (VNN), but are not thought to be involved in learning.

neighboring mitral cells are likely to be an important consequence of learning. In contrast, M/T cells in the AOB have very small secondary dendrites, and the majority of the reciprocal synapses with granule cells are made on the extensive primary dendritic trees. These synapses are therefore ideally placed to control the transmission of information along individual primary dendrites from glomeruli to the M/T cell soma and therefore to disrupt the transmission of vomeronasal information.

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Brennan and Keverne in mouse vomeronasal receptor neurons. J. Neurophys. 77:2856–2862. Mucignat Caretta, C., Caretta, A., and Cavaggioni, A. (1995). Pheromonally accelerated puberty is enhanced by previous experience of the same stimulus. Physiol. Behav. 57:901–903. Muller-Schwarze, D. (1979). Flehmen in the context of mammalian urine communication. In Chemical Ecology: Odour Communication in Animals, F. J. Ritter (Ed.). Elsevier, New York. Novotny, M. V., Weidong, M., Wiesler, D. and Zidek, L. (1999). Positive identification of the puberty-accelerating pheromone of the house mouse: the volatile ligands associating with the major urinary protein. Proc. R. Soc. Lond. B 266:2017–2022. Powers, J. B., and Winans, S. S. (1975). Vomeronasal organ: critical role in mediating sexual behavior of the male hamster. Science 187:961–963. Robertson, D. H. L., Cox, K. A., Gaskell, S. J., Evershed, R. P., and Beynon, R. J. (1996). Molecular heterogeneity in the major urinary proteins of the house mouse Mus musculus. Biochem. J. 316:265–272. Robertson, D. H. L., Hurst, J. L., Bolgar, M. S., Gaskell, S. J., and Beynon, R. J. (1997). Molecular heterogeneity of urinary proteins in wild house mouse populations. Rap. Comm. Mass Spec. 11:786–790. Rodriguez, I., Feinstein, P., and Mombaerts, P. (1999). Variable patterns of axonal projections of sensory neurons in the mouse vomeronasal system. Cell 97:199–208. Rodriguez, I., Del Punta, K., Rothman, A., Ishii, T., and Mombaerts, P. (2002). Multiple new and isolated families within the mouse super family of Vlr vomeronasal receptors. Nature Neurosci. 5:134–140. Rosser, A., and Keverne, E. B. (1985). The importance of central noradrenergic neurones in the formulation of an olfactory memory in the prevention of pregnancy block. Neuroscience 15:1141–1147. Rosser, A. E., Remfry, C. J., and Keverne, E. B. (1989). Restricted exposure of mice to primer pheromones coincident with prolactin surges blocks pregnancy by changing hypothalamic dopamine release. J. Reprod. Fertil. 87:553–559. Ryba, N. J. P., and Tirindelli, R. (1997). A new multigene family of putative pheromone receptors. Neuron 19:371–379. Salazar, I., and Sánchez Quinteiro, P. (1998). Supporting tissue and vasculature of the mammalian vomeronasal organ: the rat as a model. Micros. Res. Tech. 41:492–505. Scalia, F., and Winans, S. S. (1975). The differential projections of the olfactory bulb and accessory olfactory bulb in mammals. J. Comp. Neurol. 161:31–56. Schwarting, G. A., Drinkwater, D., and Crandall, J. E. (1994). A unique neuronal glycolipid defines rostrocaudal compartmentalization in the accessory olfactory system of rats. Dev. Brain Res. 78:191–200. Segovia, S., and Guillamón, A. (1993). Sexual dimorphism in the vomeronasal pathway and sex differences in reproductive behaviors. Brain Res. Rev. 18:51–74. Singer, A. G., and Macrides, F. (1990). Aphrodisin: pheromone or transducer? Chem. Senses 15:199–204.

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979 tance in snake vomeronasal receptor neurons. Chem. Senses 25:67–76. Trotier, D., and Døving, K. B. (1998). ‘Anatomical description of a new organ in the nose of domesticated animals’ by Ludvig Jacobson (1813). Chem. Senses 23:743–754. Trotier, D., Doving, K. B., Ore, K., and Shalchiantabrizi, C. (1998). Scanning electron microscopy and gramicidin patch clamp recordings of microvillous receptor neurons dissociated from the rat vomeronasal organ. Chem. Senses 23:49–57. Vandenbergh, J. (1969). Male odor accelerates female sexual maturation in mice. Endocrinology 84:658–660. Vandenbergh, J. G., Whitsett, J. M., and Lombardi, J. R. (1975). Partial isolation of a pheromone accelerating puberty in female mice. J. Reprod. Fertil. 43:515–523. von Campenhausen, H., and Mori, K. (2000). Convergence of segregated pheromonal pathways from the accessory olfactory bulb to the cortex in the mouse. Eur. J. Neurosci. 12:33–46. Wang, D., Chen, P., Liu, W. M., Li, C. S., and Halpern, M. (1997). Chemosignal transduction in the vomeronasal organ of garter snakes: Ca2-dependent regulation of adenylate cyclase. Arch. Biochem. Biophys. 348:96–106. Weiler, E., McCulloch, M. A., and Farbman, A. I. (1999). Proliferation in the vomeronasal organ of the rat during postnatal development. Eur. J. Neurosci. 11:700–711. Whitten, W. (1956). Modification of the oestrous cycle of the mouse by external stimuli associated with the male. J. Endocrinol. 13:399–404.

47 Trigeminal Chemosensation Richard L. Doty University of Pennsylvania, Philadelphia, Pennsylvania, U.S.A.

J. Enrique Cometto-Muñiz University of California, San Diego, La Jolla, California, U.S.A.

I.

INTRODUCTION

optic and olfactory nerves, but not the trigeminal ones. Two years later Bishop (1833) described a patient with paralysis of the trigeminal nerve with normal ability to smell, and Shaw (1833) wrote a scathing critique of Magendie’s earlier experiments, pointing out why they were invalid.* Today we know that the qualitative sensations of smell are mediated by CN I; however, we also know that most, if not all, odorous chemicals have the propensity to stimulate, directly or indirectly, free nerve endings of CN V located within the lining of the nasal vestibule and nasal chambers, producing such sensations as irritation, tickling, burning, warming, cooling, and stinging.† These sensations, which often serve to avert the organism away from

During the early part of the nineteenth century there was considerable debate regarding whether the sense of smell was mediated by the first cranial nerve (CN I, the olfactory nerve) or the fifth cranial nerve (CN V, the trigeminal nerve). Although Sir Charles Bell believed that olfaction was subserved by CN I (Bell, 1812), he erroneously thought that CN I and CN V fibers were united for a portion of their projection, as indicated in his 1811 classic Idea of a New Anatomy of the Brain (Bell, 1966). Francois Magendie, Bell’s chief French rival and the primary proponent of the theory that CN V mediated olfaction, published his major arguments in 1824, along with a series of flawed physiological animal experiments that he touted as demonstrating his point (Magendie, 1824). Magendie’s conclusions received little confirmation from others, and reports soon appeared supporting Bell’s contention that CN I mediates olfactory function. For example, in 1826 Eschricht noted that persons without olfactory nerves or with degenerate nerves were anosmic. Cruveilhier (1829) communicated a case to the Anatomical Society of Paris (of which he was president) in which a fungus of the dura matter had produced damage to the optic chiasm and had destroyed the olfactory nerves and sectors of the anterior cerebral lobes, but left the trigeminal nerves intact. This individual was both anosmic and blind. Vidal (1831) subsequently reported to the same society a case of an anosmic blind person who at autopsy had a tumor that destroyed the

* To place these observations in historical context, it should be noted that it was not until 1847 that Todd and Bowman reported that a sector of the epithelium on the upper turbinate differed in appearance from the rest of the nasal mucosa (Todd and Bowman, 1847). The different cell types within the olfactory epithelium were first described in 1855 (Ecker, 1855; Eckhard, 1855). The first descriptions and illustrations of true olfactory receptor cells and their cilia were published in 1856 (Schultz, 1856). An excellent review of these and other early studies is provided by Zippel (1993); see also the introduction to this volume.

†

There is some suggestion that the nasal mucosa may also receive some sensory fibers from the facial (CN VII), vagus (CN X), and upper thoracic spinal nerves, although their role, if any, in chemoreception is obscure. Eccles (1982) has speculated that these pathways may explain referred pain and headaches due to nasal disease. 981

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harmful sources of stimulation, are classified by some as a component of the “common chemical sense,” a term first used by Parker (1912) to describe the general chemical responsiveness of mucosal and epithelial tissue mediated via free nerve endings from a number of different nerves. Parker believed the common chemical sense to be an independent sensory system, a belief no longer viewed as viable. The term “chemesthesis” has been employed in more modern times as a replacement for the notion of a common chemical sense to describe chemosensitivity mediated via free nerve endings (Green and Lawless, 1991; Green et al., 1990). In addition to serving chemosensory functions, CN V fibers within the nasal vestibule mediate the tactile sensations of temperature and pressure, including the perception of nasal airflow during breathing (Burrow et al., 1983; Cauna and Hinderer, 1969). Importantly, CN V stimulation can reflexively influence cardiovascular responses (e.g., heart rate and blood pressure), respiration rate, nasal engorgement, epinephrine secretion, nasal secretion, and sneezing (Alarie, 1966; Allen, 1929; James and Daly, 1969, 1972; Kratschmer, 1870), and there is evidence that the trigeminal and olfactory systems interact centrally. For example, olfactory and trigeminal pathways converge on the same neural elements within the mediodorsal nucleus of the thalamus of the rat; blocking the trigeminal pathway enhances odor-induced activity in the nucleus (Inokuchi et al., 1993). Lesioning or reversibly blocking the rabbit trigeminal ganglion inhibits olfactory bulb activity (Stone et al., 1968). Electrical stimulation of the trigeminal nerve decreases bulbar activity (Stone, 1969), a phenomenon that has also been reported for electrical stimulation of the vagus nerve (Garcia-Diaz et al., 1984). Interestingly, CN V may also modulate the activity of olfactory receptor cells within the neuroepithelium via a local axon reflex associated with the release of substance P (Bouvet et al., 1987). In this chapter, we (1) review basic aspects of the anatomy and physiology of the intranasal trigeminal system, with an emphasis on humans, (2) describe basic characteristics of human trigeminal chemosensory processing, and (3) present data from psychophysical studies related to potential CN V interactions with CN I–mediated chemoreception. Recent structure-activity studies will be presented, along with a discussion of the application of psychophysical methods to environmental issues. The reader is referred elsewhere for more general reviews on trigeminal chemosensation and related topics, including the complex relationships between the trigeminal system and autonomic processes in general (Bryant and Silver, 2000; DeLong and Getchell, 1987; Doty et al., 2003; Eccles, 1990; Green and Lawless, 1991; Keverne et al., 1986; Tucker, 1963; Walker et al., 1990; Widdicombe, 1986).

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II. ANATOMY AND PHYSIOLOGY OF THE CHEMOSENSORY TRIGEMINAL SYSTEM In the human, sensory nerve endings from branches of the trigeminal nerve are found in the epithelia of the nose and sinuses, the oral cavity, the eyelids, and the cornea (Fig. 1). The anterior and lateral portions of the nasal cavity are innervated by the lateral and medial nasal branches of the ethmoidal nerve, which is derived from the nasociliary branch of the ophthalmic division of CN V arising from the trigeminal ganglion (also called the Gasserian or semilunar ganglion). This nerve enters the nasal cavity via the anterior ethmoidal foramen near the lateral margin of the cribriform plate. The posterior portion of the nasal cavity is innervated by the nasopalatine nerve, one of the four nerves that branch from the sphenopalatine ganglion. This nerve contains parasympathetic postganglionic fibers from the sphenopalatine ganglion, sympathetic postganglionic fibers from the sympathetic ganglia in the neck, and sensory fibers from the maxillary nerve that traverse the sphenopalatine ganglion. Developmentally, the trigeminal nerve is well formed in utero (Gasser and Hendrickx, 1969; Hogg, 1941). Indeed, the perioral areas supplied by the mandibular and maxillary divisions are the first embryonic regions to respond to

Figure 1 Primary branches of the trigeminal nerve that innervate the nasal and oral cavities. (From Silver, 1987.)

Trigeminal Chemosensation

cutaneous stimulation (~7.5 weeks). The ophthalmic division can be observed at 4–5 gestational weeks and is presumed to be functional by 10.5 weeks (Brown, 1974; Humphrey, 1966; Streeter, 1908). Not surprisingly, trigeminal reactivity to chemical stimulation is present at birth. Thus, when the nares of newborn infants (ranging in age from 16 to 131 hours) were confronted with vials containing either cotton or ammonium hydrochloride, they turned away from the side of the ammonia on 64 % of 304 trials (Rieser et al., 1976). A number of fiber types are found within each of the branches of the trigeminal nerve. In the case of the infraorbital nerve of the rat, both myelinated and unmyelinated axons are present, with myelinated ones ranging from 0.8 to 14.9 m in diameter and unmyelinated ones ranging from 0.3 to 1.5 m in diameter (Jacquin et al., 1984). More unmyelinated than myelinated axons are found in the ethmoidal nerve, with the preponderance of fibers ranging from 2 to 6 m in diameter (Biedenbach et al., 1975). Immunocytochemical studies demonstrate that fine unmyelinated C-fibers that innervate the nasal cavities contain SP and, in many cases, associated calcitonin gene–related peptide (Finger et al., 1990). Physiological studies suggest that unmyelinated C-fibers are responsible for irritative reactions in the nasal and respiratory passages, as well as in the body skin, although small myelinated Adelta fibers may also be involved (Jancso et al., 1967; Lundblad et al., 1983). Silver et al. (1985) noted that chronic administration of capsaicin (which depletes, to a large degree, SP from fine unmyelinated afferents) eliminated or severely reduced trigeminal nerve responses to chemicals in rats, suggesting that the small unmyelinated and possibly some myelinated fibers subserve trigeminal pain reactions. Other classes of peptidergic fibers (e.g., ones that contain vasoactive intestinal peptide and luteinizing hormone releasing hormone) are also found within the nasal epithelium, although their role in chemosensory mediation is not defined (Silver et al, 1985) (see Chapter 48). Although a few nonolfactory nerve fibers have been observed extending to the epithelial surface of the nasal epithelium (see, e.g. Lundblad et al., 1983), Finger et al. (1990), using data from electron microscopy studies, reported that the vast majority of CN V free nerve endings terminate within the lamina propria, at least in amphibia and rodents. Nevertheless, they observed a few trigeminal fibers that terminated within 1 m of the epithelial surface, just below the tight junctions. Finger et al. pointed out that for volatile chemicals to stimulate these nerve endings, they must (1) pass into the nasal cavity, (2) partition into and diffuse through the mucus, and (3) cross the epithelial cell membranes and/or intercellular tight junctions. Since most trigeminal stimulants are lipid soluble, such transit is likely.

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There is considerable evidence that trigeminal nerve endings express a number of specific receptors. Thus, vanilloid receptors, found in a subset of C-fibers, have a strong afinity for capsaicin, the pungent principle in hot peppers, as well as for closely related molecules (Szallasi, 1994). Interestingly, these receptors can also be activated by noxious heat (Caterina et al., 1997). Nicotinic acetylcholine receptors are also likely to be present on nasal trigeminal nerve endings (Alimohammadi and Silver, 2000). In fact, electrophysiological studies in rats (Walker et al., 1996), as well as psychophysical and electrophysiological studies in humans (Thürauf et al., 1999), suggest the existence of a dose-dependent stereoselective activation of the trigeminal sensory system by S()- and R()nicotine. Some reactive substances presumably act directly on mucosal tissue, or indirectly by damaging mucosal tissue (via chemical reaction), subsequently releasing endogenous chemicals which, in turn, act upon specific ion channels to produce the trigeminal response. Such endogenous compounds include ATP, H, and bradykinin (Cesare and McNaughton, 1997; McCleskey and Gold, 1999). Aside from the specific stimuli mentioned above, for which there seem to be finely tuned receptors, the human trigeminal chemoreceptive system has been shown to respond to a myriad of relatively nonreactive volatile compounds possessing varied chemical functionalities such as alcohols, esters, ketones, carboxylic acids, aldehydes, and the like, including linear and branched, saturated and unsaturated, aliphatic and aromatic molecules. All of them are capable of evoking trigeminal sensations, e.g., nasal pungency and eye irritation, when presented at high enough concentrations (see review in Cometto-Muñiz, 2001). In view of such diversity in chemical structures and functionalities, it is likely that the trigeminal impact of this board range of substances rests heavily on general physicochemical parameters that govern stimulus transport from the vapor phase to the biophase, where trigeminal chemoreceptors lie. The success of a quantitative structure-activity relationship (QSAR) based on a solvation equation to describe and predict human thresholds for nasal pungency (Abraham et al., 1998a) and for eye irritation (Abraham et al., 1998b) lends support to this perspective (see sec. III. D).

III. CHEMOSENSORY TRIGEMINAL STIMULATION: HUMAN PSYCHOPHYSICAL AND PHYSIOLOGICAL STUDIES Numerous studies have sought to determine the responses produced by volatile chemicals on the trigeminal system of humans. Such responses fall into three main categories:

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(1) changes in respiration, nasal secretion, and other physiological measures to chemosensory stimulation; (2) induction of perceptual qualities, such as cooling, burning, irritation, and pain; and (3) alterations in psychophysical measures of CN I–mediated odor perception. In this section we review studies that examine the propensity of volatile chemicals, alone or in combination, to produce nonolfactory chemical sensations and describe, among other things, the functional dissociation of the intranasal trigeminal and olfactory responses. A.

Trigeminal Stimulants and the Search for “Pure” Odorants

For many years investigators have sought to identify chemicals that produce “pure” olfactory sensations, i.e., odorants uncontaminated by trigeminal activity. Such agents would be extremely useful in human olfactory research, since they would allow for the psychophysical investigation of CN I without potential confounding influences from CN V. In light of this quest, it is of interest that a number of early workers simply assumed that a rather clear distinction could be made between “pure” odorants and more “penetrating” ones, i.e., ones that produce tactile sensations. A case in point is Zwaardemaker (1925), who felt that the majority of essences, resins, and pitches fell into the “pure” odorant class. Despite the attractiveness of this concept and the astute observations of many such workers, modern studies throw into question such a simple dichotomy of odorants, suggesting that even clean air can produce some intranasal trigeminal sensation, depending on its flow rate, temperature, and degree of humidification (Doty et al., 1978; Eccles, 1990). Nevertheless, as indicated in detail below, it is clear that some chemicals produce much less trigeminal stimulation than others, and that at low concentrations and flow rates some agents likely produce little or no trigeminal activity (or at least no more trigeminal activity than that produced by inhalation of clean air). Several major attempts to identify “pure” olfactory stimulants were made in the first third of this century. Thus, 3 years before Allen’s (1928) classic ablation study that identified the nasociliary and maxillary nerves as the source of odor-induced respiratory and cardiovascular responses in dogs,* von Skramlik (1925) listed nearly 50 “pure” odorants, as inferred from the inability of five subjects to localize them to the side of the nose to which they were presented. Among these odorants were anethole, cadinene *

It is of interest that Allen reported that his study was a replication and expansion of observations made earlier by Kratschmer (1870) and Beyer (1901) on this topic.

(juniper), eugenol, geraniol, indole, limonene, phenyl ethyl alcohol, pinene, skatol, and terpineol. Examples of “impure” odorants reported by von Skramlik were ones that produced smell  sweet sensations (bromoform, chloroform, ethyl chloride, iodoform, nitrobenzol), smell  sour sensations (acetic acid, butyric acid, propionic acid, and valerianic acid), smell  cool sensations (camphor, eucalyptol, menthol, phenol, safrol), smell  warm sensations (ethanol, pentanol, propanol), and smell  painful or prickly sensations (acetone, acetic acid, ammonia, bromine, chlorine, formic acid, iodine, nicotine, pyridine, SO2, thiophene, toluol, and xylol). In 1929 Allen performed a series of human experiments to examine the influences of a number of volatile chemicals on cardiovascular responses. In one phase of this work, he described the responses of an anosmic college student to various inhaled chemicals. This student’s anosmia resulted from a fall from a telephone pole, which produced a severe skull fracture and paralysis of the left rectus lateralis eye muscle. Although the student reported being unable to smell any of the stimuli, he indicated that a number produced clear intranasal or intraoral sensations. As described by Allen (p. 625), Fresh cat’s urine was not detected, but old decomposed cat’s urine, possessing a trace of ammonia, was said to produce a different sensation than a strong concentration of ammonia. . . . pyridin was described as a taste sensation coming from the back of the throat and tongue. Peppermint caused a cooling sensation in the nose and ether a disagreeable burning sensation appearing quickly. On the other hand, chloroform elicited a pleasant sensation in the nose. Formalin, described as a tickling sensation in the nose, was a long time in appearing. A weak concentration of acetic acid or ammonia produced a burning sensation in the nose and throat, which would have resulted in a movement of the head if inhalation had continued longer. Very singularly, the subject could inhale oil of mustard for a considerable length of time without any discomfort if the cone was held 4 or 5 cm. from the nostrils. In fact, the change in respiration appeared before the sensation was detected. Importantly, menthol, eucalyptus, camphor, peppermint, ether, chloroform, and benzol, as well as weak concentrations of the strong irritants formalin, acetic acid, and ammonia, produced augmented inspirations without altering respiration rate per se. Inhalation of the oils of bergamot, cloves, orange, lavender, rose, and wintergreen, as well as asafetida, butyric acid, xylol, and fresh cat’s urine, resulted in no such changes.

Trigeminal Chemosensation

It is noteworthy that the stimuli reportedly detected by this anosmic student corresponded well to those classified by von Skramlik as being “impure” odorants (e.g., pyridine, chloroform, acetic acid, ammonia, menthol, peppermint, and formalin), providing at least rough validation of their nonolfactory effects. Similarly, a number of the chemicals that this study reported as not influencing respiration or being detected by the subject were the same as some of von Skramlik’s “pure” olfactants [e.g., eugenol (cloves)]. Although in the years following these studies several other investigators commented on stimuli that reportedly produced no trigeminal stimulation (e.g., Elsberg et al., 1935), little systematic psychophysical research was performed on this topic until 1975, when 14 anosmics were presented with 31 chemicals in sniff bottles and asked if any intranasal sensations were detected (Doty, 1975b). Eleven of the chemicals (35%) were reported by these subjects not to be detectable: anethole, benzyl acetate, eugenol, geraniol, heptane, heptyl alcohol, hexanoic acid, nonane, octane, 2-phenyl ethyl alcohol, and -terpineol. Again, most of these chemicals fell within the set that von Skramlik reported as being “pure” olfactants. In contrast to this finding, however, were the results of a subsequent and more extensive study of 47 chemicals [which included the 31 chemicals used in the Doty (1975b) study] which incorporated a forced-choice detection paradigm (Doty et al., 1978). In this experiment, three groups of subjects (n
15/group) were evaluated: (1) anosmics lacking CN I, but not CN V, nerve function; (2) normals asked to rate only intranasal CN V sensations (termed the trigeminal focus group); and (3) normals asked to rate their overall odor experience. During testing, each subject was blindfolded and, on a given trial, was presented with two sniff bottles, one after the other in random order. One bottle contained a blank (propylene glycol) and the other an undiluted odorant. Each subject was asked to identify which of the two bottles seemed strongest. If reliable detection occurred, the subject was asked to rate the stimulus’ intensity, pleasantness, coolness, warmth, and presumptive safety on a series of anchored rating scales.*

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This study, unlike the previous one, found that nearly all (45/47, or 96%) of the chemicals were detected by at least some of the anosmics. Although differences in the rated intensities given to the stimuli were present among the three groups (e.g., the normal subjects consistently rated the stimuli as much stronger than did the other two groups), the relative rankings of the intensity responses were similar (r’s ranging from 0.92 to 0.97). The pleasantness and presumed safety ratings were inversely related to the ratings of perceived intensity in all three groups. The major point of significance of this study was its conclusive demonstration that anosmics and normal subjects could detect nearly all the chemicals presented to them via nonolfactory means when the testing was performed in a forced-choice format, thereby throwing into question the conclusions of a number of previous psychophysical studies on this topic (where subjects were simply asked whether or not they smelled something). The mean intensity ratings for each of the 47 chemicals provided by the anosmic, trigeminal focus, and normal subjects in the Doty et al. (1978) study are presented in Table 1, along with the proportion of subjects within each group who detected them. It is of interest that only one chemical, vanillin, was not detected by at least one subject from the anosmic or trigeminal focus groups, and that a number of chemicals reported to be “pure” odorants by von Skramlik and others (e.g., anethole, eugenole, geraniol, indole, limonene, phenyl ethyl alcohol, and turpineol) were detected (albeit as weak) by at least a few subjects from the anosmic and trigeminal focus groups. An electrophysiological study by Silver and Moulton (1982) adds credence to the validity of these findings. Thus, these investigators found that the relative magnitude of whole-nerve recordings from the ethmoid branch of nine rats correlated very strongly (r
0.975) with the intensity ratings from the Doty et al. study for the nine compounds they evaluated. The magnitude of this correlation is striking, particularly in light of the fact that responses of a quite different nature were being compared across species from different mammalian orders. B.

Functional Dissociation of Trigeminal and Olfactory Responses in the Nose

*

We also performed structure-activity studies to determine whether we could predict the degree of trigeminal stimulation of the chemicals. The use of 11–13 readily available and computerderived molecular descriptors in linear learning machine pattern recognition analyses separated the stimuli correctly into four discrete intensity classes. A multiple linear regression equation based on such descriptors proved successful in predicting the trigeminal intensities of 12 chemical stimuli similar in structure to members of the original stimulus set (r
0.80 between predicted and observed intensities).

From the studies discussed in the previous section, we conclude that most volatile organic compounds employed in olfactory studies are capable of stimulating both the olfactory and the trigeminal nerves. The issue of olfactory vs. trigeminal stimulation becomes, then, a matter of degree or dose, as a central concept in toxicology sustains. As a consequence, a more productive and practical approach to the problem consists in elucidating the gap between the two

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Table 1 Mean Intensity Rating Scale Values (  SD) of Anosmic, Trigeminal Focus, and Normal Experimental Groupsa Anosmic group Compound Decanoic acid Vanillin Phenyl ethyl alcohol Eugenol Coumarin Nonane Octane Indole -Terpineol Geranoil Heptanoic acid Limonene Hexanoic acid Heptane Benzyl acetate Methyl salicylate -Ionone Anethole Heptyl alcohol Guaiacol Citral Camphor 4-Methylvaleric acid Linalool n-Butyl ether Valeric acid 2,4-Pentanedione Furfural Menthol Iso-amyl acetate n-Butyl alcohol Acetaldoxime 2-Heptanone Iso-valeric acid Ethyl benzene n-Butyl acetate Ethyl acetate Methanol Benzaldehyde Cyclohexanone Toluene Butyric acid Acetal Ethyl methyl ketone Pyridine Acetone Propionic acid aMaximum possible rating
9. Source: Doty et al., 1978.

Trigeminal focus group

Proportion detecting

Intensity

Proportion detecting

Intensity

Normal group intensity

00/15 00/15 01/15 01/15 02/15 03/15 03/15 03/15 05/15 02/15 05/15 06/15 07/15 05/15 07/15 09/15 09/15 08/15 13/15 13/15 12/15 14/15 09/15 13/15 13/15 15/15 15/15 15/15 15/15 15/15 15/15 15/15 15/15 15/15 15/15 15/15 15/15 15/15 15/15 15/15 15/15 15/15 15/15 15/15 15/15 15/15 15/15

0.00(0.00) 0.00(0.00) 0.13(0.50) 0.13(0.50) 0.13(0.34) 0.27(0.57) 0.27(0.57) 0.53(1.20) 0.53(1.02) 0.60(1.54) 0.87(1.45) 0.93(1.44) 0.93(1.39) 1.00(1.86) 1.40(2.12) 1.60(1.86) 1.93(2.21) 2.73(2.86) 2.80(1.80) 2.80(1.87) 2.87(2.25) 3.53(2.09) 3.93(3.68) 4.00(2.37) 4.00(2.10) 5.00(2.16) 5.57(1.29) 6.07(1.24) 6.14(0.92) 6.67(1.19) 6.67(1.30) 6.71(0.80) 6.73(1.00) 6.73(1.24) 6.87(2.00) 7.33(1.08) 7.53(1.02) 7.67(1.14) 7.73(0.93) 7.80(1.38) 7.87(1.09) 7.87(0.96) 8.13(1.15) 8.40(0.61) 8.47(0.72) 8.53(0.88) 8.73(0.57)

01/15 00/15 04/15 02/15 02/15 05/15 04/15 05/15 07/15 04/15 03/15 08/15 04/15 04/15 08/15 10/15 10/15 07/15 09/15 09/15 07/15 12/15 06/15 09/15 12/15 14/15 14/15 14/15 14/15 13/15 14/15 14/15 15/15 14/15 14/15 13/15 15/15 15/15 15/15 14/15 14/15 15/15 14/15 14/15 15/15 15/15 15/15

0.13(0.05) 0.00(0.00) 0.80(1.51) 0.67(1.85) 0.20(0.54) 1.07(2.02) 1.13(1.86) 1.13(1.86) 1.20(1.47) 0.87(1.71) 0.33(0.70) 1.60(1.96) 1.07(2.21) 1.13(2.22) 1.80(2.34) 2.46(2.25) 2.47(2.28) 1.47(2.16) 1.93(1.88) 2.73(2.77) 1.73(2.35) 3.87(2.90) 1.07(1.84) 2.53(2.47) 3.73(2.70) 3.80(2.66) 5.27(2.65) 5.33(2.55) 5.80(2.20) 5.73(3.02) 5.87(3.01) 5.93(2.32) 6.80(2.34) 6.27(2.32) 6.60(3.34) 5.93(2.93) 7.40(1.93) 6.80(2.23) 7.87(1.36) 6.27(2.54) 7.13(2.60) 7.00(2.42) 7.87(2.28) 7.33(2.09) 8.13(2.00) 8.13(1.41) 8.27(1.73)

4.07(2.14) 4.20(1.68) 4.40(1.96) 5.20(1.56) 4.60(1.36) 4.53(2.25) 4.33(1.96) 4.60(1.99) 5.60(1.78) 5.13(1.31) 4.80(2.01) 5.40(1.86) 5.33(1.78) 4.67(2.15) 4.87(2.03) 6.27(1.88) 4.47(2.31) 5.93(1.06) 5.13(1.67) 5.93(1.34) 5.53(1.75) 6.00(1.51) 6.20(2.43) 6.00(1.82) 6.53(1.41) 6.00(2.22) 7.13(1.20) 6.00(1.93) 6.60(1.41) 6.67(1.81) 6.13(1.54) 7.00(1.41) 7.53(1.02) 7.47(1.26) 6.73(1.24) 6.93(1.48) 7.60(0.95) 6.93(1.29) 7.33(1.08) 7.40(1.25) 6.80(1.51) 7.93(1.34) 7.93(1.12) 8.40(0.71) 8.13(1.31) 7.93(1.73) 8.47(0.88)

Trigeminal Chemosensation

chemosensory responses rather than searching for “pure” odorants or “pure” irritants (both extremes, if indeed possible, no doubt being the exception rather than the rule). Both human and animal studies support the general view that the olfactory system is more sensitive to chemicals than the trigeminal system (Doty, 1975b, 1978; Henton et al., 1969; Walker and Jennings, 1991; Walker et al., 1979). Although repeated presentation of irritants to the latter system can increase the intensity of perceived irritation, CN V is still less sensitive and exhibits longer latencies to stimulation than CN I. Some attempts to separate odor responses from nasal pungency (i.e., irritant) responses entailed instructing subjects to focus on one type of sensation and ignore the other (e.g., Cometto-Muñiz and Hernández, 1990; Cometto-Muñiz et al., 1989). This strategy has merit for the study of suprathreshold sensations and finds validation in the aforementioned Doty et al. (1978) study, where the responses of anosmics and the trigeminal focus group show overall good agreement. Nevertheless, it cannot be easily applied to studies at the threshold level, especially if one decides to use a forcedchoice detection paradigm to minimize response biases. In this case, the testing of anosmic subjects becomes crucial. Assuming that, as a general rule, olfaction is more sensitive than the nasal trigeminal system, odor thresholds can be measured in normosmics, whereas trigeminal thresholds (unbiased by odor sensations) can be measured in anosmics. Within this scheme we can use the robust forced-choice procedure to measure both types of thresholds. Testing of anosmics represents an important tool in the functional separation of trigeminal and olfactory responses from the nose. We can, then, define, for any volatile odorant, the concentration below which the chemical would, on average, not be detected by smell, the concentration range in which the chemical will only evoke odor, and the concentration range above which it will evoke odor and concomitant trigeminal-related nasal pungency. This way of expressing the chemosensory potency of a substance provides very relevant information, particularly for applied issues, e.g., perfumery, food, indoor air quality, that goes well beyond the simple classification of the stimulus as “olfactory” or “trigeminal.” Another strategy to probe into trigeminal chemoreception sensitivity without olfactory interference consists in testing the ocular mucosa. A number of physiological and psychophysical techniques have been developed for the evaluation of human eye irritation, either to single compounds (e.g., Hempel-Jørgensen et al., 1997, 1999a; Kjaergaard et al., 1992) or to mixtures (e.g., HempelJørgensen et al., 1998, 1999b; Kjaergaard and Pedersen, 1989). The eyes, also served by the trigeminal nerve, can be tested for eye irritation thresholds. These thresholds

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could represent a surrogate response to nasal pungency thresholds as measured in anosmics. Comparative studies of nasal and ocular trigeminal chemosensitivity have provided a picture of overall similarity (Cometto-Muñiz and Cain, 1991, 1995, 1998; Cometto-Muñiz et al., 1998, 1999). This does not rule out that for a certain chemical or under certain conditions, the trigeminal response in the eyes might turn to be more sensitive or less sensitive than that in the nose. The issue will be treated in more detail in the next section. The point to stress is that any difference in trigeminal chemosensitivity between the nose and the eyes is dwarfed in comparison with the much higher sensitivity of the olfactory system (Cometto-Muñiz and Cain, 1995). A third experimental approach to separate the trigeminal from the olfactory response of the nose to airborne stimulants is to measure nasal localization or, perhaps more properly, nasal lateralization thresholds. In 1964, von Bèkesy concluded that localization of an odor to the right or left nostril can occur based on differences in time or intensity of presentation to the two nostrils. The stimuli employed, however, likely had considerable trigeminal impact (benzol, eucalyptus, cloves, and lavender). Early (e.g., von Skramlik, 1925), as well as more modern (e.g., Kobal et al., 1989; Schneider and Schmidt, 1967), studies clearly demonstrate that localization to one or the other nostril is only possible via trigeminal and not olfactory activation. Thus, nasal localization thresholds can be measured in normosmics and in anosmics using a forcedchoice procedure and provide a means to probe into nasal trigeminal sensitivity devoid of olfactory interference. As discussed later in the chapter, nasal localization thresholds in normosmics and anosmics fall close to each other. C.

Suprathreshold Responses to Nasal Irritants

Several lines of evidence suggest that the suprathreshold buildup in trigeminal sensation across increasing stimulus concentrations may be more rapid for the trigeminal than for the olfactory (or olfactory  trigeminal) nerve(s). For example, Doty (1975b) found the average exponent of power functions fitted to magnitude estimation data from anosmic patients to be larger than those fitted to analogous data from normal subjects for both methyl ethyl ketone (0.35 vs. 0.30) and furfural (0.55 vs. 0.45), although these differences were not statistically significant. Cain (1976) reported, for the odorant n-butanol, that the function relating perceived irritation to stimulus concentration is steeper than that relating perceived odor to stimulus concentration in normal subjects. Similarly, Murphy (1987) points out that the power function exponent for CO2 (a strong irritant at high concentrations) is 1.2, a value much larger than

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those observed for odorants commonly used in olfactory research (e.g., Berglund et al., 1971; Doty, 1975a). D.

Nasal Pungency, Eye Irritation, and Nasal Localization Thresholds Within Homologous Chemical Series

The above-mentioned strategies provide useful tools to separate trigeminal from olfactory human nasal chemosensory responses. The other component of the stimulusresponse interaction includes the myriad of diverse volatile agents that are capable of evoking odor and pungency. In order to expand our knowledge of the physicochemical basis for sensory potency of odorants and irritants, it is useful to resort to orderly arrays of stimuli for testing. Examples of such arrays can be found in homologous chemical series. Members of each of these series share a common chemical functional group (e.g., a hydroxyl group, HO, in alcohols and a carboxylic group, —COOH, in organic acids) and differ from the next one in the addition or subtraction of one carbon atom (with its corresponding hydrogens, e.g., —CH2—) to the carbon chain. One important feature within these series is that physicochemical properties (e.g., vapor pressure, solubility) change systematically across members. This allows one to correlate these orderly physicochemical changes with corresponding changes in chemosensory potency, as measured, for example, by detection threshold procedures. Carbon chain length constitutes, then, a convenient “unit of change” in reporting the results.* Employing the dual strategy of testing anosmics to separate nasal trigeminal from olfactory detection, and of employing homologous chemical series to create a continuum of physicochemical properties, Cometto-Muñiz and colleagues measured odor and trigeminal (“nasal pungency”) thresholds for homologous n-alcohols (ComettoMuñiz and Cain, 1990), acetate esters (Cometto-Muñiz and Cain, 1991), secondary and tertiary alcohols and acetates (Cometto-Muñiz and Cain, 1993), ketones (Cometto-Muñiz and Cain, 1993), alkylbenzenes (Cometto-Muñiz and Cain, 1994), aliphatic aldehydes and carboxylic acids (Cometto-Muñiz et al., 1998), and for selected terpenes (Cometto-Muñiz et al., 1998). Figure 2 shows how odor and nasal pungency thresholds change across homologous alcohols, acetates (including some secondary, tertiary, and branched members in both series), ketones, and alkylbenzenes. Figure 3 shows analogous

*

It should be noted, however, that numerous physicochemical properties co-vary (e.g., molecular weight with chain length), making it difficult to ascertain the specific element responsible for any psychophysical change.

data for homologous aldehydes and carboxylic acids, as well as for selected terpenes. It is apparent from these figures that, for all of the stimuli tested, nasal pungency thresholds lie between one and five orders of magnitude above odor thresholds along the concentration axis (log ppm by volume). Within the various homologous series, both nasal thresholds decline with carbon chain length, albeit at a faster rate for odor than for pungency (at least for the first few members of each series). In various series, odor thresholds tend to reach a plateau, whereas pungency thresholds show a “cut-off” effect. This means that, from a certain member on, a nasal pungency threshold cannot be reached with certainty in all repetitions of testing or in all the anosmics tested (even when presenting the stimulus at full vapor saturation). For the different series, the cut-off occurs at approximately the level of 1-octanol, octyl acetate, propyl benzene, octanal, and hexanoic acid (dashed lines, Figs 2 and 3). Two mechanisms have been proposed to explain the appearance of such cut-offs in homologous series (Franks and Lieb, 1990): a physical one whereby the series reaches a member whose maximum available quantity in the vapor phase falls below the threshold for the effect, and a biological one whereby the series reaches a member lacking a key property, e.g., becomes too big to fit a binding pocket or to interact with a receptor site. A systematic exploration of the extent and generality of cutoffs for nasal pungency thresholds along homologous series (e.g., Cometto-Muñiz et al., 1998) will provide a better understanding of the physicochemical basis for this chemosensory response. As mentioned above, measuring eye irritation thresholds constitutes an alternative experimental approach to probe into the trigeminal impact of volatile chemicals. As a general rule, eye irritation and nasal pungency thresholds fall well into register (Fig. 4) (Cometto-Muñiz and Cain, 1991, 1995). Nevertheless, there are cases where it is possible to measure a robust eye irritation threshold for an homolog that extends just beyond the cut-off point for nasal pungency (e.g., 1-octanol, octyl acetate) or for a certain chemical family member for which no nasal pungency threshold can be firmly measured (e.g., geraniol among the terpenes) (see Fig. 4). Also, measurement of continuous psychometric detectability functions for eye irritation (as opposed to measurement of a discrete threshold value) has indicated slightly higher eye than nasal trigeminal sensitivity for 1-butanol but no difference for 2-heptanone (Cometto-Muñiz et al., 1999). The implications and generality of these preliminary findings have not yet been investigated. Another alternative to assess trigeminal nasal chemosensitivity consists in measuring nasal localization (i.e., lateralization) thresholds. These thresholds tend to

Figure 2 Thresholds for nasal pungency (filled circles) and odor (empty circles) along homologous series of alcohols, acetates, ketones, and alkylbenzenes. Only primary and unbranched homologs are joined by a line. Stretches of dotted lines on nasal pungency thresholds show those members for which pungency begins to fade or cut off (see text). Bars, sometimes hidden by the symbol, indicate standard deviations.

Trigeminal Chemosensation 989

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Figure 3 Thresholds for nasal pungency (filled circles) and odor (empty circles) along homologous series of aliphatic aldehydes and carboxylic acids, as well as along selected terpenes. Stretches of dotted lines on nasal pungency thresholds show those members for which pungency begins to fade or cut off (see text). For six terpenes a threshold for nasal pungency could not be evoked. Only two of the remaining six terpenes (carene and cineole) evoked nasal pungency in all repetitions and all anosmics (Cometto-Muñiz et al., 1998). Bars, sometimes hidden by the symbol, indicate standard deviations.

overlap or fall slightly above nasal pungency thresholds (Fig. 5). Interestingly, roughly the same substances that occasionally or systematically fail to evoke a nasal pungency threshold (cut-off effect) also fail to produce a nasal localization threshold (Cometto-Muñiz and Cain, 1998; Cometto-Muñiz et al., 1998). When anosmics began to be systematically tested in search of nasal pungency thresholds devoid of odor biases, the question arose as to whether their trigeminal sensitivity was comparable to that of normosmics. Normosmics sometimes report considerable pungency at, or even below, the anosmics’ thresholds (Cometto-Muñiz and Cain, 1990; Gudziol et al., 2001; Kendal-Reed et al., 1998), and such reports tend to show high variability (Kendal-Reed et al., 2001). Without an experimental design that controls for response biases, this observation could reflect confusion in normosmics between strong odor and pungency, or it could reflect different trigeminal sensitivity between anosmics and normosmics. A study of chemo-somatosensory event–related potentials using the strongly trigeminal

Doty and Cometto-Muñiz

Figure 4 Thresholds for nasal pungency (in anosmics) (filled squares) and for eye irritation (in normosmics) (empty triangles) along homologous alcohols, acetates, ketones, and alkylbenzenes, and along selected terpenes. Bars, sometimes hidden by the symbol, indicate standard deviations.

Figure 5 Comparison of thresholds for odor (empty squares), nasal pungency (filled squares), and nasal localization (circles) along homologous n-alcohols and selected terpenes.

stimulus carbon dioxide found normosmics to produce a marginally (but significantly) larger peak-to-peak amplitude in the early P1N1 wave than did persons with olfactory impairment (Hummel et al., 1996). In contrast, psychophysical measurements employing forced-choice detection paradigms to minimize response biases have typically not observed significant differences between anosmics and normosmics in trigeminal chemoreception. For

Trigeminal Chemosensation

example, eye irritation thresholds measured in normosmics and anosmics employing homologous n-alcohols (ComettoMuñiz and Cain, 1998) and a group of terpenes (ComettoMuñiz et al., 1998) are similar in both groups. Detectability functions (which cover the range from chance detection to virtually perfect detection) for eye irritation evoked by 2heptanone, 1-butanol, butyl acetate, and toluene are also similar in normosmics and anosmics (Cometto-Muñiz et al., 1999, 2002). Nasal localization thresholds measured in normosmics and anosmics for n-alcohols (Cometto-Muñiz and Cain, 1998) and terpenes (Cometto-Muñiz et al., 1998) gave a picture of comparable nasal trigeminal sensitivity in both groups since a small advantage for normosmics failed to reach significance. Overall, such data suggest that any difference in trigeminal sensitivity between normosmics and anosmics, if in fact real, is likely small. Along these lines, a study of irritation-induced reflex changes in respiration in mice revealed no effect of anosmia on sensitivity to nasal irritation (Hansen et al., 1994). E. Quantitative Structure-Activity Relationships for Human Trigeminal Chemosensory Responses An important feature of the thresholds presented in Figures 2–5 is that they were all obtained with a uniform methodology. This included: (1) a two-alternative, forced-choice method with presentation of increasing concentrations (e.g., see Cometto-Muñiz and Cain, 1998), (2) a common criterion for threshold (five correct choices in a row), (3) the same stimulus-delivery technique (the commonly used “squeeze bottle” technique, a form of static olfactometry (Cain et al. 1992; Doty, 2000), and (4) calibration and direct measurement of the chemical stimulus in the vapor phase via gas chromatography. These characteristics provide a database with a robust intrinsic consistency, essential for comparative studies of quantitative structureactivity relationships (QSARs). Also of value in such studies is the selection of compounds from diverse chemical families (i.e., homologous series, terpenes) whose properties cover a wide range of values and varied systematically. 1. The Linear Solvation Model A number of descriptors have been used to correlate sensory irritation. Among them we can mention normal boiling point (Doty et al., 1978; Muller and Greff, 1984), adjusted boiling point (Roberts, 1986), saturated vapor pressure (Nielsen et al. 1990), the Ostwald solubility coefficient of the irritant (i.e., log L, where L is the concentration in solvent/concentration in gas phase) (Nielsen et al. 1992), and partition coefficients (specifically water-

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air and octanol-water) (Doty et al., 1978; Hau et al. 1999). Interestingly, all these descriptors are physicochemical parameters and do not involve the precise chemical structures of the compounds. Attempts have also been made to correlate olfactory potency (Anker and Jurs, 1990; Chastrette, 1997; Dravnieks, 1977; Egolf and Jurs, 1993; Hau and Connell, 1998; Rossiter, 1996; Schnabel et al. 1988; Shvets and Dimoglo, 1988). For the most part these relationships are difficult to interpret either chemically or mechanistically (Abraham, 1996). More recently, a linear solvation model was developed that not only provides strong statistical fit but that also conveys chemically and mechanistically meaningful information on both the stimulus (i.e., the chemical irritant) and the receptor biophase responsible for the sensory response. This model was developed by Abraham and has been successfully applied to a number of physicochemical and biochemical processes (Abraham, 1993a, b, 1996). The approach involves modeling, through a solvation equation, the transfer of the stimulus (i.e., the irritant) from the vapor phase (i.e., the air entering the nose) to a condensed (bio)phase (i.e., the nasal epithelium) and being distributed among various biophases, including the one where reception takes place (i.e., the free nerve endings of the trigeminal nerve). The equation takes the following general form: log SP
c  r·R2  s·π2H  a·H2  b·2H 1·log L16 (1) where log SP is the dependent variable. SP, in our case, is a sensory property—either the reciprocal of the nasal irritation or pungency threshold (1/NPT) or the reciprocal of the eye irritation threshold (1/EIT). We use the reciprocals simply because the larger the quantity, the more potent the irritant. The independent variables are excess molar refraction (R2), dipolarity/polarizability (πH2), overall or effective hydrogen-bond acidity ( 2H), overall or effective hydrogen-bond basicity (2H), and gas-liquid partition coefficient on hexadecane at 298 K (L16). The coefficients c, r, s, a, b, and 1 are found by multiple linear regression analysis. However, these are not simply fitted coefficients since they reflect the complementary properties of the biophase that must be receptive to the chemosensory stimulus. In this way, the independent variables provide a physicochemical characterization of the stimulus, whereas the coefficients provide a physicochemical characterization of the receptive biophase that will interact with that stimulus. The r-coefficient gives the tendency of the biophase to interact with the gaseous volatile through polarizability-type interactions, mostly via electron pairs. The s-coefficient is the measure of the

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biophase dipolarity/polarizability (because a dipolar stimulus will interact with a dipolar biophase and a polarizable stimulus will interact with a polarizable biophase). The acoefficient represents the complementary property to the chemical stimulus hydrogen-bond acidity and thus is a measure of the biophase hydrogen-bond basicity (because a stimulus that is a hydrogen-bond acid will interact with a basic biophase). Similarly, the b-coefficient is a measure of the biophase hydrogen-bond acidity (because a stimulus that is a hydrogen-bond base will interact with an acidic biophase). The l-coefficient is a measure of the biophase lipophilicity. 2. Nasal Irritation or Pungency Thresholds The values of nasal irritation or pungency thresholds depicted in Figure 2 served to develop the first version of a solvation equation specific for that sensory modality (Abraham et al., 1996). Later on, this equation proved successful not only to describe experimentally the obtained nasal pungency thresholds, but also to predict nasal pungency thresholds for volatile organic compounds (VOCs) that were not employed to develop it (i.e., new VOCs not included in the 34 used to build the equation) (ComettoMuñiz et al. 1998). These additional VOCs were the carboxylic acids and aliphatic aldehydes whose measured NPTs, shown in Figure 3, could be accurately predicted (with the exception of acetic acid). In turn, the incorporation of these additional NPTs allowed a refinement of the equation that reads as follows (Abraham et al., 1998a): H log (1/NPT)
8.519  2.154 πH 2 16 3.522 2  1.397 H 2  0.860 log L (2)

Here, NPT stands for nasal pungency threshold in ppm by volume. The number of data points (i.e., VOCs) used in the calculation of this equation was 43, the goodness of fit (r2) was 0.955, the overall SD in log (1/NPT) was 0.27, with an F statistic of 201. The respective standard deviations for each of the five coefficients were 0.274, 0.343, 0.215, 0.355, and 0.034. The t-test showed all listed coefficients (s, a, b, 1) to be significant at or below p = 0.001. In contrast, the r · R2 term of Eq. (1) failed to show significance and was discarded. In turn, Eq. (2) satisfactorily predicted NPTs for selected terpenes (Abraham et al., 1998d; Cometto-Muñiz et al., 1998) 3. Eye Irritation Thresholds The number of VOCs for which human eye irritation thresholds had been measured with a uniform methodology (Cometto-Muñiz and Cain, 1991, 1995; ComettoMuñiz et al. 1997) was considerably lower than that for

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nasal pungency thresholds. This situation hampered the development of a comparable solvation equation for the ocular trigeminal response. Nevertheless, a considerable body of data is available in terms of Draize eye scores in rabbits. Apart from the difference in species tested, the Draize test relies on testing the eyes with pure liquids, not vapors. A recent investigation described a straightforward calculation to convert Draize eye scores for pure organic liquids into scores for the corresponding vapors (Abraham et al., 1998c). With this tool at hand, it was shown, in a follow-up study, that Draize eye scores and eye irritation thresholds in humans can be combined into one QSAR (Abraham et al., 1998b), giving rise to the following equation: log (1/EIT)
7.918  0.482 R2  1.420 πH 4.025 2 H 16 H  1.219   0.853 log L (3) 2 2 where EIT stands for eye irritation threshold in ppm by volume. The number of data points (i.e., VOCs) used in the calculation of this equation was 54, the goodness of fit (r2) was 0.928, the overall SD in log (1/EIT) was 0.36, and the F statistic was = 124. The respective standard deviations for each of the six coefficients were 0.211, 0.307, 0.376, 0.404, 0.455, and 0.048. 4.

Applicability of the Solvation Equations

The equations that we have presented above for human nasal and ocular trigeminal chemoreception apply to what can be called “transport” processes, i.e. those processes where the key step is either the distribution of a stimulus between biophases or the rate of transfer of the stimulus from one biophase to another. The equations would not apply to stimuli that acted mainly through exact geometrical or conformational states, since changes of that sort in molecular structure would change the above mentioned physicochemical parameters only slightly (if at all), whereas, when relevant, stereochemical differences could change potency dramatically. Also, the described equations would underestimate the potency of chemically reactive substances (Abraham et al., 1990), i.e., those substances that can directly react with tissue via various mechanisms such as breaking of disulfide bonds, chemical reaction with a nucleophilic group, oxidation, etc. (Nielsen, 1991). In summary, the success of Eqs. (2) and (3) in correlating, and even predicting, human nasal pungency and eye irritation thresholds evoked by relatively nonreactive VOCs suggests that, indeed, the key step in the production of these chemosensory responses is the transport of the stimulus to a receptive biophase.

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F.

Trigeminal Reponses to Mixtures of Chemicals

In situations of everyday life, the chemical senses are stimulated by complex mixtures of substances, and the trigeminal system is certainly no exception. Despite this fact, little is known about how the trigeminal system, in particular, processes stimulation with chemical mixtures (for a discussion of chemical mixtures in olfaction, see Chapter 10). The topic can be addressed at the level of clearly suprathreshold stimulation and at the level of perithreshold stimulation. Again, separation of the trigeminal from the olfactory response becomes difficult to accomplish, particularly at the suprathreshold level, since almost all VOCs can stimulate both chemosensory channels. Early investigations with the pungent odorants formaldehyde and ammonia presented in binary mixtures have shown that at low, medium, and high concentrations of the components, the total nasal perceived intensity of the mixtures shifts from hypoadditivity to simple additivity and, finally, to hyperadditivity (Cometto-Muñiz et al., 1989). In other words, as the concentration of both compounds increased, the intensity of the corresponding mixtures turned out significantly lower than, equal to, and, finally, greater than the sum of their components. The outcome was interpreted to reflect a progressive trigeminal involvement, at the expense of olfactory involvement, in the overall response. A follow-up study with identical substances and mixtures, where subjects were asked to assess the olfactory (odor) and trigeminal (pungency) attributes of the evoked sensations, confirmed this interpretation since odor was always hypoadditive in mixtures whereas pungency was, mainly, additive, and even suggested hyperadditivity (ComettoMuñiz and Hernández, 1990). The approach of testing homologous chemical series for olfactory and trigeminal thresholds served as the basis for an investigation comparing odor, nasal pungency, and eye irritation thresholds for five chemical mixtures against the same thresholds for the individual components of the mixtures (Cometto-Muñiz et al., 1997). The five mixtures tested included two three-component mixtures, two six-component mixtures, and one nine-component mixture. Given the significant role that lipophilicity of the stimulus plays in both olfactory and trigeminal stimulation, the constituents of the mixtures were selected such that one of the three-component mixtures and one of the six-component mixtures contained relatively low molecular weight, low-lipophilicity homologs, whereas the remainding three-and six-component mixtures contained relatively high molecular weight, high-lipophilicity homologs. The nine-component mixture contained both types of compounds. The outcome revealed various degrees of stimulus agonism (i.e., additive effects) for the olfactory modality and for the two trigeminal modal-

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ities. Degree of agonism increased with number of components and with the lipophilicity of such components, particularly for the trigeminal responses. In fact, synergistic stimulus agonism characterized the eye irritation response to the most complex (the nine-component) and the most lipophilic (one of the six-component) mixtures. Instead of measuring a threshold according to a fixed criterion of response, a recent study focused on measuring complete detectability (i.e., psychometric) functions for 1-butanol and 2-heptanone singly and in binary mixtures of varying proportions (Cometto-Muñiz et al., 1999). Again, the chemosensory responses comprised odor, nasal pungency, and eye irritation. Knowledge of the detectability function for each individual substance allowed the

Figure 6 Showing how a single detectability function for odor (upper panel), nasal pungency (middle panel), and eye irritation (lower panel) fits the detection data for two single chemicals (2-heptanone, filled circles; 1-butanol, filled squares) and their various binary mixtures (all other symbols) when all stimuli are expressed in concentrations units of one chemical (here, 2-heptanone) via the concept of sensory equivalent concentrations (see text). The equation that best fits the experimental data for each sensory modality is shown on each graph.

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calculation of sensory equivalent concentrations, i.e., concentrations of the two chemicals that produced the same percentage of detectability. In turn, this made it possible to express all stimuli, single and mixtures, in concentration units of one (or the other) compound. The outcome showed that a single function could fit these combined data for each sensory endpoint, lending support, as a first approximation, to the notion of chemosensory agonism (in the sense of dose additivity) between the members of binary mixtures presented at perithreshold levels (Fig. 6). Very recent studies on the nasal pungency and eye irritation detectability of mixtures of butyl acetate and toluene have confirmed the outcome of complete sensory agonism in mixtures of relatively low detectability (but still above chance detection) and have indicated a partial loss of sensory agonism in mixtures of relatively high detectability (but still below perfect detection) (Cometto-Muñiz et al., 2001). G. Psychophysical Interactions between the Olfactory and Trigeminal Systems In light of the physiological interactions between the olfactory and trigeminal systems that were mentioned earlier, one might expect that stimulation of CN V might alter sensations derived from stimulation of CN I. There is some mention of this in the early literature. Thus, as noted by Cain and Murphy (1980), it was reported by Alexander Bain in 1868 that “if a current of carbonic acid accompanies an odour, the effect (odour) is arrested.” In 1930, Katz and Talbert reported that the irritation property of some odorants with both odor and irritative properties predominates at high concentrations, suggesting that CN V stimulation is masking CN I–mediated perception. More recent support for this concept comes from a study by Cain and Murphy (1980). These investigators had eight subjects judge the perceived magnitude (i.e., intensity) of four concentrations of n-amyl butyrate, four concentrations of carbon dioxide, and all 16 combinations of these stimuli. The stimuli were presented to one nostril only, and magnitude estimates were made using the method of magnitude estimation (where subjects assign numbers relative to the perceived magnitude of the stimuli) (see Chapter 10). The subjects first judged the magnitude of the overall sensory experience, and then of the odor and irritative components. A similar experiment, in which the CO2 was presented to one nostril and the odorant to the other, was also performed using 10 subjects. Overall, the perceived magnitude of the n-amyl butyrate appeared to be suppressed by the CO2, and vice versa (i.e., the magnitude of irritation was depressed by some concentrations of n-amyl butyrate), even when the odorant and irritative stimuli were presented to separate

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nares. This suggested to these authors that the locus of interaction was in the central nervous system. H.

Environmental Applications of Psychophysical Measurements

Separation of trigeminal from olfactory chemosensory responses evoked by airborne chemicals constitutes a central aspect to many environmental areas. Two of the most relevant areas involve occupational or industrial exposures and nonindustrial (including residential) exposures. Regarding industrial exposures, it has been estimated that about 40% of the threshold limit values (TLVs) established by the American Conference of Governmental Industrial Hygienists (ACGIH) (American Conference of Governmental Industrial Hygienists, 1994) for industrial and occupational exposures are based on sensory irritation (Alarie, 1981). This includes, primarily, irritation of the eyes, nose, and throat. In these environments the substance (or group of substances) that could be responsible for symptoms of sensory irritation is typically known from the materials and processes used in that particular setting. The critical information consists in establishing the concentrations at which these compounds singly or in mixture begin to evoke irritative responses, as well as noticeable odors. Once such levels are established, TLVs can be chosen that avoid untoward symptoms. An animal bioassay based on the respiratory frequency depression of mice exposed to irritants (Alarie, 1966, 1973) has been used to extrapolate probable human irritation responses (Alarie, 1981), but the suitability of the test has been questioned (Bos et al., 1992). The strategies described in this chapter to separate olfactory from trigeminal responses provide the means to assess directly in humans the irritative properties of airborne chemicals, at least for those whose health effects of concern at environmentally realistic concentrations rest on sensory irritation. Regarding nonindustrial exposures, there is growing concern about the high incidence of occurrence of certain health and toxicological effects in indoor environments such as office buildings, schools, and even residential buildings. A percentage of occupants of these indoor spaces report such symptoms as headache, difficulty in concentration, drowsiness, and lassitude, with symptoms of sensory irritation of eyes, nose, and throat figuring prominently (Cometto-Muñiz and Cain, 1992). This wide array of symptoms has been referred to as the “sick building syndrome” (Apter et al., 1994). It is likely that a number of chemical and physical agents might be involved in the production of these symptoms, but it is recognized that VOCs play an important role (Hodgson et al. 1994; Kostiainen, 1995; Rothweiler and Schlatter, 1993). Given

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the prevalence of trigeminally mediated responses in problematic indoor environments, studies of this chemoreceptor system can contribute substantially to the understanding, prevention, and control of their appearance. Among the most relevant issues are (1) understanding the physicochemical basis for the production of sensory irritation by airborne chemicals so that better and less offending materials can be employed within buildings (e.g., those used in furnishings, carpets, paints, etc.) and (2) knowledge of how individual members of complex mixtures produce their combined trigeminal impact, allowing for the translation of chemical measurements into corresponding irritative sensory responses. It should be pointed out that the trigeminal and olfactory thresholds cited here have been measured with the “squeeze bottles” (Amoore and Ollman, 1983; Cain, 1989; Doty, 2000). As shown above, this simple and convenient delivery system produced a robust picture of relative chemosensory potency within and across homologous series and chemical families. Nevertheless, the thresholds obtained might not be, in absolute terms, the values produced under environmentally realistic conditions such as whole-body exposures. A very recent investigation of nasal pungency thresholds in homologous alcohols, acetates, and ketones has found that an improved delivery system (based on glass vessels) produced thresholds uniformly lower than those produced via the plastic squeeze bottles by a factor of 4.6, although the relative potency of the homologs remained essentially unaltered (Cometto-Muñiz et al., 2000). Additional studies should provide insight into the relationship between chemosensory thresholds measured via various delivery systems and those obtained in whole-body exposures. IV.

SUMMARY

In this chapter, basic aspects of the anatomy, physiology, and chemical responsiveness of the human trigeminal system were reviewed. Additionally, studies demonstrating that ocular trigeminal sensitivity may be similar to intranasal trigeminal sensitivity were noted. Among other topics addressed were the functional dissociation of CN I and CN V responses, the application of modern structure-activity studies in predicting irritative responses of volatiles, and an assessment of potential functional interactions between CN I and CN V. It is apparent from the material reviewed in this chapter that the trigeminal system is generally less sensitive to volatile agents than the olfactory system, and very few, if any, odorants fail to stimulate CN V at high enough concentrations. It is pointed out that knowledge of the factors responsible for nasal and ocular irritation is not only of theoretical interest, but of practical interest as well, particularly in relation to environmental concerns.

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V.

ACKNOWLEDGMENTS

This work was supported, in part, by Grants PO1 DC 00161 (RLD), RO1 DC 04278 (RLD), RO1 DC 02974 (RLD), RO1 AG 27496 (RLD), R29 DC 02741 (JEC-M), and RO1 DC 02741 (JEC-M) from the National Institutes of Health, Bethesda, MD, as well as by a grant from the Center for Indoor Air Research (JEC-M). We thank Dr. Michael H. Abraham for his insights and leading role in the analysis of structure-activity relationships described in this chapter. REFERENCES Abraham, M. H. (1993a). Application of solvation equations to chemical and biochemical processes. Pure Appl. Chem. 65:2503–2512. Abraham, M. H. (1993b). Scales of solute hydrogen-bonding: their construction and application to physicochemical and biochemical processes. Chem. Soc. Rev. 22:73–83. Abraham, M. H. (1996). The potency of gases and vapors: QSARs—anesthesia, sensory irritation, and odor. In Indoor Air and Human Health, 2nd ed., R. B. Gammage and B. A. Berven (Eds.). CRC Lewis Publishers, Beca Raton FL, pp. 67–91. Abraham, M. H., Andonian-Haftvan, J., Cometto-Muñiz, J. E., and Cain, W. S. (1996). An analysis of nasal irritation thresholds using a new solvation equation. Fundam. Appl. Toxicol. 31:71–76. Abraham, M. H., Kumarsingh, R., Cometto-Muñiz, J. E., and Cain, W. S. (1998a). An algorithm for nasal pungency thresholds in man. Arch. Toxicol. 72:227–232. Abraham, M. H., Kumarsingh, R., Cometto-Muñiz, J. E., and Cain, W. S. (1998b). Draize eye scores and eye irritation thresholds in man can be combined into one quantitative structure-activity relationship. Toxicol. in Vitro 12: 403–408. Abraham, M. H., Kumarsingh, R., Cometto-Muñiz, J. E., and Cain, W. S. (1998c). A quantitative structure-activity relationship (QSAR) for a Draize eye irritation database. Toxicol. in Vitro 12:201–207. Abraham, M. H., Kumarsingh, R., Cometto-Muñiz, J. E., Cain, W. S., Rosés, M., Bosch, E., and Díaz, M. L. (1998d). The determination of solvation descriptors for terpenes, and the prediction of nasal pungency thresholds. J. Chem. Soc. Perkin Trans. 2:2405–2411. Abraham, M. H., Whiting, G. S., Alarie, Y., Morris, J. J., Taylor, P. J., Doherty, R. M., Taft, R. W., and Nielsen, G. D. (1990). Hydrogen bonding 12. A new QSAR for upper respiratory tract irritation by airborne chemicals in mice. Quant. Struct.Act. Relat. 9:6–10. Alarie, Y. (1966). Irritating properties of airborne materials to the upper respiratory tract. Arch. Environ. Health 13:433–449. Alarie, Y. (1973). Sensory irritation by airborne chemicals. CRC Crit. Rev. Toxicol. 2:299–363.

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Doty and Cometto-Muñiz Brown, J. W. (1974). Prenatal development of the human chief sensory trigeminal nucleus. J. Comp. Neurol. 156:307–335. Bryant, B., and Silver, W. L. (2000). Chemesthesis: The common chemical sense. In The Neurobiology of Taste and Smell, T. E. Finger and W. L. Silver (Eds.). Wiley-Liss, New York, pp. 73-100. Burrow, A., Eccles, R., and Jones, A. S. (1983). The effects of camphor, eucalyptus and menthol vapor on nasal resistance to airflow and nasal sensation. Acta Otolaryngol. 96:157–161. Cain, W. S. (1974). Contribution of the trigeminal nerve to perceived odor magnitude. Ann. NY Acad. Sci. 237:28–34. Cain, W. S. (1976). Olfaction and the common chemical senses: Some psychophysical contrasts. Sensory Proc. 1:57–67. Cain, W. S., and Murphy, C. (1980). Interaction between chemoreceptive modalities of odour and irritation. Nature 284:255–257. Cain, W. S. (1989). Testing olfaction in a clinical setting. Ear Nose Throat J. 68:316–328. Cain, W. S., Cometto-Muñiz, J. E., and de Wijk, R. A. (1992). Techniques in the quantitative study of human olfaction. In Science of Olfaction, M. J. Serby and K. L. Chobor (Eds.). Springer-Verlag, New York, pp. 279–308. Caterina, M. J., Schumacher, M. A., Tominaga, M., Rosen, T. A., Levine, J. D., and Julius, D. (1997). The capsaicin receptor: a heat-activated ion channel in the pain pathway. Nature 389:816–824. Cauna, N., and Hinderer, K. H. (1969). Fine structure of blood vessels of the human nasal respiratory mucosa. Ann. Otol. Rhinol. Laryngol. 78:865–885. Cesare, P., and McNaughton, P. (1997). Peripheral pain mechanisms. Curr. Opin. Neurobiol. 7:493–499. Chastrette, M. (1997). Trends in structure-odor relationships. SAR QSAR Environ. Res. 6:215–254. Cometto-Muñiz, J. E. (2001). Physicochemical basis for odor and irritation potency of VOCs. In Indoor Air Quality Handbook, J. D. Spengler, J. Samet, and J. F. McCarthy (Eds.). McGrawHill, New York, pp. 20.1–20.21. Cometto-Muñiz, J. E., and Cain, W. S. (1990). Thresholds for odor and nasal pungency. Physiol. Behav. 48:719–725. Cometto-Muñiz, J. E., and Cain, W. S. (1991). Nasal pungency, odor, and eye irritation thresholds for homologous acetates. Pharmacol. Biochem. Behav. 39:983–989. Cometto-Muñiz, J. E., and Cain, W. S. (1992). Sensory irritation. Relation to indoor air pollution. Ann. NY Acad. Sci. 641:137–151. Cometto-Muñiz, J. E., and Cain, W. S. (1993). Efficacy of volatile organic compounds in evoking nasal pungency and odor. Arch. Environ. Health 48:309–314. Cometto-Muñiz, J. E., and Cain, W. S. (1994). Sensory reactions of nasal pungency and odor to volatile organic compounds: The alkylbenzenes. Am. Ind. Hyg. Assoc. J. 55:811–817. Cometto-Muñiz, J. E., and Cain, W. S. (1995). Relative sensitivity of the ocular trigeminal, nasal trigeminal, and olfactory systems to airborne chemicals. Chem. Senses 20:191–198. Cometto-Muñiz, J. E., and Cain, W. S. (1998). Trigeminal and olfactory sensitivity: Comparison of modalities and methods of measurement. Int. Arch. Occup. Environ. Health 71:105–110.

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Doty and Cometto-Muñiz Keele, C. A. (1962). The common chemical sense and its receptors. Arch. Int. Pharmacodyn. Ther. 139:547–557. Kendal-Reed, M., Walker, J. C., and Morgan, W. T. (2001). Investigating sources of response variability and neural mediation in human nasal irritation. Indoor Air 11:185–191. Kendal-Reed, M., Walker, J. C., Morgan, W. T., LaMacchio, M., and Lutz, R. W. (1998). Human responses to propionic acid: I. Quantification of within- and between-participant variation in perception by normosmics and anosmics. Chem. Senses 23:71–82. Kevern, E. B., Murphy, C. L., Silver, W. L., Wysocki, C. I., and Meredith, M. (1986). Nonolfactory chemoreceptors of the nose: Recent advances in understanding the vomeronasal and trigeminal systerns. Chem. Senses 11:119–133. Kjærgaard, S. K., and Pedersen, O. F. (1989). Dust exposure, eye redness, eye cytology and mucous membrane irritation in a tobacco industry. Int. Arch. Occup. Environ. Health 61:519–525. Kjærgaard, S. K., Pedersen, O. F., and Mølhave, L. (1992). Sensitivity of the eyes to airborne irritant stimuli: Influence of individual characteristics. Arch. Environ. Health 47:45–50. Kjærgaard, S. K., Pedersen, O. F., Taudorf, E., and Mølhave, L. (1990). Assessment of changes in eye redness by a photographic method and the relation to sensory eye irritation. Int. Arch. Occup. Environ. Health 62:133–137. Kobal, G., Van Toller, S., and Hummel, T. (1989). Is there directional smelling? Experientia 45:130–132. Kostiainen, R. (1995). Volatile organic compounds in the indoor air of normal and sick houses. Atmos. Environ. 29:693–702. Kratschmer, F. (1870). Über Reflexe von der Nasenschleimhaut auf Athmung und Kreislauf. Sitz. Acad. Wiss. Wien. 62:147–170. Lehman, C. (1991). The effect of anesthesia of the chorda tympani nerve on taste perception in humans. Doctoral dissertation, Yale University, New Haven, CT. Lundblad, L., Lundberg, J. M., Brodin, E., and Anggard, A. (1983). Origin and distribution of capsaicin-sensitive substance P-immunoreactive nerves in the nasal mucosa. Acta Otolaryngol. 96:485–493. Lundblad, L., Brodin, E., Lundberg, J. M., and Anggard, A. (1985). Effects of nasal capsaicin pretreatment and cryosurgery on sneezing reflexes, neurogenic plasma extravasation, sensory and sympathetic neurons. Acta Otolaryngol. 100:117–127. Magendie, F. (1824). Le nerf olfactif est-il i’organe de i’odorat? Experiences sur cette question. Magendies J. Physiol. Exp. Pathol. 4:169–176. Martin, J. H., and Jessell, T. M. (1991). Modality coding in the somatic sensory system. In Principles of Neural Science, 3rd ed. E. R. Kandel, J. H. Schwartz and T. M. Jessell (Eds.). Elsevier, New York, pp. 341–352. McCleskey, E. W., and Gold, M. S. (1999). Ion channels of nociception. Annu. Rev. Physiol. 61:835–856. Muller, J., and Greff, G. (1984). Recherche de relations entre toxicité de molécules d’intérèt industriel et propriétés physicochimiques: test d’irritation des voies aériennes supérieures appliqué à quatre familles chimiques. Food Chem. Toxicol. 22:661–664.

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48 The Structure and Function of the Nervus Terminalis Marlene Schwanzel-Fukuda and Donald W. Pfaff The Rockefeller University, New York, New York, U.S.A.

I. A.

INTRODUCTION

study the embryonic history of the nervus terminalis. Locy observed that as it developed it extended as a “group of closely packed cells” between the epithelium of the olfactory pit and the rostral forebrain “on each side of the neuropore, in close connection with elements of the disappearing neural crest.” He suggested that the neural crest cells may contribute, at least in part, to its formation. The name nervus terminalis was given to it by Locy (1905), who observed that the central terminations of this nerve are nearly always found in the lamina terminalis, the site of closure of the anterior neuropore. The largest of the ganglia of the nervus terminalis, the ganglion terminale, is usually seen near the point of entry of the central roots of this nerve into the forebrain. It was named by Huber and Guild (1913), whose study in rabbits provided one of the first and most comprehensive descriptions of the nervus terminalis in mammals (Fig. 1). The nervus terminalis was studied intensively from the time of its discovery in the late nineteenth century through the early part of this century. Comparative anatomists, who were interested in vertebrate cephalogenesis, recognized in the nervus terminalis the features of a phylogenetically ancient nerve (Brookover, 1910; Ayers, 1919). The nervus terminalis remains remarkably constant in its anatomy and in its position on the nasal septum and forebrain, across all species of vertebrates including fishes (Sheldon, 1909; Brookover, 1910; Brookover and Jackson, 1911; Demski, 1986; von Bartheld and Myer, 1986; Demski et al., 1987, Munz and Claas, 1987), amphibia (Herrick, 1909; McKibben, 1911; Wirsig and Getchell, 1986; Muske

Historical Background

The nervus terminalis, or terminal nerve, is a plexiform, ganglionated cranial nerve of unknown function found in the nose of most, if not all, vertebrates, including humans. Located rostral to the 12 cranial nerves and discovered after they were named, it is also called the zeroeth cranial nerve. In most vertebrates, the nervus terminalis shares a common origin with the vomeronasal nerve in the epithelium of the medial olfactory pit, which gives rise to the vomeronasal organ. The peripheral part of the nervus terminalis, in the form of a loose plexus, crosses the nasal mucosa and courses through the cribriform plate of the ethmoid bone medial to the olfactory and vomeronasal nerves (Fig. 1). It can be distinguished from these nerves by the presence of ganglia at nodal points in the nerve plexus and ganglion cells intermingled among the nerve fibers along its course. The intracranial part of the nervus terminalis lies along the ventromedial surfaces of the olfactory bulbs. The central part, consisting of three or four fine rootlets, enters the forebrain caudal to the medial sides of the olfactory bulbs. The first description of the nervus terminalis is attributed to Gustav Fritsch (1878), who noted it, briefly, as a “supernumerary cranial nerve” in the brain of a shark (Galeus canis). Felix Pinkus (1894) gave the first detailed description of the “new nerve” in Protopterus, and for many years it was known as the nerve of Pinkus. William Locy (1905), in Squalis acanthas embryos, was the first to 1001

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Figure 1 Graphic reconstruction of the right side of the olfactory bulb and stalk, olfactory and vomeronasal nerves, and the nervus terminalis, based on a series of sagittal sections of a head of a one-day-old rabbit. The nervus terminalis (NT) is shown in jet black, and the ganglion cells along its course are indicated as small open circles. The olfactory (ON) and vomeronasal nerves (VN) are given in outline. F, forebrain; OB, olfactory bulb; AOB, accessory olfactory bulb; CP, cribriform plate; NT, nervus terminalis; GT, ganglion terminale; RNT, roots of the nervus terminalis; SNT, septal distribution of the nervus terminalis; VNO, vomeronasal organ; VN, vomeronasal nerve; ON, olfactory nerve. (Adapted from Huber and Guild, 1913.)

and Moore, 1988; Hoffman and Myer, 1989), and mammals (Johnston, 1913, 1914; Huber and Guild, 1913; Larsell, 1918, 1950; Bojsen-Moller, 1975; SchwanzelFukuda et al., 1985, 1987), including aquatic mammals (Buhl and Oelschlager, 1986; Demski et al., 1990) and humans (DeVries, 1905; Dollken, 1909; Johnston, 1913, 1914; Brookover, 1914; McCotter, 1915; Pearson, 1941; O’Rahilly, 1972; Bossy 1980; Oelschlager, 1987). It is even present in animals that do not have an olfactory or a vomeronasal system, such as the porpoise (Johnston, 1914), and in chickens (von Bartheld et al., 1987; WirsigWiechmann, 1990; Norgren and Lehman, 1990) and bats (Brown, 1987; Oelschlager, 1988), which lack only the vomeronasal system. The fact that the nervus terminalis is observed in adult animals indicates that it is not a transient embryological structure, and its presence in humans suggests that it is a highly conserved part of the nervous system (Fig. 2).

B. Early Speculations on the Function of the Nervus Terminalis The function of the nervus terminalis is still the subject of speculation, much as it was 100 years ago (for a review see

Demski, 1993). It has always been difficult to isolate and test for function (Tucker, 1971), due in part to the close association of its branches with those of the vomeronasal, olfactory, and trigeminal nerves. Most of the functions postulated for this nerve were based on morphological observations rather than experimental evidence. Both sensory and autonomic functions have been proposed for the nervus terminalis, based on its origin from the olfactory epithelium and the resemblance of its ganglion cells to sympathetic neurons (Huber and Guild, 1913; Pearson, 1941) (see Figures 1, 3, 4, 5). Hardesty (1914) suggested that the sympathetic cells migrated into the ganglia of the nervus terminalis from a forward growth of the sympathetic trunk into the region of the head. Stewart (1920), using albino rats, examined this possibility and that of a possible origin from the sphenopalatine (pterygopalatine) ganglion, since this ganglion receives preganglionic fibers from the facial nerve and distributes postganglionic (parasympathetic) fibers to the nasal cavity. He concluded that all of the ganglion cells of the nervus terminalis arose from the epithelium of the olfactory pit. Larsell (1918), suggested that the ganglion cells may migrate into the nasal mucosa along branches of the trigeminal nerve. Several investigators (Johnston, 1914; Larsell, 1918, 1950; Pearson, 1941; Bojsen-Moller, 1975)

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considered an autonomic (vasomotor) function for some of the ganglion cells on the basis of the close proximity of nervus terminalis fibers to Bowman’s glands and to the blood vessels of the nasal mucosa. Observations of afferent-type terminations of the nervus terminalis in the olfactory epithelium, including that of the vomeronasal organ, led to the speculation that it contains a sensory component as well (Johnston, 1913; Larsell, 1918, 1950; Pearson, 1941; Bojsen-Moller, 1975). The presence of the nervus terminalis in animals that lack both an olfactory and a vomeronasal system appears to rule out a main or accessory olfactory function for this nerve. Since a function could not be determined by experimental means for the nervus terminalis, interest in its study waned. It came to be considered the vestige of an ancient nerve whose function was lost or superseded by other parts of the nervous system over the long course of vertebrate evolution (Locy, 1905; Huber and Guild, 1913). Reference to the nervus terminalis was dropped from many neuroanatomy and embryology textbooks after 1950. C. Immunocytochemical Techniques and New Data on the Nervus Terminalis The renewal of interest in the nervus terminalis can probably be attributed to new information about it gained through modern immunocytochemical techniques. The discovery, in mammals, that a population of ganglion cells and fibers of the nervus terminalis contain luteinizing hormone–releasing hormone (LHRH), an essential hormone for reproductive function, brought to light a system of LHRH neurons located outside of the hypothalamus and preoptic area (Schwanzel-Fukuda and Silverman, 1980; Jennes and Stumpf, 1980; Schwanzel-Fukuda et al., 1981; Witkin and Silverman, 1983; Witkin, 1987), and provided a marker for the nervus terminalis. The startling discovery that the cells that synthesize and release LHRH actually originate in the nose, in the same part of the olfactory pit as that which gives rise to the vomeronasal and terminalis nerves, and that the LHRH cells migrate into the brain along branches of these nerves (Schwanzel-Fukuda and Pfaff, 1988, 1989; Wray et al., 1989a, b) reinforced experimental evidence of a strong link between the olfactory and reproductive systems (Parkes and Bruce, 1961; Keverne, 1978; Keverne and de la Riva, 1982; Singer et al., 1987). These data, associating the nervus terminalis with a physiologically important neuropeptide and the evidence that these findings are generalized across species, including rats (Schwanzel-Fukuda et al., 1985; Zheng et al., 1988; Daikoku-Ishido et al., 1991), opossums (Schwanzel-Fukuda et al., 1987; Zheng et al., 1990a, b), chickens (Norgren and Lehman, 1991; Murakami et al.,

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1991; Sullivan and Silverman, 1991; Kuenzel and Blahser, 1991), fish (Halpern-Sebold and Schreibman, 1983; Munz and Claas, 1987; Oka and Ichikawa, 1990; Demski and Wright, 1991; Wright and Demski, 1991; Parhar et al. 1995, 1996), amphibia (Wirsig and Getchell, 1986; Muske and Moore, 1987, 1988, 1990; Murakami et al., 1992), dolphins (Demski et al., 1990), and primates (Witkin, 1987; Ronnekleiv and Resko, 1990), including humans (Schwanzel-Fukuda et al., 1989, 1996; Patel et al., 1991; Boehm et al., 1994; Kim et al., 1999) (See Fig. 2), have stimulated new research on this interesting cranial nerve. An exception to the generalization about the presence of a terminal nerve with associated GnRH immunoreactive cell bodies and axons in all vertebrate species may be found in the silver lamprey (Eisthen and Northcutt, 1996). These investigators observed GnRH immunoreactivity in the brain but not in the terminal nerve or in any structure within the nasal regions. Tobet and coworkers (Tobet et al., 1997) made a similar observation in adult lampreys, and they cited research that identified two distinctive forms of GnRH that stimulate the pituitary-gonadal axis (Deragon et al., 1994). Interestingly, Tobet speculates that the form of GnRH found in the central nervous system in lamprey may

Figure 2 Inferior surface of the adult human brain illustrating the components of the olfactory system. The tract of the nervus terminalis (NT) lies medial to the tract of the olfactory nerve (OT) coursing along the surface of the gyri recti. Anteriorly, it is intimately associated with the olfactory bulb (OB). (From Kim et al., 1999.)

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represent a phylogenetically older pattern of GnRH control of the pituitary-gonadal axis. He speculates that in other species, including mammals, the newer forms of GnRH may have come to be expressed in neurons that arise from the olfactory placode (Tobet et al., 1997).

II. THE ANATOMY OF THE NERVUS TERMINALIS A. Histochemical and Immunocytochemical Procedures The nervus terminalis can be distinguished from the vomeronasal, olfactory, and trigeminal nerves by using histological techniques that differentially stain the nervus terminalis fibers a deep blue-black. These methods include a modified pyridine-silver method (Huber and Guild, 1913; Larsell, 1918; Pearson, 1941) and cholinesterase methods or staining with osmium tetroxide and periodic acid (Bojsen-Moller, 1975). Immunocytochemical procedures with antiserum to LHRH reveal a population of ganglion cells and fibers in the nervus terminalis and can be used to trace the course of this nerve from the nose into the forebrain (SchwanzelFukuda and Silverman, 1980, Schwanzel-Fukuda et al., 1985; Schwanzel-Fukuda et al., 1987). LHRH, a decapeptide, is a highly conserved molecule, virtually unchanged through 500 million years of vertebrate evolution. Certain amino acid sequences in the LHRH molecule are found in common with those seen in alpha yeast mating factor (Sherwood, 1987). Alpha yeast mating factor has been shown to evoke the release of luteinizing hormone (LH) and follicle-stimulating hormone (FSH) from gonadotropes of the anterior pituitary gland (Loumaye et al., 1982). In some nonmammalian species of vertebrates, including teleosts (Stell et al., 1984; Ekstrom et al., 1988), amphibians (Wirsig and Getchell, 1986), and chickens (Wirsig Weichmann, 1990), the tetrapeptide FMRF-amide has been localized in cells and axons of the nervus terminalis. Stell and coworkers (Stell et al., 1984) showed in teleosts that FMRF-amide is colocalized with LHRH in ganglion cells of the nervus terminalis and that some of these neurons project to the retina. FMRF-amide (molluscan cardioexcitatory peptide) is a tetrapeptide that was first isolated from molluscan ganglia (Price and Greenberg, 1977). Similar to LHRH, FMRF-amide is a highly conserved molecule. It is present in cells and fibers of the nerve net of coelenterates, which represents the simplest form of nervous system in the animal kingdom (Grimmelikhuijzen et al., 1992).

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B. The Nervus Terminalis in Mammals: General Description The nervus terminalis, throughout its course in the nose, is located just lateral to midline and medial to the vomeronasal and olfactory nerves, i.e., deeper in the mucosa covering the nasal septum than these nerves. Fibers of the nervus terminalis, in the form of a loose plexus and accompanied by ganglion cells, can be traced from the epithelium of the vomeronasal organ into the septal mucosa (Huber and Guild, 1913; Larsell, 1918, 1950; Bojsen-Moller, 1975). Many of these fibers closely parallel the branches of the vomeronasal nerve and send communicating branches to this nerve as it courses through the septal mucosa (Fig. 1). Other branches of the nervus terminalis in the nose are distributed to the dorsal and anterior parts of the septal mucosa, where they accompany the anterior ethmoidal branch of the trigeminal nerve (Bojsen-Moller, 1975). In mice, rats, and hamsters, Gruneberg (1973) described a small ganglion in the anterior part of the nasal mucosa, near the vestibule, which he believed to be a part of the nervus terminalis. Small ganglia are seen at nodal points of the nervus terminalis plexus in the nose, and single cells or small clusters of ganglion cells are found interspersed among the fibers of this nerve in the septal mucosa (Larsell, 1950). Many of these fibers are found in close proximity to the nasal glands and blood vessels. The nervus terminalis emerges from the nose through the cribriform plate of the ethmoid bone medial to the olfactory and the vomeronasal nerves, either side of the midline. It forms a plexus along the medial surface of the olfactory bulb, which leads into the major ganglion of the nervus terminalis, the ganglion terminale, usually found just caudal to the ventromedial surface of the olfactory bulb (Fig. 1). From this point, a branch of the nervus terminalis joins the vomeronasal nerve as it runs to its terminus in the accessory olfactory bulb. The remaining fibers of the plexus organize into three or four fine rootlets that enter the forebrain caudal to the medial part of the olfactory bulb, following perforating branches of the anterior cerebral artery (Larsell, 1918, 1950). These central roots have been traced into the olfactory tubercle, the septal, the precommissural and the preoptic areas of the brain (Huber and Guild, 1913; Larsell, 1950; Bojsen-Moller, 1975). Larsell (1918, 1950) described bipolar and multipolar cells in the ganglia of the nervus terminalis. Using pyridine silver and gold chloride techniques, he observed that the axons of these cells showed two types of endings in the walls of blood vessels. Type I consisted of delicate, unmyelinated, varicosed fibers that penetrated the muscular walls of the blood vessels and ended in small

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varicosities on the smooth muscle cells. He considered these to be vasomotor. The endings that distinguished the broad, short, Type II axons consisted of terminal knobs, which ended at various levels throughout the muscular coat of the artery. Larsell believed these endings to be sensory. A third type of nervous terminalis axon terminal, which he also believed to be sensory, consisted of free nerve endings that appeared to innervate the epithelium of the septal mucosa and the vomeronasal organ. C.

Functional Studies of the Nervus Terminalis

Studies to determine the function of the nervus terminalis, using electrical stimulation or lesioning techniques, suggest that it may mediate some chemosensory responses to so-called pheromones (Kyle et al., 1987). Demski and Northcutt (1983) have shown, in teleosts, that electrical stimulation of the medial olfactory tract (which includes the central roots of the nervus terminalis) causes sperm release. Wirsig and Leonard (1987) demonstrated, in hamsters, that cutting the central roots of the nervus terminalis results in deficits in mating behavior. However, cutting the central roots of the nervus terminalis in newborn or early postnatal rats did not affect their ability to find their home nest (Schwanzel-Fukuda and Pfaff, 1983). The anatomical basis for these effects may lie in a complex interaction between afferent and efferent components in the ganglia of the nervus terminalis. White and Meredith (1987) examined the nervus terminalis in elasmobranchs and concluded from their electrophysiological studies that the ganglion terminale is tonically active and that this activity is suppressed by efferent impulses from the brain. In a teleost, the dwarf gourami, Oka and Matsushima (1993) used whole-brain preparations to examine spontaneous electrical activities of LHRH-immunoreactive terminal nerve cells. They reported that the axons of these cells have a wide distribution throughout the brain but do not project to the pituitary gland. The results of their electrophysiological data and anatomical observations led Oka and Matsushima (1993) to hypothesize that the LHRH-immunoreactive axons of the terminal nerve have a neuromodulatory function on target neurons throughout specific brain regions which mediate the animal’s response to hormonal or environmental conditions. Wirsig-Wiechmann and Lepri (1991) discovered LHRH-immunoreactive cells and fibers (branches from the nervus terminalis) in the pterygopalatine (sphenopalatine) ganglia of prairie voles. They postulate that these LHRH neurons may be part of a neural system that “modulates the control of the vascular pump of the vomeronasal organ, thereby modulating stimulus access to the vomeronasal organ.” The pterygopalatine ganglion is the cranial

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autonomic ganglion that innervates the palate, nasal mucosa, and paranasal sinuses, and it controls the vascular pumping mechanism of the vomeronasal organ (Eccles, 1982). Moore and coworkers (Moore et al., 1987) introduced the interesting consideration that the LHRH content of the nervus terminalis may be an important factor in the function of this nerve. Studying the effects of LHRH on sexual behaviors in newts, they proposed that these effects may be mediated by the actions of LHRH on cells of the nervus terminalis, to transduce olfactory signals, rather than on cells of the hypothalamus and the pituitarygonadal axis. There is evidence that LHRH functions as a neurotransmitter in the sympathetic ganglia of frogs (Jan et al., 1979; Jones et al., 1984). Recent studies by Eisthen and coworkers (Eisthen et al., 2000) in the mudpuppy (Necturus maculosus) used voltage-clamped whole cell recordings to examine the effects of 0.5–50 m GnRH on voltage-activated currents in olfactory receptor neurons in epithelial slices. These investigators, after verifying that the terminal nerve of the mudpuppy is GnRH immunoreactive, tested the hypothesis that the terminal nerve has a neuromodulatory function. They discovered that GnRH increased the excitability of olfactory receptor neurons and concluded that the terminal nerve functions to modulate the odorant sensitivity of olfactory receptor neurons. This report is the first physiological demonstration that GnRH, a decapeptide present in the terminal nerve, modulates activity of olfactory receptor cells (Eisthen et al., 2000). These exciting data open new areas of research into the function of this interesting and still enigmatic cranial nerve. III.

THE NERVUS TERMINALIS IN HUMANS

A. General Description: The Nervus Terminalis in Adult Humans Johnston (1914) examined 14 adult human brains and found the nervus terminalis present in all of them. Similar findings were reported by Brookover (1914) and McCotter (1915). The peripheral, intracranial, and central fibers of the nervus terminalis follow a generally similar distribution in humans to that seen in other mammals. Since the olfactory bulbs are comparatively small in humans, the intracranial course of this nerve differs somewhat from that seen in macrosmatic animals. The nervus terminalis emerges from the septal mucosa through the cribriform plate and forms a plexus on either side of the crista galli. The intracranial plexus of the nervus terminalis courses beneath the pia mater over the orbital surface of the gyrus rectus medial to the olfactory bulb and tract and enters the

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forebrain in the “region of the fissura prima, either upon, in front of or behind the medial olfactory tract” (Johnston, 1914; Kim et al., 1999) (Fig. 2). A recent study in adult humans using antibodies to GnRH and immunocytochemical procedures (Kim et al., 1999) provides a comparison between the distribution of fetal GnRH neurons seen along the developing migration route from the medial olfactory placode and the GnRHimmunoreactive neurons observed in the adult human brain. The data collected by these investigators confirm the presence of GnRH-immunoreactive cell bodies and axons in all parts of the adult olfactory system and in remnants of the nervus terminalis, seen medial to the olfactory nerve on the gyrus rectus (Fig. 2) B. Development of the Nervus Terminalis in Humans: Early Studies The earliest report of a nervus terminalis in human embryos is attributed to De Vries (1905), who considered it, together with the vomeronasal nerve, equivalent to the nervus terminalis of fishes. Dollken (1909) regarded the nervus terminalis as the “special nerve of the vomeronasal organ,” and he traced fibers from the ganglion terminale of a human embryo into the forebrain. In sagittal sections of a 31 mm (but not 9.5 mm) human embryo, Johnston (1913) made the observation that the nervus terminalis and vomeronasal nerve are separate nerves. He described the origin of the nervus terminalis from the epithelium of the vomeronasal organ and its connection by means of several fine rootlets with the region of the anterior neuropore in the forebrain. He presented the notion that the nervus terminalis and the olfactory nerve may be regarded as “two components of a segmental nerve, analogous to the gustatory and general cutaneous components which exist together in the facial or glossopharyngeal nerves in some fishes.” Brookover (1914) examined the nasal regions of two human fetuses, near term, and found a peripheral distribution of the nervus terminalis “so large in man that it may be said to be hypertrophied as compared to the known development in other mammals.” He counted, in addition to the many cells in the ganglion terminale, about 1500 cells in the nasal mucosa associated with branches of the nervus terminalis. Examination of serial, 8 m, sagittal sections of the brain and nasal regions of three 19 week old human fetuses in this laboratory corroborates Brookover’s description of the prominence of the peripheral and intracranial parts of the nervus terminalis in the human fetus (Schwanzel-Fukuda et al., 1989). Pearson (1941) carried out an elegant, detailed study of the development of the nervus terminalis in human

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embryos, using pyridine silver techniques (Figs. 3–5). He found the anlage of the ganglion terminale in a 14 mm (but not 9 mm) embryo, noting that both the ganglion terminale and the vomeronasal nerve arise from the medial border of the olfactory placode, the same region that gives rise to the vomeronasal organ. The cells that made up the ganglion terminale in the 14 mm embryo formed “irregular masses along the medial side of the olfactory nerves.” The fibers of the nervus terminalis could not be distinguished from the vomeronasal nerve at this early stage of development. In contrast, the olfactory nerves were set apart because they developed from the lateral parts of the olfactory placode and no ganglion cells were seen in connection with them. In a 17 mm embryo Pearson saw the nervus terminalis as a broad band of cells and fibers extending from the dorsal surface of medial olfactory placode to the wall of the forebrain. At the 24 mm stage (Fig. 3), the ganglion terminale appeared to be fused to the wall of the forebrain and the external limiting membrane of the brain had disappeared at the site of fusion, allowing the fibers of the nervus terminalis to continue into the forebrain. Pearson noted that many cells at this junction appeared to form a stream through the ganglion terminale. He interpreted this to be a migration of cells from the forebrain into the ganglion terminale and concluded that the nervus terminalis is made up of cells from both the olfactory placode and the forebrain. After formation of the olfactory bulbs in a 38 mm human embryo (Fig. 4), Pearson traced the olfactory nerves into the developing olfactory nerve layer of the olfactory bulb. He observed the ganglion terminale on the medial wall of the olfactory bulb, adjacent to the olfactory nerve layer, as “still a fairly compact mass of cells and fibers” at this age. A few nerve fibers, which he believed to be the vomeronasal nerve, coursed from the more lateral parts of the ganglion terminale into an area he identified as the accessory olfactory bulb, or formation. A similar structure was seen in the human embryo by Humphrey (1940). The remaining fibers from the ganglion terminale entered the medial forebrain brain caudal to the developing olfactory bulb (Fig. 4). In a 78 mm human embryo (Fig. 5) Pearson showed the peripheral and intracranial distribution of the nervus terminalis. The ganglion terminale appeared more distinctive at this age, and it was separated laterally from the olfactory nerves. Pearson speculated that the nervus terminalis is functional in mammals and that it contains both autonomic and sensory components. The sensory components, he believed, were derived from the olfactory placode and represented by the bipolar and unipolar cells seen in the ganglia and along the course of the nervus terminalis. The multipolar cells found in ganglia of the

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Figure 3 A transverse view through the forebrain of a 24 mm human embryo, pyridine silver preparation. The lateral margin of the nervus terminalis is still in close relation to the olfactory nerve. The caudal end of the ganglion terminale has begun to separate from the ventromedial wall of the forebrain. Many cellular elements are present among the fibers of the nervus terminalis where they enter the brain. N. term., nervus terminalis; gang. term., ganglion terminale; nasal epi., nasal epithelium; N. olf., olfactory nerve. (From Pearson, 1941.)

nervus terminalis, which Pearson thought resembled sympathetic ganglion cells, represented the autonomic components. More recent studies carried out toward the end of the twentieth century (described in the following sections) built on many of the experiments described above to further probe the structure of the terminal nerve. These studies used immunocytochemical procedures with antibodies to LHRH and other proteins found in the axons and cell bodies of the terminal nerve or hybridization procedures with highly specific probes to various molecular components found in the composition of the nervus terminalis. IV. EMBRYOLOGY OF THE NERVUS TERMINALIS IN MICE STUDIED WITH ANTIBODIES TO LHRH AND IMMUNOCYTOCHEMICAL PROCEDURES A. LHRH Immunoreactivity in Cells and Axons of the Nervus Terminalis The development of the nervus terminalis is very closely associated with the development of the olfactory and

vomeronasal nerves. Evidence demonstrating that the LHRH neurons share a common origin in the epithelium of the medial olfactory pit with the nervus terminalis and the vomeronasal nerves (Schwanzel-Fukuda and Pfaff, 1989; Wray et al., 1989a, b; Muske and Moore, 1989, 1990; Daikoku-Ishido et al., 1990; Norgren and Lehman, 1991, Ronnekleiv and Resko, 1991) casts a new light on the nervus terminalis and warrants some discussion here. Immunocytochemical procedures, using antibodies to LHRH, reveal that a population of the ganglion cells and axons of the nervus terminalis contain LHRH immunoreactivity. In most animals, including humans, LHRH immunoreactive axons are found in the peripheral and intracranial plexuses and in the central roots of the nervus terminalis (Fig. 6). LHRH-containing ganglion cells accompany these axons and the branches of the nervus terminalis originating from the epithelium of the medial olfactory pit. The ganglion terminale contains both LHRH-immunoreactive and non–LHRH-immunoreactive cells (Fig. 7), the LHRH neurons being generally fewer in number than the non–LHRH-immunoreactive cells (Schwanzel-Fukuda and Silverman, 1980; Schwanzel-Fukuda et al., 1985; Jennes and SchwanzelFukuda, 1992).

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Figure 4 A composite drawing made from several consecutive cross sections through the forebrain of a 38 mm human embryo, copper protargol preparation. The ganglion terminale is situated close to the layer of olfactory nerve fibers of the medial wall of the olfactory bulb. The ganglion at this age is still a fairly compact mass of cells and fibers. The course of the nervus terminalis caudad to its place of entrance into the brain lies close to an area that is thought to be the accessory olfactory bulb (or formation). As the fibers from the ganglion terminale are followed caudad in this embryo, fiber bundles turn laterad to enter the bulb in the region of the accessory olfactory bulb. These fibers are considered to be part of the vomeronasal nerve. The remaining fibers that enter the brain caudad to the accessory olfactory bulb belong to the nervus terminalis. N. term., nervus terminalis; gang. term., ganglion terminale; bulb olf., olfactory bulb; bulb olf. acc., accessory olfactory bulb; N. vn., vomeronasal nerve; lam. fib., lamina fibrosa (olfactory nerve layer of the olfactory bulb). (From Pearson, 1941.)

Figure 5 Diagram showing the peripheral course of the nervus terminalis in a 78 mm human embryo, copper protargol preparation. At this age the ganglion terminale is seen as a loose mass of fibers and cells in the region medial to the rostral end of the olfactory bulb. a. N. term., anterior branches of the nervus terminalis; r. N. term., roots of the nervus terminalis; vn. o., vomeronasal organ; N. vn., vomeronasal nerve; gang. term., ganglion terminale; bulb olf., olfactory bulb. (From Pearson, 1941.)

B. The Origin of LHRH Neurons and Development of the Nervus Terminalis and Vomeronasal Nerve from the Medial Olfactory Placode In embryonic mice, by day 9 of gestation the ectoderm on the lateral sides of the head thickens and forms the

olfactory placodes. Soon after, the olfactory placodes sink beneath the surface and develop into simple olfactory pits, either side of midline, separated by the developing nasal septum. By day 10 of embryonic life, the medial part of either olfactory pit evaginates to form a secondary recess. The epithelium of this recess, which forms the anlage

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Figure 6 LHRH-immunoreactive fibers (central roots of the nervus terminalis) crossing branches of the anterior cerebral artery (A) at the junction of the septum and the olfactory lobe. A small cluster of LHRH-immunoreactive neurons (arrow) is seen in the ventromedial septum (S). Adult male guinea pig, 6 m sagittal section, lightly counterstained with cresyl violet. (From Schwanzel-Fukuda and Silverman, 1980.)

Figure 7 The ganglion terminale, caudal to the olfactory bulb on its medial aspect. Two LHRH-immunoreactive neurons are seen among the nonimmunoreactive cells in the ganglion. Several LHRH-containing axons are evident among the ganglion cells and can be traced from the ganglion into the surrounding neuropil (arrows). Adult female mouse, 8 m sagittal section, lightly counterstained with cresyl violet. (From Schwanzel-Fukuda et al., 1987.)

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of the vomeronasal organ, gives origin to both the vomeronasal and terminal nerves and is the site of origin of LHRH neurons, seen on about day 11 in embryonic mice (Fig. 8). LHRH-immunoreactive neurons are never seen in the epithelium of the olfactory placode before formation of this medial part of the olfactory pit (SchwanzelFukuda and Pfaff, 1989). C. The Role of the Nervus Terminalis in the Migration of LHRH Neurons Soon after their appearance, by about day 11.5, the LHRH neurons begin to migrate out of the epithelium of the olfactory pit into the mesenchyme of the developing nasal mucosa. These cells are comparatively widely separated in the epithelium of the olfactory pit, but as they migrate out they aggregate into cords (Figs. 9, 10) along fibers of the nervus terminalis and vomeronasal nerves, which are difficult to distinguish from each other, at these ages (Figs. 3, 4). By days 12 and 13 of embryogenesis, large numbers of LHRH neurons are seen in cords on the nasal septum (Fig. 10). On about day 14 of gestation, the majority of LHRH neurons are seen crossing from the nasal septum into the forebrain (Fig. 11). As they enter the forebrain, the

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cords of LHRH-immunoreactive neurons begin to separate and follow one of two courses, seen as a Y-shaped branching of the LHRH cell cords, caudal to the medial parts of the developing olfactory bulb. In one limb, LHRH neurons (probably by means of a branch of the nervus terminalis to the vomeronasal nerve) course over the dorsal-medial surface of the olfactory bulb to the accessory olfactory bulb. In the other and larger limb, LHRH-immunoreactive neurons enter the forebrain (probably by means of the central roots of the nervus terminalis). By day 16 of embryogenesis the migration of the LHRH neurons is largely over in mice and most of these cells are now seen arching through the forebrain, from the olfactory tubercle, through the medial septum and the medial preoptic areas into the hypothalamus. However, some LHRH-immunoreactive neurons continue to be seen along the migration route (in ganglia of the nervus terminalis) throughout the life of the animal. Just as Pearson (1941) described in human embryos (Figs. 3–5), the nervus terminalis can be distinguished, in mice, from the vomeronasal and olfactory nerves after a certain period of development. After the olfactory bulbs are formed, the olfactory and vomeronasal nerves can be traced into the main and accessory olfactory bulbs,

Figure 8 The medial olfactory pit (the anlage of the vomeronasal organ) of an 11-day-old embryonic mouse. The LHRHimmunoreactive neurons, oriented perpendicular to the apical (luminal) surface (A) of the developing vomeronasal organ, are seen among nonimmunoreactive cells in the apical, intermediate, and basal layers of the epithelium. 8 m sagittal section, lightly counterstained with cresyl violet. (From Schwanzel-Fukuda and Pfaff, 1990.)

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Figure 9 At 11.5 days of embryogenesis, LHRH-immunoreactive neurons are seen leaving the epithelium of the medial olfactory pit (MOP) and migrating onto the nasal septum (NS), towards the forebrain. These neurons appear to accompany axons of the nervus terminalis and vomeronasal nerves, which are also derived from the epithelium of the medial part of the olfactory pit. 8 m sagittal section, lightly counterstained with cresyl violet. (From Schwanzel-Fukuda and Pfaff, 1990.)

respectively. The ganglion terminale can now be seen either side of midline caudal to the olfactory bulb, suspended between the intracranial plexus along the medial surface of the olfactory bulb and the central roots of the nervus terminalis which enter the medial forebrain (Figs. 7, 11). In the 18-day-old fetal mouse the ganglion terminale is formed and both LHRH-immunoreactive and non–LHRH-immunoreactive cells are seen within it (Fig. 11). Throughout life and in old age [personal observations of several 3-year-old C 57/BI/J6 mice, Jackson Laboratories, and several 3-year-old opossums (Monodelphis domestica) provided by Dr. Barbara Fadem], LHRH-immunoreactive neurons are seen along

the course of the nervus terminalis through the nasal regions and in the ganglia of this nerve. In human embryos, the development of the nervus terminalis and the origin and migration of LHRH neurons from the epithelium of the medial olfactory pit is similar to that seen in mice. The epithelium of the medial olfactory placode gives rise to olfactory Schwann cells (Chuah and Au, 1991) and axons of the vomeronasal and terminal nerves. These cells and axons begin to emerge from the epithelium by day 28 of gestation and make up the LHRH cell migration route from the olfactory placode to the forebrain. The LHRH-immunoreactive neurons, however, are not detected until approximately

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D. Other Studies on LHRH-Immunoreactive Neurons in Ganglia of the Nervus Terminalis

Figure 10 Cords of LHRH-immunoreactive neurons are seen on the nasal septum, coursing toward the forebrain with branches of the terminalis and vomeronasal nerves, from the epithelium of the medial olfactory pit. 8 m sagittal section of the nasal septum of a 13-day-old embryonic mouse, lightly counterstained with cresyl violet.

42 days of gestation (Boehm et al., 1994; SchwanzelFukuda et al., 1996). Two interesting studies by Dellovade and coworkers (Dellovade et al., 1998a, b) in the small eye (Sey) mouse demonstrated that the olfactory placode and olfactory bulbs are altered in this mouse and the LHRH hormone system does not develop. The phenotype in the small eye mouse is due to a point mutation in the developmental control gene Pax-6 and results in failed development of the eye and the olfactory placodes in homozygous (Sey/Sey) embryos and various eye abnormalities in heterozygotes (Sey/). The absence of LHRH immunoreactivity in the nasal regions and brain of homozygous (Sey/Sey) embryos support and extend the evidence that LHRH cells originate in the olfactory placode in mice.

Using tritiated thymidine and LHRH immunocytochemistry, researchers determined that most of the LHRH neurons are “born” on about day 10 of embryonic life in the epithelium of the medial part of the olfactory pit (Wray et al., 1989; Schwanzel-Fukuda and Pfaff, 1990). These findings were substantiated by the results of experiments using hybridization and specific probes for LHRH mRNA (Wray et al., 1989a,b; Zheng et al., 1990a,b,c; Schwanzel-Fukuda et al., 1992a,b). Zheng and associates (Zheng et al., 1991) further characterized the LHRH-immunoreactive neurons, migrating and in ganglia of the nervus terminalis, by immunocytochemistry and electron microscopy. Both LHRH-immunoreactive and non–LHRH immunoreactive cells were seen in clusters and cords on the nasal septum and in the ganglion terminale. Zheng et al. (1991) measured the distance the LHRH immunoreactive cells migrate, along the nervus terminalis, from the epithelium of the medial olfactory pit to the forebrain during the 24-hour stage between days 11 and 12 of embryogenesis. They estimated that the greatest distance that the LHRH cells travel at this stage is 950 m and that they migrate at the rate of about 40 m per hour. The LHRH immunoreactive and non–LHRH-immunoreactive cells were very similar in appearance, the difference between them being the content of LHRH immunoreactivity. In the epithelium of the olfactory pit and on the nasal septum, LHRH-containing neurons showed accumulations of reaction product around the nuclear envelope. No secretory vesicles were detected in these young cells, suggesting that these cells had not yet attained their target structure. More recent immunocytochemical studies on the development of the LHRH systems in embryonic monkeys (Quanbeck et al., 1997) led to the discovery by these investigators that two populations of LHRH neurons emerge from the epithelium of the medial olfactory placode. One population of LHRH-immunoreactive cells, detected by antiserum to LHRH GF-6 (provided by Dr. Nancy Sherwood), are described as “early” cells, since they migrate out of the placodal epithelium several days earlier (at E 30) than the migration of “late” LHRH cells (at E 32). The two populations of LHRH cells are distinctive. The early LHRH cells were only detected by antiserum GF-6, while the late LHRH cells were immunoreactive to all LHRH antisera tested except GF-6. In addition, the early LHRH cells differ morphologically from the late cells, appearing smaller in size and showing a somewhat different distribution in the brain. The authors hypothesize, based on the pattern of final distribution of these two populations of LHRH cells in the brain, that the late cells are the “bona fide LHRH neurons that regulate the pituitary-gonadal axis.” Quanbeck and coworkers continue to study these cells in order to

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Figure 11 At 18 days of gestation, the ganglion terminale (arrow) can be clearly seen caudal to the olfactory bulb, suspended between the intracranial plexus along the medial surface of the olfactory bulb (OB) and the central roots of the nervus terminalis (NT), which enter the medial forebrain. Both LHRH- and nonimmunoreactive cells are seen in the ganglion terminale. 8 m sagittal section, lightly counterstained with cresyl violet.

determine the molecular form of the early LHRH cells and, in turn, to discover their function in the primate brain. E. Cell Culture: Explants of Embryonic Olfactory Epithelium Containing LHRH Neurons Further confirmation for the hypothesis that LHRH neurons originate in the epithelium of the medial olfactory pit comes from studies that explanted embryonic neurons from medial olfactory epithelia of embryonic rats (Terasawa et al., 1993). These investigators succeeded in developing a primary cell culture system of LHRH neurons, clearly derived from the olfactory placode. Similarly, in explants of embryonic LHRH cells harvested from the nasal epithelium of mice (Schwanzel-Fukuda et al., 1992), relevant perhaps to the notable tendency of LHRH neurons to aggregate during their migration, it was observed that LHRH neurons in culture survived better if they were in close contact with one another. Cell culture offers many advantages for the study of the chemical and mechanical factors involved in LHRH cell migration. In embryonic mice, using hybridization procedures, Wray and coworkers (Wray et al., 1996) detected glutamate decarboxylase (GAD) mRNA in the olfactory epithelium and in the epithelium of the ventral olfactory pit during the period of LHRH cell migration, specifically from day 11.5 through 13.5. GAD is the enzyme that

synthesizes gamma-aminobutyric acid (GABA). They noted that GAD mRNA was significantly reduced in the epithelium of the medial olfactory pit by day 16.5 of embryogenesis, which is about the time that the migration of LHRH cells into the brain is largely over in mice. Using antibodies to GAD and GABA and immunocytochemical procedures, Wray and coworkers observed “immunopositive axonal-like tracts” on the nasal septum of mice on day 12.5, but saw a decrease in GABA-ergic staining by day 13.5. In this same study, utilizing embryonic explants of the nasal septum to examine possible synaptic interactions between LHRH cells and the GABA-ergic neurons, the authors discovered synaptic contacts between LHRH neurons and the GABA-immunoreactive cells. More recent studies from this same laboratory at the National Institutes of Health, Fueshko et al. (1998) demonstrated that GABAergic neurons actually inhibit the migration of LHRH neurons and decrease the distance that LHRH cells migrate. These important studies provide valuable clues for the mechanisms of LHRH cell migration. The authors suggest that GABA may “act as an embryonic signal, regulating LHRH cell entrance into the central nervous system.” Additional evidence for the notion that GABA-ergic neurons and LHRH neurons may interact to influence certain critical stages of development of the LHRH cell migration route into the brain is found in the work of Tobet and coworkers (Tobet et al., 1997). In the prolarval

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lamprey, LHRH- and GABA-immunoreactive cells were found in proximity to each other, with the GABA-ergic cells appearing earlier in gestation (10 days after fertilization) than the LHRH cells, detected some 20 and 30 days after fertilization. The authors postulated that GABA-ergic cells, which appeared to surround the LHRH neurons, may influence development of LHRH neurons in lamprey. They postulate that while these embryonic in interactions are transient, the primary interactions between GABA-ergic and LHRH cells may be a lifelong and a more direct olfactory modulation of LHRH secretion (Tobet et al., 1997). V.

IMMUNOCYTOCHEMICAL STUDIES OF THE CHEMICAL MAKEUP OF THE NERVUS TERMINALIS

A. Neural Cell Adhesion Molecule Immunoreactivity in the Nervus Terminalis of Developing Mice The chemical makeup of the migration route, of which the nervus terminalis is an important part, was examined in this laboratory with antibodies to different substrate molecules, including laminin, cytotactin-binding (CTB) proteoglycan, fibronectin, and neural cell adhesion molecule (Schwanzel-Fukuda et al., 1992). Neural cell adhesion molecule (NCAM) immunoreactivity (but not immunoreactivity for the other compounds) was found on the central processes of the nervus terminalis and the olfactory and vomeronasal nerves. The development of these nerves is preceded by an apparent migration of NCAM-immunoreactive cells from the epithelium of the simple olfactory pit, soon after its formation from the olfactory placode. These NCAM-immunoreactive cells form an aggregate in the nasal mesenchyme between the olfactory pit and the forebrain. The olfactory and vomeronasal nerves and the nervus terminalis grow into this cellular aggregate, which, as development proceeds, becomes continuous with the rostral forebrain, forming in effect a scaffolding along which the LHRH neurons migrate into the brain (Figs. 12, 13). The lateral part of the cellular aggregate receives the ingrowing axons of the olfactory nerves, while the medial part receives the central processes of the vomeronasal and terminalis nerves accompanied by migrating LHRH-immunoreactive neurons (Fig. 13). The LHRH neurons, after they leave the epithelium of the medial olfactory pit, are never seen independent of the NCAM-immunoreactive axons of the nervus terminalis and the vomeronasal nerves until they enter the forebrain (Figs. 14, 15). A few fine fibers, probably the central roots of the nervus terminalis, accompany the LHRH cells some distance into the parenchyma of the developing forebrain (Fig. 15).

Figure 12 This 8 m sagittal section is lateral to midline and shows the developing olfactory bulb (OB) and the NCAMimmunoreactive fibers of the scaffold along which LHRH neurons migrate into the brain. NCAM-immunoreactive cells are seen throughout the epithelium of the olfactory pit (OP). NCAMimmunoreactive olfactory nerve fibers merge into fascicles (long arrow), which end in the NCAM-immunoreactive cellular aggregate that caps the ventral surface of the developing olfactory bulb. LHRH-immunoreactive neurons (shorter arrows) are seen among the deeply stained NCAM-immunoreactive axons of the nervus terminalis and vomeronasal nerves, on the nasal septum, and along the caudal margin of the cellular aggregate. (From Schwanzel-Fukuda et al., 1992.)

The NCAM-immunoreactive cellular aggregate we describe (Figs. 12, 13) may be the “olfactory ganglion” described by Disse (1897) or the “blastema” Lejour (1967) referred to in her study of the development of the olfactory nerves in rats. Similarly, His (1889) reported in a 5-weekold human embryo an aggregate of cells “connected with the central nervous system on one hand and the olfactory epithelium on the other.”

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Figure 13 An 8 m transverse section through the forebrain (F) and nasal regions of a 13-day-old embryonic mouse. At this age, NCAM-immunoreactive cells are seen in the middle or intermediate zone of the epithelia of the olfactory pit. The NCAM-immunoreactive fibers coursing from the lateral and dorsal parts of the epithelium of the olfactory pit into the NCAMimmunoreactive cellular aggregate probably are the central processes of the olfactory nerves and those seen medially (long arrows) are the nervus terminalis and vomeronasal nerves. Clusters of LHRH cells (short arrows) are seen only in the medial parts of the NCAM-immunoreactive cellular aggregates, and with the NCAM-immunoreactive fibers of the nervus terminalis and vomeronasal nerves, either side of midline. (From SchwanzelFukuda et al., 1992.)

The lateral parts of the cellular aggregate, which receive the ingrowing axons of the olfactory nerves (Fig. 13), eventually form the olfactory nerve layer of the olfactory bulb and possibly glial and sheath cells of the olfactory nerves (Pearson, 1941; Lejour, 1967). The medial parts develop into the ganglion terminale and central roots of the nervus terminalis (Pearson, 1941) and provide the route of entry for LHRH neurons into the brain (Figs. 13, 15).

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Figure 14 NCAM-immunoreactive cells are seen in the epithelium of the medial olfactory pit (MOP), and NCAM-immunoreactive nerve fibers of the nervus terminalis and vomeronasal nerves emerge from it into the nasal mesenchyme. Cords of LHRH neurons (arrows) course toward the forebrain in and among these NCAM-immunoreactive neurites and are never seen independent of them. OP, olfactory pit; bv, blood vessel. 8 m sagittal section, 13-day-old embryonic mouse. (From SchwanzelFukuda et al., 1992.)

The LHRH-immunoreactive neurons that are seen in the ganglia of the nervus terminalis in older animals appear to be left behind on the migration route from the nose to the forebrain. We have no data on the fate or function of these cells, whether they remain in place or continue to migrate into the brain. There is evidence that the olfactory epithelium of a number of forms may continue to generate new receptor cells throughout life (Graziadei and Monti Graziadei, 1978, 1983). Studies by Breipohl and others (e.g., Breiphol) qualify these data and show that the turnover of olfactory epithelial cells is not “automatic,” but is dependent upon, and subject to, modulation from hormonal, metabolic, or environmental factors (see Chapter 5). The olfac-

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Figure 15 Thick cords of LHRH-immunoreactive neurons are seen entering the ventral surface of the forebrain, caudal to the developing olfactory bulb. In the substance of the brain, the LHRHimmunoreactive cells appear to disaggregate and they are no longer seen in cords. Singly or in pairs these cells appear to follow an arch of fine NCAM-immunoreactive fibers (arrows) to their final destinations. These fine fibers correspond in position to the central roots of the nervus terminalis. 14-day-old embryonic mouse, 8 m sagittal section. (From Schwanzel-Fukuda et al., 1992.)

tory epithelium is subject to damage from the inhalation of toxins or viruses and is often not repaired in older individuals (see Chapter 16). It remains to be seen, in future studies, if the medial part of the olfactory epithelium shares these remarkable capacities and hazards, and the effect, if any, on the origin and migration of LHRH neurons. B. Role of Polysialic Acid Component of N-CAM in Migration of LHRH Neurons from Medial Olfactory Placode to Forebrain in Mice Antibodies to the polysialated form of N-CAM (PSA-NCAM, generously provided by Dr. Genevieve Rougon

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and Dr. Urs Rutishauser) revealed in early human embryos and in embryonic mice a discrete localization of this isoform in parts of the LHRH cell migration route on which the cells appeared to be in transit (SchwanzelFukuda et al., 1996). These areas included the axons of the vomeronasal and terminal nerves in the nasal mesenchyme just outside of the olfactory placode and the caudal margin of the “scaffold” along which LHRH neurons enter the forebrain, medial and caudal to the developing olfactory bulb (Fig. 12). Yoshida and coworkers (Yoshida et al., 1999) demonstrated in mice that enzymatic removal of PSA at embryonic day 12 (a peak day of LHRH cell migration in this species) “significantly inhibited the migration of nearly one half of the LHRH cell population” without affecting the migration route itself. Observations made in chicks (Murakami et al., 2000) demonstrated the strength of this phenomenon across vertebrate species. Murakami and coworkers observed PSA-N-CAM immunoreactivity on both the LHRH neurons and on somatostatin-positive axons of the migration route. Using a polysialic acid–specific endoneuraminidase, they found that PSA removal did not alter the appearance or behavior of the somatostatin-positive olfactory fibers with the migration route. However, it did induce a significant deviation of migrating LHRH neurons from the regular path into the forebrain. These workers postulated that PSA contributes to a specific pattern of LHRH cell migration into the forebrain by “limiting the interaction of the LHRH neurons with their surrounding environment.” In the case of the chick, PSA-N-CAM is present on both the LHRH neurons and the axons of the migration route. VI. A HUMAN FETUS WITH KALLMANN’S SYNDROME: LHRH-IMMUNOREACTIVE NEURONS REMAIN IN GANGLIA OF THE NERVUS TERMINALIS IN THE NOSE A. Kallmann’s Syndrome: Hypogonadotropic Hypogonadism with Anosmia Kallmann’s syndrome (hypogonadotropic hypogonadism with anosmia) is a rare genetic disorder characterized by a failure of development of the reproductive tracts and the absence of a sense of smell (Kallmann et al., 1944). It is also known as De Morsier’s olfactogenital dysplasia and may accompany midline cranioencephalic dysraphias (De Morsier, 1962). The hypogonadism is attributed to an absence of LHRH in the brain, and the anosmia is due to bi- or unilateral agenesis of the olfactory bulbs (see Chapter 28).

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B. LHRH Immunoreactivity in the Nervus Terminalis of a Kallmann Fetus Compared to Fetuses of the Same Age and Sex We had the opportunity to examine, by immunocytochemical procedures and antiserum to LHRH, the brain and nasal regions of a 19-week-old male fetus that had Kallmann’s syndrome and to compare the results with that seen in three normal fetuses of the same age and sex (Schwanzel-Fukuda et al., 1989). A normal distribution of LHRH-immunoreactive cells and fibers was seen in the brain and nasal regions of each of the three control fetuses. The ganglion cells and fibers of the nervus terminalis showed a few LHRHimmunoreactive cells and fibers (Fig. 16). In contrast, the brain of the Kallmann fetus showed no LHRH immunoreactivity whatsoever. Examination of the nasal regions of this fetus showed thick fascicles of LHRH-immunoreactive fibers and clumps of LHRH-immunoreactive cells in the peripheral and intracranial parts of the nervus terminalis (Figs. 17, 18). The clumps of LHRH-immunoreactive ganglion cells seen in the intracranial plexus were deep to the meninges and seen on each side of the crista galli (Fig. 17). The olfactory bulbs were absent in the Kallmann fetus. The central processes of the olfactory nerves emerged from the cribriform plate and formed neuromas on the dorsal surface of the plate, lateral to the plexuses of the nervus terminalis (Fig. 18A, B, D). The neuromas, like the clumps of LHRH-immunoreactive ganglion cells of the nervus

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terminalis (Figs. 17, 18C), were deep to the meninges. No contact was seen between the olfactory nerves or the nervus terminalis and the forebrain. It appears that development from the olfactory placode proceeded normally to some extent. The olfactory and vomeronasal nerves and the nervus terminalis, accompanied by LHRH-immunoreactive ganglion cells, were present in the nose (Fig. 18). The absence of the olfactory bulbs may be due to a failure of the olfactory nerves to make contact with the brain and induce their formation (Burr, 1916). The absence of LHRHimmunoreactive neurons in the brain of the Kallmann fetus may be due to the failure of the migration route into the brain provided by the central roots of the nervus terminalis. The recent discovery of a candidate gene for X-linked Kallmann’s syndrome (Franco et al., 1991; Bick et al., 1992; Legouis et al., 1992), which encodes a putative adhesion molecule, is consistent with our hypothesis that a defect of LHRH neuronal migration (the NCAMimmunoreactive pathway of the nervus terminalis) may explain the etiology of Kallmann’s syndrome. Development of an antibody to the Kal-1 encoded protein, anosmin-1, visualized with immunohistofluorescence and immunoelectron microscopy showed that anosmin-1 is a transient and regionally restricted component of the extracellular matrices during organogenesis in human embryos (Hardelin et al., 1999). An interesting report by Schwob (1993) describes an examination of the olfactory mucosa, harvested by

Figure 16 Normal human fetal brain, 19 weeks of gestation. An LHRH-immunoreactive neuron is seen, intermingled with axons and ganglion cell bodies of the nervus terminalis in the nasal cavity. 8 m sagittal section, lightly counterstained with cresyl violet. (From Schwanzel-Fukuda et al., 1989.)

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Figure 17 Nineteen-week-old fetus with Kallmann’s syndrome. Thick clusters of LHRH-immunoreactive neurons are seen on the dorsal surface of the cribriform plate, deep to the meninges, in structures corresponding to the axons and ganglia of the nervus terminalis, either side of midline. A few LHRH-immunoreactive neurons and fine fibers are seen among nonimmunoreactive axons as they course upward through the cribriform plate. 8 m sagittal section, lightly counterstained with cresyl violet.

means of an intranasal biopsy, from an adult male patient with Kallmann’s syndrome. He found evidence that olfactory neurons may be turning over more rapidly in Kallmann’s syndrome, without fully maturing, and the olfactory mucosa resembled that seen in experimental animals that had survived a long period following ablation of the olfactory bulbs. Similar to our conclusions in the Kallmann’s fetus, Schwob concluded that the absence of the olfactory bulbs in their Kallmann’s patient was due not to a failure of the olfactory epithelium to form, but to a failure of the olfactory nerves to contact the forebrain and induce formation of the olfactory bulbs. Recent studies that examined the effects of transplantation (Daikoku-Ishido et al., 1990) or removal (Murakami et al., 1992) of the olfactory placode on the migration of LHRH neurons have shown that these cells do not appear in the brain if this structure and its derivatives are not in place. In this laboratory, 1 L injections of antiserum to NCAM into the area of the olfactory pits of 10-day-old embryonic mice resulted in few LHRH-immunoreactive neurons in the epithelium of the medial olfactory pits and retarded the migration of these cells into the brain (Schwanzel-Fukuda et al., 1991).

VII. THE NERVUS TERMINALIS AS A TRANSPORT ROUTE FROM THE NOSE TO THE FOREBRAIN The nervus terminalis is very important in early development. It is a “superhighway” for the migration of LHRH neurons from the nose to the brain, and it is the only route in mammals by which these neurons pass into the brain. During the interval of development in which the migration route of the nervus terminalis is “open” for LHRH neurons, the migration route could also be a means by which other substances of various sorts may be delivered efficiently to the forebrain. This could include medicinal substances of potential therapeutic value. However, this privileged nasal-forebrain route of the nervus terminalis might also constitute a means by which toxic substances could enter the basal forebrain.

VIII. SUMMARY AND SOME THOUGHTS FOR FUTURE STUDY The nervus terminalis, distinct from the vomeronasal and olfactory nerves, is the only cranial nerve that projects

Figure 18 Microprojection drawings in the sagittal plane of selected 8 m paraffin sections through the brain and nasal cavity of a 19-week-old fetus with Kallmann syndrome, showing the distribution of LHRH-expressing neurons (each dot represents one LHRH neuron) and axons (thick lines represent an LHRH-immunoreactive axon bundle). (A) Section 813. The tangle formed by the central processes of the olfactory nerves on the left side of the cribriform plate (CP) is seen in this section, slightly lateral to midline. A few LHRH-immunoreactive cell bodies and axons appear in ganglia and axons of the nervus terminalis (NT) medial to the olfactory nerve bundle on the cribriform plate and in the nasal cavity. The optic nerve (on) and a part of the pituitary gland (p) are also visible in this section. (B) Section 908. No LHRH immunoreactivity is seen in either the median eminence (ME) or in the organum vasculosum of the lamina terminalis (OVLT) or in the preoptic area (POA). This section, closer to midline than A, shows LHRH cells and axons with branches of the nervus terminalis, medial to the olfactory nerves, on the cribriform plate and on the nasal septum.

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Figure 18 (Continued) (C) Section 1026. The crista galli (cg) is shown in this section, just on midline. A thick fascicle of LHRH-immunoreactive axons, that appears to be a part of the nervus terminalis (NT), emerges from the nasal septum onto the cribriform plate, accompanied by a dense cluster of LHRH-expressing cells. LHRH-immunoreactive cell bodies and axons are seen near blood vessels and glands on the nasal septum and in the nasal cavity. (D) Section 1273. On the right side of the cribriform plate, lateral to the midline, the “tangle” formed by the central processes of the olfactory nerves encompasses a part of the cartilage of the crista galli (cg). A few LHRH-immunoreactive axons are seen on the medial surface of this structure.

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directly from the nose to the septal and preoptic areas of the brain (Fig. 6). Originating from the epithelium of the vomeronasal organ, it is in a position to receive information from the external environment, to relay and integrate this information through its ganglia, and to convey it to the central nervous system. Along the course of the nervus terminalis, from the nose to the forebrain, its axons and ganglion cells are found in close proximity to blood vessels and to the cerebrospinal fluid. It may be possible that these axons are sensitive to blood or cerebrospinal fluid–borne substances, and this association is retained throughout the life of the individual. In light of these observations, at the present time we are examining the possible role of vasculogenesis on development of the migration route for LHRH neurons from the epithelium of the medial olfactory pit to the forebrain. Early in gestation, shortly following the formation of the olfactory pit, and coinciding with the appearance of NCAMimmunoreactive cells in the olfactory placodal epithelium, a rich vascular field develops in the nasal mesenchyme between this epithelium and the forebrain. The NCAM–immunoreactive cells migrate out of the epithelium into the nasal mesenchyme and they aggregate around the developing blood vessels. This takes place on or about day 10 in embryonic mice and day 28 in human embryos. Soon after the migration route or “cellulovascular strand” is formed the forebrain is, in effect, moored to the olfactory epithelium. The LHRH cells then emerge from the epithelium and aggregate into cords, which course across the nasal mesenchyme on or encompassed by NCAM–immunoreactive cell bodies and axons of the terminal and vomeronasal nerves that make up the “cellulovascular strand” or migration route. A recent study in this lab using an antibody to a matrix metalloproteinase (MMP2) demonstrated that metalloproteinase is present not only in the cytoplasm of the endothelial cells that line the developing blood vessels, but in the cell bodies and axons of the migration route. It does not appear to be co-localized with the LHRH-immunoreactive neurons. Endothelial cell–associated proteinases or other compounds on or in the cell membrane may be able to locally disrupt the basement membrane of the forebrain and facilitate the passage of LHRH cells into the brain (Schwanzel-Fukuda, 1999).

IX.

CONCLUSIONS

The nervus terminalis has been found in most species of vertebrates in which it has been sought, including humans. It can be distinguished from the olfactory and vomeronasal nerves by LHRH-immunoreactivity in its axons and in a population of ganglion cells associated with it.

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The cells that synthesize and release LHRH, a critical hormone in the regulation of reproductive function, share a common origin with the nervus terminalis in the epithelium of the medial olfactory pit and migrate into the brain along branches of the nervus terminalis. This phenomenon has been shown to be generalized across all species of vertebrates. The nervus terminalis is not a vestigial nerve. It is present in adult animals of all species, including humans. LHRH immunoreactivity is seen in cells and axons of the nervus terminalis throughout life. The function of the nervus terminalis, other than its role in the migration of LHRH neurons, remains an enigma. Given its close proximity to the vomeronasal system, as well as its association with it, the nervus terminalis may well play an important role in chemosensory processes in a number of forms. Hopefully, researchers, armed with the newer techniques of immunocytochemistry and molecular biology, will continue (or be inspired) to find this ancient and highly conserved cranial nerve worthy of study. REFERENCES Ayers, H. (1919). Vertebrate cephalogenesis. J. Comp. Neurol. 30:323–342. Bick, D., Franco, B., Sherins, R. S., Heye, B., Pike, L., Crawford, J., Maddalena, J., Incerti, B., Pragliola, A., Meitinger, T., and Ballabio, A. (1992). Brief report: Intragenic deletion of the Kalig-1 gene in Kallmann’s syndrome. N. Engl. J. Medi. 326:1752–1755. Boehm, N., Roos, J., and Gasser, B. (1994). Luteinizing hormone-releasing hormone (LHRH)-expressing cells in the nasal septum of human fetuses. Dev. Brain Res. 82:175–180. Bojsen-Moller, F. (1975). Demonstration of terminalis, olfactory, trigeminal and perivascular nerves in the rat nasal septum. J. Comp. Neurol. 159:245–256. Bossy, Y. (1980). Development of olfactory and related structures in staged human embryos. Anat. Embryol. 161:225–236. Breipohl, W., Mackay-Sim, A., Grandt, D., Rehn, B., and Darrelmann, C. (1986). Neurogenesis in the vertebrate main olfactory epithelium, In Ontogeny of Olfaction. W. Breipohl (Ed.). Springer-Verlag, New York, pp. 21–33. Brookover, C. (1910). The olfactory nerve, the nervus terminalis and the preoptic sympathetic system in Amia calva. L. J. Comp. Neurol. Psych. 20:49–119. Brookover, C. (1914). The nervus terminalis in adult man. J. Comp. Neurol. 24:131–135. Brookover, C. (1917). The peripheral distribution of the nervus terminalis in an infant. J. Comp. Neurol. 28:349–360. Brookover, C., and Jackson, T. (1911). The olfactory nerve and the nervus terminalis of Ameiurus. J. Comp. Neurol. 21:237–260. Brown, J. W. (1987). The nervus terminalis in insectivorous bat embryos and notes on its presence during human ontogeny. In

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1025 Tucker, D. (1971). Nonolfactory responses from the nasal cavity: Jacobson’s organ and the trigeminal system. Handbook Sensory Physiol. 4:151–181. Von Bartheld, C. S., and Myer, D. L. (1986). Tracing of single fibers of the nervus terminalis in the goldfish brain. Cell Tissue Res. 245:143–158. White, J., and Meredith, M. (1989). Immunocytochemical and histochemical evidence for two neurotransmitter systems in the elasmobranch nervus terminalis ganglion. Soc. Neurosci. Abstr. 19:31. Wirsig, C. R., and Getchell, T. V. (1986). Amphibian terminal nerve: distribution revealed by LHRH and AchE markers. Brain Res. 385:10–21. Wirsig, C. R., and Leonard, C. M. (1987). Terminal nerve damage impairs the mating behavior of the male hamster. Brain Res. 417:293–303. Wirsig-Wiechmann, C. R. (1990). The nervus terminalis in the chick: an FMRF amide-immunoreactive and AChE-positive nerve. Brain Res. 523:175–179. Wirsig-Wiechmann, C. R., and Lepri, J. J. (1991). LHRHimmunoreactive neurons in the pterygopalatine ganglia of voles: a component of the nervus terminalis? Brain Res. 568:289–293. Witkin, J. W. (1987). Luteinizing hormone-releasing hormone (LHRH) in olfactory systems in primates. In The Terminal Nerve. Structure. Function and Evolution, L. S. Demski and M. Schwanzel-Fukuda (Eds.). Ann. NY Acad. Sci. 519:174–183. Witkin, J. W., and Silverman, A.-J. (1983). Luteinizing hormonereleasing hormone (LHRH) in rat olfactory systems. J. Comp. Neurol. 218:426–432. Wray, S., Nieburgs, A., and Elkabes, S. (1989a). Spatiotemporal cell expression of luteinizing hormone-releasing hormone in the prenatal mouse: evidence for an embryonic origin in the olfactory placode. Dev. Brain Res. 46:309–318. Wray, S., Grant, P. and Gainer, H. (1989b). Evidence that cells expressing luteinizing hormone-releasing hormone mRNA are derived from progenitor cells in the olfactory placode. Proc. Natl. Acad. Sci. USA 86:8132–8136. Wray, S., Fueshko, S. M., Kusano, K., and Gainer, H. (1996). GABAergic neurons in the embryonic olfactory pit/vomeronasal organ:maintenance of functional GABAergic synapses in olfactory explants. Dev. Biol. 180:631–645. Wright, D. E., and Demski, L. S. (1991). Gonadotropin hormonereleasing hormone (GnRH) immunoreactivity in the mesencephalon of sharks and rays. J. Comp. Neurol. 307:49–56. Yoshida, K., Rutishauser, U., Crandall, J. E., and Schwarting, G. A. (1999). Polysialic acid facilitates migration of luteinizing hormone-releasing hormone neurons on vomeronasal axons. J. Neurosci. 19(2):794–801. Zheng, L.-M., Caldani, M., and Jourdan, F. (1988). Immunocytochemical identification of luteinizing hormone-releasing hormone-positive fibers and terminals in the olfactory system of the rat. Neuroscience 24:567–578. Zheng, L.-M., Pfaff, D. W., and Schwanzel-Fukuda, M. (1990a). Synaptology of luteinizing hormone-releasing hormone (LHRH)-immunoreactive cells in the nervus terminalis of the

1026 gray short-tailed opossum (Monodelphis domestica). J. Comp. Neurol. 294:2–12. Zheng, L.-M., Pfaff, D. W., and Schwanzel-Fukuda, M. (1990b). Terminations of the LHRH-immunoreactive fibers in the subfornical organ of the gray short-tailed opossum: an ultrastructural study. Neuroendocrinology 51:413–424. Zheng, L.-M., Schwanzel-Fukuda, M., Hejtmancik, J. F., Gibbs,

Schwanzel-Fukuda and Pfaff R. B., and Pfaff, D. W. (1990c). Properties of neuroendocrine cells migrating from the olfactory placode into the basal forebrain. Soc. Neurosci. Abstr. 16:953. Zheng, L.-M., Pfaff, D. W., and Schwanzel-Fukuda, M. (1991). Electron microscopic identification of luteinizing hormonereleasing hormone-immunoreactive neurons in the medial olfactory placode and basal forebrain of embryonic mice. Neuroscience 46:407–418.

Author Index

AAAAI (American Academy of Allergy, Asthma and Immunology), 527, 543 Aaron, J. I., 884, 885, 899, 900 Aars, H., 647, 649 Aase, A., 554, 568 Aaserud, O., 608, 612 Abate-Shen, C., 116, 138 Abbasi, A. A., 893, 893, 894 Abbate, R., 62, 71 Abbé, E., 653 Abbey, H., 528, 545 Abbondanzo, S., 27, 41, 123, 150, 131, 155, 396, 397 Abbott, J., 320, 322, 327, 356, 383 Abbott, M. W., 212, 214, 225 Abbott, P., 356, 378 Abd al-Hady, M. R., 962, 965 Abdallah, L., 888, 894 Abdul-Karin, F. W., 595, 608 Abe, K., 663. 673, 709, 711, 715, 716, 723, 727, 728 Abelman, E., 495, 496, 951, 954 Abi-Nader, F., 444, 457 Abogadie, F. C., 81, 85, 86 Abolmaali, N., 472, 477 Abraham, A., 212, 223, 492, 494, 495, 501 Abraham, J., 893, 899 Abraham, M. H., 983, 987, 988, 990–992, 994, 995, 995, 997 Abraham, S., 1003, 1012–1016, 1018, 1024, 1025 Abraham-Fuchs, K., 241, 244, 248 Abramovich, D. R., 311, 327 Abu-Hamdan, D. K., 893, 899 Acampora, D. 116, 131, 135 Accordini, C., 661, 677 Aceto, A., 58, 66 Acharya, J., 950, 954 Acharya, V., 950, 954

Ache, B. W., 24, 26, 40, 43, 81–82, 86, 87, 89, 218–220, 223, 226, 227, 807, 817, 821, 822 Acheson, A., 773, 782 Acker, W., 602, 612 Ackerman, B. H., 951, 954 Ackerman, S., 923, 928 Ackroff, K., 683, 701, 744, 755, 906, 924 ACOEM (American College of Occupational and Environmental Medicine) 527, 543 Acosta, W. C., 1003, 1025 ACP (American College of Physicians) 527, 543 Acree, T. E., 714, 729 ACS (American Chemical Society), 527, 543 Acuna, G., 723, 728 Adachi, A., 888, 900, 915, 924 Adamek, J. P., 147, 148, 163, 167–169, 171, 172, 176, 180, 268, 272 Adamo, P., 983, 998 Adams, C. E., 365, 380 Adams, D., 831, 840 Adams, D. R., 17, 24, 40, 57, 58, 66 Adams, G. K., 55, 71 Adams, L., 267, 269 Adams, P. R., 1005, 1023 Adamson, J. E., 441, 453 Addicks, K., 26, 49 Adelman, L., 22, 42, 124, 137, 505, 506, 509, 523 Adjodah, D., 82, 85 Adkins, C. J., 281, 293 Adlard, P. A., 511, 518 Adler, A. P., 422, 437 Adler, C.H., 495, 495 Adler, E., xxv, xxxvii, 709, 710, 711, 717, 723, 724–727, 798, 798, 848, 858

1027

Adler, G., 887, 888, 897, 898 Adler, H. T., 35, 48 Adour, K. K., 938, 942, 954 Adrian, E. D., 145, 154, 186, 194 Adrian, T. E., 76, 85 Affani, J. M., 30, 42 Affifi, A., 551, 557, 570–571 Agarwal, N., 716, 730 Agasandyan, Kh. V., 33, 40, 47 Aggerbeck, H., 554, 564 Aggleton, J. P., 680, 682, 700, 703 Aghajanian, G. K., 170, 177–179 Agnew, J., 581, 585, 588, 590 Agosta, W. C., 364, 369, 381 Agranoff, B. W., xxviii, xliii Agrawal, U., 215, 225 Agrin R. 269, 271, 479, 491, 499, 515, 522 Aguero, A., 915, 927 Aguilar-Baturoni, H. U., 187, 196, 982, 997 Aguirre, A., 647, 648, 649 Aguirre, G. K., 258, 269, 273 Ahlers, C., 644, 645, 648, 649, 658, 676 Ahlgren, P., 596, 608 Ahlskog, J. E., 485, 488, 495 Ahlstrom, R., 542, 543, 585, 587 Ahmed, M. S., 442, 444, 457 Ahne, G., 193, 198, 231, 247, 259, 264, 266, 271, 699, 702, 791, 798 Ahokas, A. J., 882, 897 Aiba, T., 102, 107 Aika, Y., 140, 151, 158, 163 Aiken, G. R., 34, 46 Aikman, S. N., 882, 899 Ailabouni, A., 642, 649 Aisa, M. C., 32, 47 Aiuchi, T., 713, 730 Ajdukovic, D., 785, 789, 791, 799 Ajilore, O., 492, 500

1028 Akabas, M. H., 709, 724, 737, 753 Akaishi, T., 888, 894 Akano, Y., 961, 966 Akaoka, H., 154, 162 Akashi, A., 153, 161 Akasofu, K., 364, 381 Akbarian, S., 516, 518 Akella, G. D., 856, 857, 858 Akerlund, A., 463, 467, 473 Akert, K., 680, 702 Akeson, R., 123, 131, 560, 564 Akeson, R. A., 560, 569, 664, 676 Akhtar, S., 444, 453 Akil, H., 560, 570 Akil, M., 517, 519 Akizuki, M., 553, 561, 562, 571 Akopian, A. N., 722, 725 Aksoy, I. A., 59, 73 Ala, T. A., 551, 553, 556, 567, 572 Alani, S., 686, 700 Alarie, Y., 982, 991, 992, 994, 995, 996, 999 Alavi, A., xxviii, xli, 516, 520, 593, 594, 608 Alavi, J., 910, 929 Al-Awqati, Q., 709, 737, 753 Albers, K. M., 772, 773, 780 Albert, M., 600, 608 Albert, V. R., 564, 565, 618, 623 Alberts, J. R., 310–312, 314, 322, 326, 351, 355, 361, 362, 372, 380, 618, 622, 908, 928 Albinus, B. S., 655, 659, 670 Albone, E. S., xxix, xxxiii, 352, 372 Albonetti, M. E., 357, 372 Alborn, A., 93, 108 Albrecht, R. M., 662, 672, 713, 726 Alcock, B., 666, 667, 670 Alcorta, E., 334, 340 Alda, M., 491, 499 Aldinger, S. B., 81, 88 Aldren, C., 468, 475 Aldrich, H. C., 81, 89 Aldrige, V. J., 233, 249 Aletta, P., 99, 110 Alev, N., 747, 748, 754 Alexander, B. K., xxxi, xxxiv, 410, 427 Alexander, S., 795, 803 Al-Hadlaq, S. M., 762, 777, 778 Alheid, G. F., 420, 426 Alho, K., 230, 248 Ali, S. F., 564, 567 Ali, Z., 304, 306 Alicke, T., 410, 432 Alimohammadi, H., 983, 996 Alkawaf, R., 829, 840 Allen, C., 419, 429 Allen, C. M., 939, 940, 941, 954 Allen, D., 915, 924 Allen, D. M., 151, 154, 185, 194 Allen, E., xxviii, xxxiii Allen, J. M., 76, 85 Allen, K. D., 831, 840

Author Index Allen, S., 884, 901 Allen, W. F., xxiv, xxxiii, 312, 322, 410, 413, 426, 982, 984, 996 Allen, W. K., 123, 131, 560, 564 Alleva, A. M., 882, 902 Alleva, E., 356, 362, 372, 374, 422, 428 Alliger, R., 491, 492, 498 Allin, J. T., 309, 322, 360, 372 Allin, M., xxix, xlv, 256, 259, 266, 267, 273 Allingstrup, L., 786, 802 Allison, A. C., 140, 143, 145, 154, 268, 269, 855, 858 Allison, S. D., 392, 399, 583, 590 Allison, T., 230, 244, 248 Allman-Farinelli, M. A., 893, 900, 950, 956 Alloro, M. C., 367, 375 Alloway, T. M., 905–906, 927, 930 Allphin, A. L., 595, 608 Almkvist, O., 480–482, 495, 500 Almli, C. R., 775, 777, 780 Alonso, J. R., 154, 156 Alonso, M. J., 554, 563, 567 Alpers, D. H., 291, 293, 642, 649 Alpert, N. M., 686, 702 Alsop, D. C., 259, 264–266, 273, 344, 599, 613 Altar, C. A., 564, 561, 564 Altbäcker, V., 315, 322, 355, 370, 373, 833, 834, 838, 840 Alte, C., 82, 85 Althoff, H., 444, 455 Althoff, L., 63, 72 Altman, J., xxviii, xxxiii, 128, 129, 131, 136, 143, 154, 760, 764, 774, 778 Altman, L. L., 533, 539, 541, 546 Altmannsberger, M., 32, 48, 124, 138 Alvarez, P., 174, 175, 178, 192, 193, 198, 412, 414, 418, 419, 426, 429, 433 Alvarez-Buylla, A., xxviii, xxxix, 128, 134, 143, 156, 159, 620, 624 AMA (American Medical Association, Council of Scientific Affairs), 527, 543, 634, 637, 963, 965 Amador, X., 493, 499 Amano, H., 712, 729 Amaral, D. G., 168, 173, 176, 261, 263, 272, 414, 438, 602, 612, 680, 700, 701 Amaravi, R. K., 514, 520 Amato, G., 56, 57, 61, 64, 65, 70 Ambach, E., 330, 340 Amberla, K., 482, 499 Ambros-Ingerson, J., 410, 425, 426 Amedee, R. G., 444, 445, 454, 468, 476 Amend, D., 535, 538, 543 American Conference of Governmental Industrial Hygienists, 576, 587, 994, 996 Amieva, M. R., 104, 110 Amir, S., 172, 174, 177, 367, 375 Amit, Z., 914, 928

Ammini, A. C., 472, 474 Amoore, J. E., 204, 207, 223, 227, 277, 279, 293, 296, 306, 314, 327, 333, 344, 469, 473, 576, 587, 995, 996 Amsel, A., 412, 430 Amsterdam, J. D., 482, 495, 496, 951, 954, 961, 966 Amundsen, M. A., 542, 543 Amzel, L. M., 78, 86, 280, 293 Anand-Kumar, T. C., 61, 66 Anastasopoulos, I., 514, 522 Anchan, R., 62, 73 Andermann, F., 268, 270, 424, 431 Andersen, H. B., 888, 894 Andersen, I. B., 4, 15, 55, 71, 997 Andersen, M. B., 417, 427 Andersen, S. R., 554, 568 Anderson, A. E., 472, 475 Anderson, A. J., 145, 157, 182, 196, 622, 624 Anderson, B. P., 193, 196, 410, 425, 430 Anderson, C. O., 310, 327 Anderson, D., 600, 611 Anderson, D. J., 121, 133, 760, 780 Anderson, E., 413, 433 Anderson, G. H., 882, 884, 895, 898, 900 Anderson, I. B., 22, 47 Anderson, J. A., 462, 473, 476 Anderson, J. J. B., 885, 894, 903 Anderson, K. T., 34, 40 Anderson, M. W., 60, 62, 63, 66, 553, 565 Anderson, N. H., 203, 210, 223 Anderson, S., xxvii, xxxiv, 130, 131 Anderson, V., 491, 492, 493, 496 Anderson, W. R., 920, 932 Andervont, H. B., 368, 372 Andiné, R., 542, 543 Andoh, S., 938, 949, 957 Andonian-Haftvan, J., 992, 995 Andre, J. M., 950, 954 Andreasen, N. C. 269, 491, 496 Andreini, I., 26, 33, 42, 78, 84, 86, 126, 134 Andreotti, G., 642, 650 Andres, K. H., xxvii, xxxiii, 23, 30, 33, 40, 93, 107, 615, 623, 660, 676 Andressen, C., 26, 49 Andrew, C., xxix, xlv, 256, 259, 266, 267, 273, 687, 702 Andrew, R. J., 919, 926 Andrews, K., 829, 830, 841 Andrews, L. S., 583, 588 Andrews-Labenski, J., xxxi, xliii, 863, 878 Andriason, I., 171, 178 Andrieni, I., 565, 566 Andrieu, J., 102, 109 Andringa, A., 33, 45, 394, 398, 556, 567, 581, 583, 584, 588, 589 Andy, O. J., 230, 245, 246, 261, 269 Angele, A., 27, 45, 83, 89 Angelucci, S., 58, 66 Anger, W. K., 564, 565 Anggard, A., 450, 453, 983, 998

Author Index Anholt, R. R. H., xxvi, xliii, xliv, 76, 79–80, 83, 85, 90, 334, 340, 342 Anisko, J. J., xxx, xxxv, 404, 407 Anker, L. S., 991, 996 Anliker, J. A., 831, 840, 858, 858 Anonymous 359, 372, 645, 648, 893, 894 Ansari, K. A., 479, 485, 489, 496 Anselmi, M., 443, 457 Antczak, A. A. A., 788, 793, 802 Antonello, R. M., 489, 501 Antonini, L., 514, 518, 521 Antonucci, R. F., 744, 755 Antony, M. M., 539, 546 Anttila, S., 57, 70 AOEC (Association of Occupational and Environmental Clinics) 525, 527, 528, 535, 536, 543 Aoki, C., 557, 568 Aoki, J., 723, 728 Aou, S., 174, 178, 182, 197, 680, 688, 701, 702, 704 Aoyama, T., 61, 73 Apanius, V., 321, 322 Aparicio, R., 305, 306 Apfelbach, R., 392, 397, 552, 564, 582, 587, 588 Apfelbaum, M., 882, 896 Apostoli, P., 581, 589 Applebaum, S. L., xxxi, xxxv, 107, 108, 213, 216, 225, 266, 270, 332, 341, 461, 474 Appleman, L. M., 582, 588, 591 Aprison, M. H., xxviii, xliii Apter, A. J., 466–467, 473, 994, 996 Arabie, P., 740, 756 Arai, K., 366, 372, 952, 957 Arai, S., 709, 711, 715, 716, 723, 727, 728 Arai, Y., 1003, 1016, 1018, 1023 Araki, L., 330, 343 Araneda, R. C., 221, 223 Araneda, S., 152, 153, 154–156, 162, 561, 565 Arbour, P., 447, 454 Arcaro, K. F., 320, 322 Archambo, G., 795, 797, 800 Archer, T., 911, 914, 917, 927 Archilei, G., 243, 244 Arctander, S., 277, 278, 293 Arden, M. A., 345, 372 Arey, L., 656, 670, 670, 784, 798 Arglebe, C., 789, 799 Argüelles, J., 831, 840 Arimondi, C., 31, 42 Aristotle, 651 Armelagos, G., xv, xxxvi Armstrong, C., xxvi, xxxiii, 556, 565 Armstrong, R. N., 58, 66 Arnedo, M., 915, 927 Arner, L. D., 392, 397 Arneric, S. P., 620, 624 Arnhold, S., 26, 49 Arnhold-Schneider, M., 946, 954 Arnold, A. P., 919, 924

1029 Arnold, C., 891, 899, 910, 929 Arnold, F., 659, 670 Arnold, R., 887, 899 Arnold, S. E. xxxii, xliv, 269, 273, 491–493, 498, 499, 505, 508–518, 519–521, 523 Arnold, S. F., 923, 929 Arnold, S. M., 789, 799 Arnold, W., xxii, xxxvii, 550, 569 Arnott, L., 356, 381 Arnould, C., 315, 316, 326, 355, 358, 372, 381 Aron, C., 348, 372 Aroniadou-Anderjaska, V., 149, 151, 155, 156, 158, 183, 187, 194, 196 Aronsohn, E., 217, 218, 223 Arriza, J. L., 27, 42, 47, 84, 87, 90 Arters, J., 394, 396, 397 Árvay, A., 369, 372 Arvidson, K., 658, 661, 663, 670, 671, 825, 840 Aryan, M., 483, 501 Asa, C. S., 363, 376 Asai-Tsuboi, H., 182, 200 Asami, T., 661, 673 Asano-Miyoshi, M., 709, 711, 716, 727 Asanuma, N., 24, 26, 41, 711, 716 Asbury, A. K., 489, 501, 936, 954 Asch, R. H., 554, 566 Asenbaum, S., 485, 499 Ashbaugh, N., 815, 817, 821 Ashe, J. H., 911, 930, 916, 918, 930 Ashford, N. A., 525–530, 532–534, 536, 537, 543, 545 Ashihara, M., 124, 136 Ashmead, D. H., 828, 840 Ashmore, M., 644, 648 ASHRAE (American Society of Heating, Refrigeration and Air Conditioning Engineers), 532, 543 Ashton, E. H., 368, 397 Aspen, J., 861, 863, 876 Asselbergs, W. F., 392, 397 Assheuer, J., 259, 271 Astbäck, J., 663, 670 Astic, L., 36, 41, 46, 55, 69, 100, 105, 107, 110, 123, 130, 131, 145, 155, 158, 182, 190, 195, 197, 388, 398, 551, 557, 565, 569 Aston-Jones, G., 152, 160 Astwood, E. B., 919, 927 Atassi-Sheldon, S., 816, 822 Atema, J., 185, 196, 660, 670, 731, 757, 807, 814, 821 Athanassiadou, A., 514, 522 Atiyah, R. A., 597, 612 Atkinson, C., 893, 902 Atkinson, J. C., 646, 647, 648, 650 Atkinson, J. K., 300, 306 Atkinson, M. E., 774, 781 Atkinson, R. C., xv, xxxiii Atkinson, S. A., 839, 842 Atlas, S. W., 599, 608

ATSDR (Agency for Toxic Substances and Disease Registry) 527, 543 Attali, J. R., 554, 572 Attneave, F., 795, 799 Attramadal, H., 27, 42 Au, C., xxvii, xl, 104, 107, 108, 122, 123, 125, 132, 1011, 1022 Aubert, I., 154, 159 Auerbach, A., 121, 133 Auerbach, S. H., 960, 965 Auffermann, H., 234, 244 Augustijns, P., 562, 572 Augustinack, J. C., 512, 523 Augustine, J. R., 685, 700 Augustiniak, J., 392, 399 Aujla, R., xxvi, xlii Aungst, B. J., 553, 562, 568 Aurora, S., 621, 624 Austin, C. E., 443, 453 Austin, J. H., 470, 473, 477 Austin, S., 463, 476 Autuori, M., 736, 757 Avantaggiato, V., 116, 131 Avenet, P., xxv, xxxiii, 711, 714–716, 718, 720, 721, 725, 726 737, 738, 753, 755 Averback, P., 512, 519, 600, 608 Avery, M. E., 7, 16 Avila, J., xxvii, xli, 29, 47, 105, 106, 111, 112 Avila, K. J., 57, 72 Aviram, A., 950, 954 Awasthi, Y. C., 58, 70 Axel, R., xxv, xxvi, xxvii, xxxiii, xxxiv, xxxix, xliv, 24, 26, 28, 32, 35–37, 41, 44, 48, 49, 78–79, 82, 85–87, 89, 90, 99, 100, 107, 113, 126, 130, 131, 134, 135, 137, 138, 145, 146, 155, 157, 160, 164, 221, 224, 289, 294, 296, 306–307, 334, 340, 350, 351, 354, 373, 376, 379, 387, 391, 397, 560, 566, 572, 723, 725, 971, 972, 973, 976, 977 Axell, T., 647, 649 Ayabe-Kanamura, S., 221, 226, 235, 244, 783, 787, 793, 797, 801 Ayer, R. K., 335, 340 Ayer-LeLievre, C., 99, 101, 107, 110, 1017, 1022 Ayers, H., 1001, 1021 Ayers, J. A., 582, 590 Ayers, J. W. T., 321, 322 Ayyub, C., 334, 335, 340 Azen, E. A., 644, 648, 658, 671, 709, 710, 725, 726 Aziz, Q., 686, 700 Azzali, G., 656, 661, 664, 666, 671 Azzalina, J. D., 215, 225, 792, 800 Baars, A. M., 417, 433 Baba, M., 509, 522 Baba, S., 467, 476, 629, 637 Babbel, R. W., 595, 609

1030 Babigian, H. M., 923, 928 Babu, R., 469, 474 Baby, R. E. 306 Bachinger, T., 305, 306 Bachmanov, A. A., 717, 724, 727, 830, 840, 885, 894 Bacigalupo, J., 75, 88 Backman, L., 482, 499 Backus, C., 761, 762, 767, 780 Bacon, A. W., xxxii, xxxiii, 482, 483, 496 Bacon Moore, A. S., 482, 496 Baddeley, A., 222, 223 Bader, R. F. W., 291, 293 Badie-Elder, N., 885, 886, 898 Baehr, W., 76, 81–84, 89 Baer, L., 686, 702 Bagla, R., xxxi, xxxv, 170, 177, 184, 195, 389, 392, 398, 413, 428, 484, 485, 494, 497, 515, 520, 785, 789, 797, 800, 801, 804, 937, 955 Bahar, A., 145, 157 Bahrami, F., 553, 559, 565 Bailey, B. J., 7, 15, 450, 457 Bailey, M. S., 100, 111, 109, 127, 129, 130, 131 Bailey, R. P., 551, 554, 555, 565 Bailey, K. C., xix, xxxiii Baim, D., 143, 160, 512, 522 Baimbridge, K. G., 83, 85 Bain, A., 994, 996 Bain, G., 78, 86, 280, 293, 621, 623 Baird, J. C., 211, 223 Baird, J. P., 691, 700 Bairoch, A., 58, 70 Baisi, E., xxxiii Bajaj, J. S., 61, 66 Bajaj, S., 472, 474 Bajgrowicz, J. A., 276, 293 Bakalyar, H. A., 24, 41, 44, 80, 85 Bakardjiev, A., 104, 107 Bakarich, A. C., 915, 931 Bakay, L., 605, 608 Bakay, R. A., 620, 627 Baker, A. F. H., 817, 821 Baker, C. V. H., 760, 778 Baker, D. L., 910, 932 Baker, E. M., 343 Baker, H., 27, 29, 41, 48, 99, 107, 111, 123, 124, 131, 131, 133, 150–152, 155, 163, 184, 194, 396, 397, 511, 514, 515, 519, 523, 551, 554, 556, 559–561, 564, 565, 566, 570, 572, 573, 617, 618, 623, 625, 626 Baker, H. T., 103, 107 Baker, J. L., 124, 135 Baker, K. B., 486, 500 Baker, L. P., 27, 49 Baker, R., 551, 556, 572, 573 Baker, T. A., 551, 565 Baker, T. B., 920–921, 925 Bakhit, C., 564, 561, 564 Bakin, R. E., 24, 26, 44, 123, 135 Bakke, J. E., 63, 67, 550, 552, 559, 566, 577, 582, 588

Author Index Bakre, M. M., 710, 716 Balaban, C. D., 797, 802 Balas, G., 116, 133 Balazs, J. M., 422, 427 Balbes, L. M., 276, 293 Balboni, G. C., 62, 66, 73 Baldaccini, N. E., 76, 89, 560, 565 Baldwin, B. A., 423, 431 Baldwin, C. M., 526, 535, 538, 543, 544 Baldwin, M., 581, 589 Balfour, R., 343 Balin, B. J., 509, 521, 551, 556, 558, 565, 560, 561, 566 Balkan, B., 887, 902 Ball, A. K., 1004, 1025 Ball, M. J., 600, 608 Ballabio, A., 1001, 1017, 1021, 1022 Ballantine, J. A., 346, 375 Ballard, C., 513, 521 Ballard, P. A., 487, 499 Ballesteros, M. A., 915, 924 Balmaceda, J. P., 554, 566 Balogh, R. D., 318–320, 322, 325, 339, 343 Balsiger, D., 356, 373 Baltes, H., 299, 307 Balzarini, A., 891, 901 Bamberger, C., 664, 676 Bammler, T. K., 58, 68 Bancaud, J., 950, 955 Bancewicz, J., 686, 700 Bancroft, J., 359, 374 Bandettini, P. A., 258, 269 Banger, K. K., 58, 64, 66 Banki, M., 790, 790, 802 Bankovi, G. 341, 858, 859 Banks, E. M., 309, 320, 322, 323, 325, 356, 360, 372, 380, 404, 407 422, 431 Banks, S., 887, 900 Bannister, L. H., xxxi, xxxvi, 5, 15, 23, 30, 34–36, 38, 41, 42, 61, 68, 104, 108, 118, 119, 123, 124, 132, 463, 475, 560, 565, 616, 623, 644, 648, 665, 677 Banoczy, J., 939, 954 Banvoetz, J. D., 439, 441, 458 Bao, H., 778, 778 Bao, Z., 57, 72 Baptista, M. A., 366, 375 Baraban, S. C., 76, 85 Baran, D., 404, 407 Baranczakowa, Z., 960, 962, 965, 966 Baraniuk, J. N., 54, 71 Barat, J. L., 595, 608 Barazangi, N., 154, 156 Barbacid, M., 767, 771, 779 Barbas, H., 169–172, 176, 680, 695, 700 Barbe, C. D., 218, 219, 220, 227 Barber, P. C., 28, 41, 560, 565 Barbry, P., 719, 727 Barceloux, D. G., 559, 565 Bard, J., 318, 320, 322, 327, 340, 344, 356, 383, 410, 437, 835, 840

Bardana, E. J., 941, 954 Barde, Y. A., 763, 782 Bardea, A., 553, 563, 568 Bardo, M. T., 420, 433 Bardoni, B., 127, 133, 1001, 1017, 1022 Bardoni, R., 149, 155 Bare, J. K., xxxi, xli, 387, 399 Bargmann, C. I., xxv, xxxiii, 76, 78, 87, 90 Bargmann, W., 19, 23, 24, 30, 47 Baril, L. L., 906–907, 931 Barkai, E., 190, 191, 194, 196, 412, 425, 435 Barker, B., 366, 379 Barker, E., 835, 840 Barker, G., 366, 379 Barker, H. L. M., 911, 930 Barker, J., xxvii, xliv, 98, 113 Barker, L. M., 691, 696, 703, 905, 908, 911, 913–914, 924–927 Barker, P. B., 595, 611 Barker, S., 217, 228, 580, 584, 585, 590 Barkovich, A. J., 38, 48, 599, 608 Barlow, H. B., 616, 623 Barlow, J., 686, 700 Barlow, L. A., 670, 671, 909, 933, 767, 778 Barneoud, P., 416, 417, 432 Barnes, A., 923, 930 Barnes, C. D., 688, 691, 694, 695, 703 Barnes, C. L., 512, 521 Barnett, E. M., 551, 557, 565 Barnett, S., 104, 111, 108, 111 Barnett, S. A., 404, 407, 908, 924 Barnett, S. C., 28, 29, 42 Barni, T., 103, 108 Barocka, A., 983, 999 Baron, G., xxxiii, 142, 155 Baron, J., 57, 58, 63, 64, 66, 73 Baroncelli, G. I., 54, 68, 101–103, 109 Baroody, F. M., 12, 14, 599, 608 Barr, E. B., 62, 72, 582, 590 Barr, R. G., 827–829, 831, 833, 840, 842, 844 Barraclough, C. A., 923, 924 Barragan, E., 192, 193, 195, 268, 269, 479, 497, 563, 567 Barrantes, I. B., 760, 780 Barratt-Fornell, A., 798, 800, 848, 849, 852, 853, 856–858, 859 Barrera, S. E., 127, 134, 603, 610, 1016, 1023 Barres, B. A., 620, 625 Barreto, S.M., 443, 452, 457 Barrow, A., 553, 562, 565 Barrow, C. S., 54, 55, 71, 582–584, 588, 591 Barrows, W. M., xxix, xxxiii, 403, 407 Barry, M. A., 797, 799 Bars, P., 790, 793, 799 Barsel, G. E., 362, 378 Barsky, A., 536, 545 Bartalena, G., 244, 244 Bartana, A., 341, 828, 842 Bartanski, T. J., 291, 294

Author Index Barth, A., 116, 137 Barth, D. S., 241, 244 Barth, K. A., 116, 135 Bartha, L., 528, 543 Bartholomew, S., 423, 431 Bartido, S. M., 557, 568 Bartko, J., 472, 477 Bartlett, P. F., xxvii, xxxix, 103, 110, 619, 626 Bartlett, P. N., 297, 300, 306 Bartolomei, J. C., 140, 152, 155 Barton, A. J., 469, 474, 510, 520 Bartoshuk, L., xv, xx, xxxi, xxxiii, xxxviii, xlv, 35, 37, 42, 119, 132, 207, 208, 210–212, 216, 224, 227 479, 480, 497, 588 600, 602, 609 639, 648, 656, 660, 674, 709, 710, 713, 715, 725, 726, 728, 729, 732, 740, 741, 747, 748, 751, 752, 753, 754, 755, 756, 759, 765, 768, 773, 780, 783, 784, 787–791, 793, 794, 798, 799–804, 805, 807, 812, 820, 822, 831, 840, 847–858, 858–860, 885, 889, 894, 896, 918, 924, 937, 938, 943, 945, 951, 952, 954–957, 961, 962, 963, 965, 997, 999 Bartter, F. C., 470, 475, 784, 801 Barz, S., 232, 234, 236, 238, 239, 244, 246, 444, 445, 455, 463, 476, 485, 496, 990, 998 Basaric-Keys, J., 119, 121, 124, 137 Basco, E., 670, 674, 767, 781 Bascom, R., 442, 456, 527, 538, 539, 543, 587, 588 Bassett, A. S., 491, 499 Bassilana, F., 721, 730 Bastianelli, E., 26, 41, 83, 85 Bates, P. R., xxvii, xl, 36, 46, 93, 98, 103, 111 Bate Smith, E. C., xv, xxxvii Batsell, W. R. J., 406, 407 Batson, J. D., 910, 924 Battey, J. F., xxv, xxxvii, 713, 714, 717, 726 Battjes, J. E., 582, 590 Batty, J., 367, 372 Baudoin, C., 335, 340, 392, 398 Baudry, M., 413, 433, 436 Bauer, I., 82, 85 Bauer, J., 241, 248 Bauer, L. O., 232, 244 Bauer, M., 82, 85, 517, 521 Baum, B. J., 639, 646, 647, 648, 650, 658, 674, 768, 781, 787, 804, 952, 957 Baum, J. D., 885, 903 Baum, M. J., 919, 933, 972, 975, 977 Baumgarten, H. G., 142, 162 Baumgarten, R., 93, 113 Baumstark, C., xxv, xli, 287, 294 Baumzweiger, W., 528, 543 Baxter, M. G., 170, 178 Bayer, S. A., 128, 129, 131, 760, 764, 774, 778

1031 Bayley, D., 723, 725 Baylis, G. C., 680, 695, 699, 700, 701 Baylis, L. L., 192, 193, 199, 680, 688, 690, 691, 693, 694, 699, 700, 703, 741, 742, 745, 757, 948, 956 Bayliss, R., 923, 928 Bazer, G. T., 551, 554, 555, 565 Bazin, B., 789, 804 Beach, F. A., xxx, xxxiii, 352, 355, 372, 403, 404, 407 Beach, J., 526, 546 Beachy, P. A., 116, 132 Beal, M. F., 555, 569 Bealer, S. L., 746, 747, 754, 757, 873, 876 Beam, K. G., xxiv, xxxvii, 721, 727, 737, 755 Bean, N. J., xxx, xxxiii, 419, 430 Bear, M., 347, 372 Beard, M. D., 97, 106, 110, 471, 474, 620, 623, 625 Bearden, C. E., 516, 519 Beatty, J., 241, 244 Beatty, W. W., 918–919, 924 Beauchamp G. K., xxx, xliv, 61, 66, 68 217, 224, 228311, 314, 317, 318, 320–322, 322–324, 326, 329, 331, 333, 340, 344, 825, 826, 828–839, 840, 841, 843–845, 353, 355–357, 372, 373 379, 394, 398, 404, 407, 410, 417, 422, 427, 433, 437, 469, 477, 717, 724, 727, 735, 751, 754, 755, 788, 795, 799, 801, 803, 857, 858, 881, 883–886, 893, 894, 895, 899, 903 Beaumont, P. J. V., 359, 373 Beauvais, B., 586, 589 Beavo, J. A., 27, 41, 43, 49, 82–83, 85, 86, 88, 90, 716, 730 Bechar, M., 553, 563, 568 Bechgaard, E., 553, 565 Bechtold, W. E., 582, 590 Beck, A., 32, 36, 48, 126, 137 Beck, M., 357, 373, 376, 378, 419, 430 Beck, S., 710, 729 Becker, A., 536, 545, 619, 627 Becker, D. P., 629, 634, 637, 959, 965 Becker, E., 236, 244, 491, 496 Becker, Y., 554, 557, 565 Beckingham, E., 441, 443, 450, 452, 455, 456, 458 Beckman, M. E., 737, 754 Beckmann, H., 517, 521 Beckstead, R. M., 679, 685, 700, 948, 954 Becquemin, M. H., 449, 453 Bedard, M., 882, 893 Bedard, Y., 831, 840 Bedlack, R. S., 483, 496 Bedoukian, 276, 293 Beeker, T. G., 716, 729 Beeman, C. S., 854, 860 Beemer, A., 557, 570 Beer, A. E., 321, 322 Beer, C. 844, 907, 931

Beesley, J. S., 116, 133 Beets, M. G. J., 292, 293, 296, 306, 715, 725, 783, 799 Begbie, J., 760, 778, 779 Beglinger, C., 887, 898 Behan, M., 173, 176, 187, 188, 192, 197 Behbehani, M. M., 149, 161, 183, 198, 736, 756 Behe, P., 714, 716, 725 Behl, C. R., 553, 563, 566 Behnisch, P., 552, 564 Behnke, J. M., 909, 933 Behrendt, H. J., xxv, xliv Beidler, L. M., xv, xxiv, xxvii, xxxiii, xl, 17, 31, 43, 75, 76, 89, 400, 552, 558, 568, 643, 648, 663, 671, 711, 713, 727, 729, 737, 741, 756, 75, 769, 773, 778, 779, 799, 801, 807, 808, 812, 820, 848, 856, 859, 939, 954., 996 Beikirch, R. J., 920, 925 Beis J. M., 950, 954 Beizer, M., 467, 475 Belanger, A., 58, 70 Belanger, S., 581, 589 Belecky, T. L., 732, 733, 754, 767, 778, 866, 876 Belekhova, M. G., 148, 155 Belinsky, S. A., 60, 62, 63, 66, 553, 565 Belitz, H.-D., 991, 999 Belknap, E., 213, 226 Bell, C., xxi, xxxiii, 710, 729, 784, 799, 791, 798, 803, 981, 996, 999 Bell, D. D., 923, 924 Bell, D. N., 942, 954 Bell, E. T., 115, 131, 922, 931 Bell, G., 884, 901 Bell, G. A., 145, 163, 183, 194, 218, 220, 223, 228, 391–394, 400 Bell, K., xxxii, xxxv, •• Bellas, D. N., 949, 954 Bellini, L., xx, xxxiii, 651, 652, 671 Bellisle, F., 953, 954 Belliveau, J. W. 269, 270 Bellringer, J. F., 365, 373 Belluscio, L., xxv, xxvi, xxxiii, xxxvii, 24, 26, 35, 37, 41, 79, 85, 130, 131, 145, 155, 350, 351, 373, 972, 973, 976 Bellussi, L., 443, 457 Belluzzi, O., 139, 140, 149, 152, 155, 162 Bels, V., 661, 676 Beltman, J., 82, 85 Beltramino, C. A., 420, 426 Belyavsky, A. V., 116, 138 Ben-Arie, N., 58, 66, 145, 159 Ben-Boutayab, N., 174, 175, 179 Bencherif, M. 983, 999 Bende, M., 204, 215, 227, 462–463, 467, 473, 475, 477, 791, 801 Benes, F. M., 516, 517, 519, 522, 603, 608 Benhatov, R., 492, 493, 501

1032 Benheim, A. E., 784–786, 792, 804 Benignus, V. A., 232, 234, 248, 584, 590 Benjamin, R. M., xxiv, xxxvi, 263, 269 Bennett, M. H., 190, 194, 410, 427 Benoliel, R., 947, 957 Benos, D. J., 26, 29–31, 33, 45., 719, 721, 725, 729, 776, 777, 779, 782 Benovic, J. L., 84, 85, 88, 90 Benowitz, N. L., 554, 563, 568 Bensimon, J. L., 17, 48 Benson, J. M., 56, 67, 582, 583, 588, 590 Benson, T. E., 145, 157, 564, 565, 617, 623 Bentham, P., 482, 498, 513, 520 Bentley, J. K., 27, 49, 82, 90 Benvenuti, C., 553, 563, 566 Beraldo, K. E. A., 826, 828, 840, 883, 894 Berbs, L., 833, 842 Berciano, J., 960, 965 Bercowicz, D. A., 149, 155 Bereiter-Hahn, J., 660, 675 Berenbaum, M. C., 807, 820 Berendse, H. W. B., 480, 484, 486, 496, 501, 514, 519, 523 Beréziat, J. C., 64, 67 Berg, H. W., 206, 227 Berg, T., 647, 649 Berg, W. A., 858, 859 Bergamaschi, E., 581, 589 Bergamasco, N. H. P., 826, 828, 840, 883, 894 Bergasa, N. V., 893, 894, 950, 954 Bergen, H., 1013, 1025 Berger, B., 515, 520 Berger, H., 229, 244, 244 Berger, P. A., 483, 501 Berger, U., 444, 455 Berger-Sweeney, J., 394, 396, 397 Berggren, B., 887, 901 Berghard, A., 24, 36, 46, 972, 976 Berglund, B., 4, 15, 204, 210, 218, 219, 223, 273, 480–482, 495, 500, 525, 542, 543, 544, 585, 587, 807, 814, 820, 988, 996 Berglund, H., 263, 272, 350, 381 Berglund, U., 204, 210, 218, 223, 267, 273, 542, 543, 585, 587, 807, 814, 820, 988, 996 Bergman, K., 552, 559, 567 Bergman, U., 64, 66, 553, 559, 565 Bergmann, F., 829, 843 Bergmans, P. L., 480, 486, 501, 514, 519, 523 Bergquist, C., 554, 563, 565 Bergrnan, K., 62, 69 Berk, G., 907, 931 Berkhoudt, H., 661, 671 Berkinshaw, E. R., 441, 453 Berkeley, M. A., xxix, xliii, 400, 869, 876 Berkowicz, D. A., 183, 184, 194 Berkowitz, R. I., 883, 902 Berlin, R., 511, 515, 519

Author Index Berliner, D. L., 311, 324, 969, 978, 979 Berman, E. J., 169, 175, 180, 393, 400, 410, 436 Berman, R. F., 920–921, 925 Bermudez-Rattoni, F., 420, 421, 427, 429, 905, 916, 918, 924, 925, 930 Bernard, C., 385, 387, 397, 397 Bernard, J. F., 685, 700 Bernard, R., xxx, xliv Bernard, R. A., 891, 895 Bernardi, P., 659, 661, 675 Bernbaum, J. C., 826, 843, 883, 899 Berndt, A., 942, 954 Berne, C., 888, 896 Bernemann, D., 946, 954 Bernhardt, P. C., 359, 373 Bernhardt, S. J., 710, 716, 725 Berns, L., 311, 317, 323 Bernstein, D. I., 60, 71, 551, 557, 570 Bernstein, I. L., 562, 565, 766, 776, 782, 830, 831, 833, 838–840, 841, 909–911, 915–921, 924, 925, 929, 931, 932 Bernstein, J. J., 36, 45, 93, 109, 616, 625 Berrettini, W., 922, 929 Berridge, K. C., 670, 671, 824, 826, 829, 840, 865, 866, 876, 882, 898, 907, 913, 917, 924, 928 Berridge, M. J., 81, 85 Berry, H., 526, 546 Berry, M., 894, 897 Berschauer, R., 102, 105, 109 Bertelsen, A., 332, 341 Berthommier, F., 185, 186, 194 Berthoud, H. R., 680, 682, 702, 703 Bertino, M., 735, 754, 886, 894, 895 Bertollo, D. N., 491, 496 Bertsche, B., 420, 436 Berven, B. A. 995 Beschi, R. J., 471, 477, 892, 901, 950, 956 Beschorner, R., 82, 85 Besson, J. A. O., 602, 612 Besson, J. M., 685, 700 Best, A. R., 618, 627 Best, D. J., 218, 226 Best, M. R., 905, 908, 911, 913–914, 924–927, 930 Best, P. V., 602, 612 Bester, H., 685, 700 Bestmann, H. J., 319, 323, 341 Betarbet R., 564, 565, 620, 627 Bethe, A., 345, 373 Bethel, M. A., 101–103, 111, 112, 619, 626 Bettens, F., 321, 327, 343 Beuerman, R. W. 983, 996 Bever, T., 359, 377 Beyer, C., 343 Beyer, H., 984, 996 Beynon, R. J., 970, 971, 977, 978 Bézirard, V., xxiv, xxxiv Bezprozvanny, I., 511, 519

Bhagavan, S., 185, 186, 200 Bhalla, V., 849, 855, 858 Bhama, J. K., 57, 62, 68 Bhandary, K., 642, 647, 648 Bharani, N. K., 38, 48 Bhasin, N., 21, 44 Bhatnagar, K. P., xxx, xxxv, xxxiii, xliii, 3, 15, 17, 48, 142, 143, 155, 160, 512, 522, 616, 625, 969, 976, 978 Bianchet, M. A., 78, 86, 280, 293 Bianchi, L. M., 762, 779 Biasi, E., xxv, xxvi, xxxiii Bibas, A., 946, 954 Biberfeld, P., 103, 109 Bice, D. E., 60, 70 Bice, P. J., 885, 886, 898 Bick, D., 127, 137, 716, 730, 1003, 1006, 1017, 1021, 1024 Bickford, L., 444, 453 Bickford, R. G., 450, 458 Bieber, S. L., 739, 740, 743, 747, 748, 749, 750, 751, 755, 757 Biedenbach, M. A., 983, 996 Biel, M., 80, 90 Bielavska, E., 358, 376 Bienenstock, J., 538, 539, 544 Biffo, L., 124, 131 Bigelow, D. C., 797, 800 Biggs, J. J., 116, 137 Bigi, S., 362, 374, 422, 428 Bigiani, A., 721, 725 Bilaniuk, L. T., 593, 594, 604, 612 Bilek, F. S., 910, 932 Bilgen, E., 447, 454 Bilker, W. B., 37, 49, 259, 264–266, 273, 344, 470, 478, 495, 500, 512, 523, 593, 594, 599, 603, 613, 631, 633, 638 Bilko, A., 315, 322, 355, 370, 373, 833, 834, 838, 840 Billard, M., 392, 399 Billecke, S., 538, 539, 544 Biller, H. F., 598, 612 Billing, J., xix, xxxiv Billinghurst, L. L., 621, 624, 627 Billington, C. J., 826, 829, 844 Bilstad, J., 645, 648 Binding, N., 582, 588 Bindra, D., 173, 176, 182, 194 Binet, A., 403, 407 Bingaman, K. D., 515, 522 Binkley, K. E., 539, 542, 544, 546 Birbaumer, N., 261, 269 Birch, G. G., 807, 820, 884, 898 Birch, L. L., 831, 836, 838, 840, 841, 845, 882, 895, 908–909, 924 Birch, M. C., 352, 373 Bird, E. D., 603, 608 Bird, T. D., 509, 513, 522 Birnholtz, J. C., 833, 841 Birrell, G. B., 26, 29–31, 33, 45 Birsdal, N. J. M., 154, 162 Bisaga, A., 416, 434

Author Index Bishop, A., 356, 376 Bishop, E. R. Jr., 472, 474 Bishop, J., 981, 996 Bitgood, M. J., 765, 778 Bitterman, M. E., 354, 373 Biziere, K., 416, 426, 434 Bizzini, L., 480, 481, 495, 500 Bjaalie, J. G., 152, 156 Bjerke, T., 444, 456, 457 Bjork-Eriksson, T., 93, 108 Bjorklund, A., 261, 272, 515, 520 Bjorkman, S., 553, 562, 565 Bjornson, C. R., 621, 623 Bjornsonbenson, W. M., 886, 897 Bjorvell, H., 887, 903 Bjuro, T., 440, 458 Black, A., 449, 454 Black, A. H., 911, 926 Black, D. W., 532, 533, 535, 539, 541, 544, 546 Black, F. L., 340, 341 Black, I. B., 124, 132 Black, R. M., 882, 895 Black, S., 449, 456 Black, W. C., 504, 597, 611 Blackhart, G. C., 342 Blackwell, H. R., 206, 223 Blackwood, D. H. R., 603, 613 Blahser, S., 1003, 1023 Blais, C. A., 886, 895 Blaisdell, J. A., 61, 70 Blake, B. L., 56, 68, 553, 559, 567 Blakemore, L. J., 151, 163 Bläker, M., 644, 645, 648, 649, 658, 676, 698, 701 Blakeslee, A. F., 469, 474, 847, 848, 850, 856, 858 Blanchard, J. C., 416, 417, 432 Blanchard, K. T., 65, 67 Blancher, A., 291, 294 Blank, J., 418, 430 Blankenvoorde, M. F. J., 645, 650 Blaschuk, O., 125, 126, 132 Blasco-Ibanez, J. M., 154, 156 Blass, E. M., 310, 315, 320, 322, 323, 325, 326, 355, 356, 360, 376, 380, 382, 759, 779, 826, 828, 829, 841, 843–846, 907, 929 Blau, J. N., 950, 954 Blaustein, J., 919, 924 Bleecker, M. L., 538, 542, 544, 585, 590 Blendy, J. A., 420, 432 Blessing, W. W., 557, 565 Blitzer, A., 9, 16 Blizzard, D. A., 717, 725 Bloch, R. M., 530, 532, 538, 545 Bloch, S., 366, 367, 373 Block, F., xxviii, xxxiv, 253, 269 Blomhoff, H. K., 62, 69 Blomlof, J., 771, 772, 781 Blomquist, A. J., xxiv, xxxvi Blon, F., xxiv, xxxiv Blondheim, S. H., 888, 897

1033 Bloodgood, R. A., 19, 41 Bloom, F. E., 153, 162, 450, 458, 516, 519 Bloom, G., 19, 42 Bloom, L. M., 864, 878 Bloom, S. R., 76, 85 Bloomfeld, R. S., 893, 895, 950, 954 Blough, D., 869, 876 Blough, P., 869, 876 Blozis, G. G., 939, 940, 954 Blum, M., 861, 876 Blum, S. L., 356, 365, 373, 377, 404, 408 Blumberg, B. S., 855, 858 Blumcke, I., 93, 112 Blumer, L., 392, 399 Blumsack, J. T., 910, 932 Blundell, J. E., 882, 895, 897 Bluthe, R. M., 415, 416, 426, 427, 428, 430, 434, 909, 929 Blystad, A. K., 62, 69 Boakes, R. A., 916, 918, 932 Bobillier, P., 148, 152, 155, 156, 561, 565, 566 Boccuzzi, S., 62, 71 Bock, G. R., 36, 42 Bock, K. W., 58, 70 Bock, R., 80, 86, 711, 719, 727 Bocskei, Z., 970, 976 Boda, K., 919, 929 Bodforss, E., xxviii, xxxviii Bodian, D., xxvi, xxxiv, 556, 565 Bodner, L., 643, 648, 887, 895 Bodor, N., 920, 932 Bodurtha, J., 342 Body, G. D., 238, 245, 490, 498 Bodyak, N., 390, 392, 394, 397, 400 Boehm, N., 1003, 1012, 1021 Boehnke, M., 848, 850, 856, 860 Boekhoff, I., xxv, xxvi, xxxiv, xli, 26, 27, 33, 41, 46–4876, 79–81, 83–84, 86, 89, 90, 287, 294, 710, 729, 972, 977 Boelens, M. H., 276, 280, 282, 286, 293, 296, 306 Boerjesson, T., 305, 306 Boesen, F., 606, 608 Boes-Hansen, S., 553, 566 Boff, E., 914, 927 Bogart, L. M., 785, 802 Bogdanffy, xxvii, xxxviii, M. S., 52, 54, 56, 59, 60, 67, 71, 553, 566, 582, 583, 588, 589 Bogerts, B., 515, 518, 519, 603, 609 Boggess, K. A., 330, 340 Bohm, S., 100, 112, 117, 120, 121, 137 Bohme, G. A., 416, 417, 432 Bo-Hong, X., 580, 591 Bohus, B., 416, 432 Boillat, M. A., 586, 589 Boissy, A., 367, 375 Boistel, J., xxix, xxxiv Bojsen-Møller. J., 1002–1004, 1021 Bojsen-Muller, F., 450, 453

Boklage, C. E., 330, 340 Boland, P., 762, 779 Bolgar, M. S., 971, 978 Bolger, W. E., 3, 6, 15, 16 Bolhuis, J. J., 422, 427 Bolla, K. I., 581, 585, 588, 590 Bolla-Wilson, K., 538, 542, 544 Boller, F., 680, 702 Bolles, R. C., 906, 914, 924 Bolling, J. P., 469, 475 Bolner, A., 661, 677 Bolon, B., 392, 399, 552, 567, 583, 590 Bolze, M. S., 891, 895 Bolzoni, D., 318, 321, 326 Bombardieri, E., 891, 901 Bomsel-Helmreich, O., 330, 340 Boncinelli, E., 116, 132, 137 Bond, J. A., 52, 62–64, 67 Bondi, M. W., xxxii, xxxiii, 482, 483, 496 Bondy, C., 101, 107 Bone, I., 786, 791, 801 Bonett, D., 516, 522 Bonfanti, L., 128, 131 Bonhoeffer, T., 145, 160 Bonifacino, J. S., 31, 41 Bönigk, W., 27, 46, 80, 86 Bonino, M., 149, 155, 157 Bonnet-Font, C., 561, 566 Bons, N., 169, 178 Bonsall, R. W., 346, 363, 373, 375, 379 Bonte, B. L., 601, 608 Bonthius, D. J., 129, 131 Booij, J., 486, 501, 514, 519, 523 Boom, H. B., 241, 247 Boorman, G. A., 578, 583, 591 Boorsma, F., 416, 432 Boorstin, D. J., xvii, xxxiv Boot, L. M., xxix, xxxviii, 346, 378 Booth, A., 359, 373, 379 Booth, D. A., 218, 223, 321, 324, 350, 378, 420, 434, 830, 842, 884, 886, 897, 914, 925 Booth, M. C. A., 680, 700 Bootzin, R. R., 538, 544 Bora, R., 392, 399 Boraas, M., 891, 899, 910, 929 Boran, T. V., 172, 178 Borchard, A. C., 511, 515, 520 Borg, G., 210, 227, 667, 671, 856, 860 Borg-Neczak, K., 551, 566 Borhani, N. O., 886, 895 Boring, E. G., xv, xxii, xxxii, xxxiv, 805, 820 Borisy, F. F., 27, 41, 42, 79, 82, 86, 88 Born, J., 240, 247, 554, 563, 571 Bornfeld, N., 30, 31, 41, 100, 107 Borror, M. J., 617, 623 Borson, S., 909, 924 Bos, K. E. O., 885, 892, 901 Bos, P. M. J., 994, 996 Bosch, E., 992, 995 Bose, K., 828, 835, 842

1034 Bosma, J. F., 761, 764, 765, 767, 768, 775, 778, 780, 841, 843, 844 Bossert, W. H., 346, 383 Bossy, J., 35, 41, 115, 118, 120, 122, 131, 311, 322 Bossy, Y., 1002, 1021 Bostantjopoulou, S., 485, 496 Bothwell, M., 762, 781 Bothwell, M. A. 111, 113 Bottey, J. A., 713, 714, 717, 729 Böttger, B., 660, 672 Bouchard, T. J. Jr., 331, 332, 342 Bouchikhi, A., 449, 453 Boudreau, J. C., 734, 747, 748, 754 Boudriot, F., 660, 671 Bougara, A., 443, 455 Boughter, J. D. Jr., xvi, xliii, 661, 675, 664, 670, 676, 709, 710, 718, 725, 726, 737, 740, 752, 754, 755 Bougueleret, L., 1017, 1023 Boulet, M., 172, 174, 176 Bouloux, P. M. G., 1003, 1012, 1016, 1025 Bouman, J., 891, 900 Bouna, S., 153, 155 Bour, T. C., 776, 780 Bouras, C., 483, 498 Bouret, S., 192, 194 Bourgeais, L., 685, 700 Bourne, D. W., 554, 563, 571 Bourne, G. H., 56, 67 Bourne, H. R., 291, 294 Bouton, M. E., 420, 427, 907, 926 Bouvet, J. F., 84, 85, 388, 397, 982, 996 Bouwer, C., 472, 477 Bovbjerg, D. H., 838, 845 Bovolin, P., 101, 111, 149, 157 Bowdler, D., 946, 954 Bowen, D. V., 364, 381 Bowen, J. D., xxxii, xxxvii, 341, 483, 498 Bowenpope, D. F., 101, 110 Bower, J. M., xxxi, xxxiv, 187, 188, 190–193, 194, 196, 201, 266, 269, 410, 413, 425, 426, 430, 486, 501 Bowers, A. A., 259, 264–266, 270 Bowers, R. L., 917, 921, 925 Bowery, N. G., 183, 194 Bowler, R., 581, 589 Bowman, J. R., 392, 399 Bowman, K., 890, 903 Bowman, W., xxii, xliv, 18, 48, 981, 999 Bowtell, R., 193, 198, 259, 264, 266, 271, 698, 699, 700, 702, 797, 802 Boxer, P. A., 587, 588 Boyd, D. B., 276, 293 Boyd, I., 891, 895 Boyd, J. C., 517, 521 Boyd, M. R., 583, 590 Boyd, W. C., 857, 858, 858 Boyer, D. M., 124, 132 Boyer, R., 514, 522 Boyle, A. G., 81, 88 Boynton, R. M., 750, 751, 754

Author Index Boyse, E. A., 318, 320, 322, 327, 337, 340, 344, 356, 372, 383, 410, 437, 840 Boyse, J., 320, 327 Bozza, T., 126, 138 Bozza, T. C., 182, 185, 194 Bozzao, A., 602–603, 609 Braak, E., 152, 161, 510, 513, 515, 519, 522 Braak, H., 512–515, 519, 522, 563, 570 Bracilovic, A., 116, 133 Bracker, A., 994, 996 Brackin, H. B. Jr., xxxii, xliii, 480, 482, 501 Bradley, A., 621, 623 Bradley, E. A., 491, 492, 496 Bradley, J., 80, 86, 639, 640, 643, 644, 648, 658, 659, 663, 665, 666, 671, 658, 663, 672, 736, 754, 755, 758, 762, 764, 765, 768, 769, 775, 777, 778, 778–782, 784, 799, 824, 825, 841, 844 Bradway, D. M., 920, 925 Brady, S. T., 558, 566 Braffman, B. H., 599, 602, 608 Bragadin, L. M., 489, 501 Braget, D. J., 920–921, 924 Bragt, P. C., 994, 996 Brain, D., 362, 365, 374, 375 Brain, P. F., 362, 373 Brainerd, H. G., 483, 496 Brake, S. C., 361, 378 Brala, P. M., 882, 895 Bramerson, A., 462, 477 Brämerson Lidén, E., 204, 215, 227 Brammer, M. J., xxix, xlv, 256, 259, 266, 267, 273, 687, 702 Brand, G. 269 Brand, J. G., 20, 45, 221, 227, 243, 248, 644, 648, 660, 672, 709, 710, 712, 715, 718, 720, 723, 725, 729, 730 Brand, M., 116, 137 Brandemihl, A., 670, 674, 771, 781, 782 Brandl, U., 244, 244 Brandt, I., 63, 67, 550, 552, 553, 559, 566, 577, 582, 588 Brandt, J., 489, 496, 602, 612 Brannon, P. M., 888, 895 Braren, M., 239, 248 Brasch, R. C., 594, 608 Brashear, H. R., 147, 148, 153, 164, 170, 180, 413, 438 Bratter, P., 511, 522 Braun, J. J., xxxi, xxxiv, 33, 34, 41, 387, 394, 397, 917–918, 925 Braunewell, K. H., 84, 86 Brauning, H. 996 Braus, H., 656, 671 Braveman, N. S., 905, 911, 925 Braver, D., 311, 317, 323, 833, 842 Bray, G. A., 883, 892, 901 Brazier, J. L., 315, 326 Brazier, M. A., 916, 918, 925

Brazis, P. W., 469, 475 Brecht, G., 472, 476 Breckenridge, L. J., 36, 37, 41, 47 Bredt, D. S., 82, 90, 619, 626, 709, 727 Breedlove, S. M., 919, 924 Breen, M. F., 310, 322, 359, 373 Breer, H., xxv, xxvi, xxxiv, xli, 24, 26, 27, 32, 33, 36, 41, 46–48, 76–77, 79, 81, 83–84, 86, 88–90, 100, 110, 112, 126, 134, 137, 182, 197, 287, 293, 294, 333, 340, 709, 710, 729, 971, 972, 977 Breipohl, W., 2, 16, 24, 30–32, 35, 38, 41, 45–47, 62, 66, 73, 94–96, 97, 99–101, 107, 110, 112, 122, 135, 217, 226, 256, 271, 400, 494, 499, 590, 616, 625, 999, 1015, 1021 Breiter, H. C., 686, 702 Brennan, P. A., 187, 194, 320, 322, 368, 373, 413, 427, 970, 972, 975, 976, 976, 977 Brennand, C. P., 950, 955 Brenneman, D. E., 553, 563, 568, 578, 588 Brent, R. L., 863, 864, 867, 870, 871, 878 Bresee, J. S., 883, 900 Breslin, P. A., 788, 789, 795, 797, 798, 799, 800, 803, 857, 858, 865, 866, 873–875, 876, 878 Bressler, S. C., 975, 977 Bressler, S. L., 186, 194, 411, 427 Breton, B., 76, 86 Brett, L. P., 913, 925, 931 Brew, B., 605, 612 Brewer, D. W., 439, 441, 458 Brewer, E. D., 985, 997 Brewer, J. B., 262, 269, 270 Brewer, W. J., 491–493, 496, 500 Brezinka, C., 340 Brezinski, D. A., 64, 67 Brezmes, J., 305, 306 Brian, J. A., 828, 840 Briand, L., xxiv, xxxiv Brichet, A., 440, 456 Brick, P., 280, 293 Bridger, G. P., 4, 15, 441, 453 Bridger W. H., 837, 841 Brierley, T., 32, 36, 47 Briggle, T., 585, 588 Briggs, J. E., 826, 829, 844 Briggs, M., 579, 588 Briggs, W. A., 893, 899 Brightman, M. W., xxiv, xxxi, xxxv, xli, 142, 144, 162, 186, 199 Brightman, V. J., 216, 224, 440, 454, 629, 635, 637, 787, 788, 790, 791, 795, 799, 890, 894, 895, 896, 935, 938, 945, 950, 951, 954, 959, 961, 963, 965 Brignol, M. J., 828, 844 Brime, J. I., 831, 840 Briner, H. R., 462, 477 Brining, S. K., 732, 733, 754, 866, 876

Author Index Brinkley, L., 642, 649, 670, 674, 939, 954 Brinner, L. J., 891, 899 Brinon, J. G., 154, 156 Briois, L., 192, 194 Brisibe, F., 832, 844 Brittebo, E. B., 38, 44, 58, 61–65, 66–68, 550, 552, 553, 558, 559, 565, 566, 577, 582, 588 Brizzee, K., 216, 228, 803 Broad, K. D., 423, 424, 428, 431 Broadhurst, P. L., 367, 378 Broadus, K., 392, 399 Broadwell, R. D., 165, 171, 173, 176, 551, 556, 558, 560, 561, 565, 566 Broberg, D. J., 910, 925 Broca, P., xxviii, xxxiv Brock, J., 392, 399 Brock, S., 620, 627 Brockes, J. P., 767, 779 Brockstedt, U., 560, 569 Brodin, E., 983, 998 Brodin, P., 647, 649 Brody, D., 495, 496, 605, 608, 891, 895 Brody, J. A., 487, 500 Broeders, G., 554, 563, 571 Broekkamp, C. L. E., 920–921, 926, 931 Broffman, J., 241, 244 Broggio, E., 482, 496 Broich, G., 236, 247646, 647, 650 Broillet, M.-C., 149, 155 Broitman, S. T., 356, 380 Bromley, S. M., xxxii, xxxv, 238, 245, 256, 268, 270, 462, 474, 484–486, 495, 497, 586, 590, 601, 609, 935, 936, 938, 951, 954 Broms, P., 451, 453 Brondel, L., 888, 898 Bronen, R. A., 167, 178 Bronner-Fraser, M., 760, 778 Bronson, F. H., xxix, xliv, 349, 352, 358, 364–366, 368, 373, 374, 376, 379, 404, 407 Bronson, G., 908, 925 Bronshtein, A. A., xxv, xxxiv, Bronstein, P., 905, 925 Bronzert, D. A., 291, 293 Brookhart, J. M., 913, 930 Brookover, C., 1001, 1002, 1005, 1006, 1010, 1021 Brooks, C. D., 550, 453 Brooks, D. J., 601, 608 Brooks, J. W., 512, 520 Brooksbank, B. W., 363, 375 Brookshire, G. S., 603, 610 Broom, D. M., 318, 324 342 Brossut, R., 369, 379 Brosvic, G. M., xxxi, xxxiv, 184, 195, 392, 396, 397, 398, 470, 471, 484, 485, 494, 497, 474, 514, 515, 520, 620, 623, 863, 867–870, 872, 874, 876, 892, 895 Brouette-Lahlou, I., 358, 373388, 397 Brouillard, M., 38, 39, 41 505, 519

1035 Brousseau, K 483, 496. Brouwer, J. N., 713, 725 Brower, L. P., 913, 925 Brown, A., xxvi, xliv, 557, 572, 983, 998 Brown, A. C., 983, 996 Brown, A. S., 516, 523 Brown, C. E., 124, 135 Brown, C. J., 1017, 1022 Brown, C. W., 413, 427 Brown, D., 27, 4156, 67 Brown, G. G., 556, 564, 568 Brown, J., 209, 223, 646, 649 Brown, J. L., 618, 623 Brown, J. W., 311, 322 983, 996, 1001, 1002, 1017, 1021, 1022 Brown, K. W., 330, 343 Brown, K. S., 207, 224, 333, 337, 340, 342 Brown, M. J., 894, 901 Brown, R. E., xxx, xxxiv, 318, 320, 322, 326, 336, 340, 352, 356, 357, 373, 381, 394, 400, 405, 407, 410, 417, 422, 427, 435 Brown, R. J., 4, 5, 16 Brown, T. S., 406, 408, 410, 427 Brown, W. M., 263, 273 Brown-Grant, K., 923, 925 Browning, A. S., 192, 193, 199, 688, 695, 697–699, 700, 704, 829, 844, 948, 956, 959, 966 Brownlee, R. G., 346, 373 Brownson, E. A., 907, 926 Brownstein, M. J., 1005, 1023 Brubaker, R. F., 469, 475 Bruce, B. K., 542, 543 Bruce, D. G., 888, 895 Bruce, G., 190, 198 Bruce, H. M., xxix, xxxiv, 346, 349, 365–367, 373, 374, 380, 970, 977, 1003, 1024 Bruch, R. C., xxvi, xxxiv, 24, 27, 33, 45, 101–103, 108, 110, 124, 131 Brucke, T., 485, 499 Bruckel, J., 888, 897 Bruder, G. E., 472, 477619, 626 Bruera, E., 890, 895 Brugger, W. E., 234, 245, 984–987, 991, 997 Brulet, P., 116, 131 Brun, B., 606, 608 Brun, G., 96, 111 Brunch, R. C., 75, 80–81, 85, 86, 88 Brune, K., 238, 245 Brunelli, C., 891, 901 Brunelli, S. A., 310, 326 Brunelli, T., 62, 71 Bruner, H. D., xxviii, xxxviii Brunet, J-F., 760, 778 Brunet, L. J., xxvi, xxxiv, 24, 26, 27, 36, 41, 44, 76, 80–81, 86, 97, 103, 108, 126, 130, 131, 134 Brunjes, P. C., 118, 129, 131, 133, 135 139, 143, 151, 155, 156, 161, 184,

194, 310, 311, 322, 355, 372, 564, 567, 616–619, 623, 624, 626, 627 Brunken, W. J., 184, 195 Bruno, J. P., 310, 322 Brunzell, J. D., 892, 896 Brustle, O., 621, 623 Bryan, E. M., 330, 340 Bryant, B. P., 81, 90, 660, 672, 982, 996 Bryant, D. N., 233, 247 Bryant, S. H., 24, 43 Bucciarelli, T., 58, 66 Bucher, J. R., 578, 583, 591 Bucher, O., 14, 15 Buchheit, K., 64, 65, 73, 98, 113 Buchholz, J., 98, 101, 102, 108 Buchholz, J. A., 119, 132 Buchman, V. L., 762, 779 Buchner, K., 552, 558, 560, 561, 573 Buchsbaum, M. S., 480, 481, 491–493, 495, 496, 501 Buchwald, C., 596, 608 Buchwald, D. S., 533, 534, 544 Buck, F., 644, 648 Buck, L., xxv, xxxiv, xxxix, 24, 26, 28, 32, 36, 41, 47, 75, 76, 78, 80, 86, 90, 99, 100, 107, 112, 117, 120, 121, 130, 136, 137 139, 145, 155, 162, 163, 182, 185, 194, 198, 221, 224, 259, 269, 280, 287, 288, 293, 296, 306, 334, 340, 350, 351, 374, 379, 381, 387, 391, 397, 551, 560, 566, 568, 571, 572, 709, 710, 713, 714, 717, 723, 725, 728, 798, 802, 972, 976, 978 Buck, R., 942, 954 Buck, S. H., 907, 925 Buck, T.M., 297 Buckland, M. E., 28, 43, 101, 102, 107, 619, 623 Buckley, L. A., 582, 583, 588 Buckner, R. L., 258, 269, 269, 270 Buckpitt, A., 583, 590 Buckwalter, J. G., 480, 498 Budd, K. C., xxxi, xlii, 788, 803 Budge, D., xvi, xxxiv Buee, L., 488, 496 Buee-Scherrer, V., 488, 496 Buell, G., 719, 725 Bufe, B., 722, 729 Buffone, A., 130, 131 Buhl, E. A., 311, 325, 1002, 1022, 1023 Buiakova, O. I., 27, 41, 123, 131, 150, 155, 396, 397, 505, 522 Bujas, Z., 785, 789, 791, 799, 864, 876 Bulcavage, L., 909, 910, 928 Buletic, Z., 798, 800 Bulfone, A., xxvii, xxxiv, 117, 136 Bull, T. R., 667, 671, 789, 799, 945, 954 Bullinger, A., 312, 325, 355, 380 Bullingham, R. E., 554, 563, 569 Bullmore, E. T., xxix, xlv, 256, 259, 266, 267, 273, 687, 702 Bullock, T. H., 748, 756, 1001, 1022

1036 Bunegin, L. J., 538, 546 Bunge, M. 111, 112 Bunney, Jr., W. E., 516, 518 Bunsey, M., 419, 420, 427, 436 Bunzow, J. R., 560, 570 Buonviso, N., 185, 186, 188, 194, 418, 427 Burch, R. E., 893, 895 Burcharth, F., 888, 894 Burchell, B., 58, 69, 70 Burd, G. D., 151, 152, 155, 156, 552, 558, 566 Burdach, K. J., xxxi, xxxiv, 216, 224, 463, 474 Burdi, A. R., 3, 16, 969, 979 Bures, J., 416, 429, 905, 915–918, 925, 928 Buresova, O., 915–918, 925 Burge, J. C., 893, 895 Burgen, A. S. V., 154, 162 Burger, B. V., 356, 374 Burger, P. C., 602, 609 Burghard, G. M., 315, 322, 968, 977 Burich, A. J., 1313, 1025 Burish, T. G., 909–910, 926 Burk, J. A., 174, 180, 194, 201, 394, 400, 411, 414, 415, 426, 433, 438 Burke, J. P., 63, 66 Burke, R. J., 207, 215, 228, 388, 400 Burket, W., xvi, xxxiv Burkl, W., 658, 671 Burks, T. F., 907, 925 Burley, V. J., 882, 901 Burman, D. M., 552, 567 Burnet, F. M., xxvi, xxxiv Burns, F., 82, 86 Burova, T. V., 77, 86 Burr H. S., 115, 127, 130, 131, 1017, 1022 Burritt, E. A., 906, 925 Burrow, L., 891, 895 Burrows, A. M., 3, 16, 969, 979, 982, 996 Burt, D. G., 62, 72, 582, 590 Burton, H., 680, 686, 688, 692, 700, 703 Burton, M. J., 688, 703, 916, 918, 932 Burton, P. R., 19, 23, 41 Burton, S., 419, 427 Buscher, D. S., 528, 543 Busenbark, K. L., 484, 486, 495, 496 Bush, G., 334, 343 Bush, K. T., 81, 86 Bushell, G. R., xxvii, xxxix, xl, 36, 46, 93, 98, 103, 110, 111 Busk, L., 56, 64, 65, 68, 70 Büssem, H., 826, 830, 841 Butcher, L. L., 153, 161 Butcher, M. J., 828, 843 Butcher, S., 185, 197 Butera, P. C., 920, 925 Butler, A. B., 167, 172, 176 Butler, J. A. V., 643, 648 Butler, R., 554, 563, 566 Butter, C. M., 882, 898

Author Index Butterfield, N. J., 831, 840 Butters, N., 209, 211, 214, 226, 227, 424, 431, 435, 495, 497, 602, 604, 610, 612, 886, 896, 897, 898 Butterworth, B. E., 582, 589 Buttery, R. G., 204, 225 Buttrick, R. L., 713, 725 Butzin, C. A., 6, 15 Buxton, R., 942, 954 Bylsma, F. W., 489, 496 Bystrova, M. F., 54, 71 Cabanac, M., 690, 691, 696, 700, 882, 888, 892, 895, 898, 907, 925 Cabezas, M. 306 Cabib, S., 359, 375 Cabrera, T., 314, 322 Cadiou, H., 26, 41 Cagan, R. H., xxv, xli, 24, 34, 47, 76, 78–79, 90, 660, 672, 712, 713, 725, 746, 758 Caggana, M., 55, 57, 72 Caggiano, M., 33, 41, 75, 76, 86, 106, 108, 119, 131 Caggiula, A. R., 887, 903 Cahill, C., 516, 520 Cahill, L., 414, 425, 427 Cahn, H., 813, 822 Cai, X., 24, 26, 44, 75, 90, 123, 135 Caicedo, A., 664, 671 Caillaud, C., 551, 567 Cain, D. P., 148, 155, 173, 176, 182, 194 Cain, W. S., xv, xxxiv, 100, 108, 204, 206, 207, 212, 215–219, 224, 226–228, 236, 247, 256, 268, 269, 270, 388, 398, 466–468, 472, 476, 477, 494, 496, 499, 576, 588, 629, 633, 634, 637, 709, 726, 783, 784, 786, 788, 790–792, 799, 801, 803, 805, 807, 817, 820–822, 856, 859, 889, 896, 983, 987, 988, 990–997 Caine, E. D., 601, 610 Cairns, N. J., 509, 512, 521, 522 Cairns, R. B., 362, 374 Cajal, R. S. xxii, xxxiv, 139, 142, 146, 162 Calas, A., 152, 153, 154, 155 Caldani, M., 154, 164, 1003, 1025 Caldarelli, D., 468, 475 Calder, A. J., 687, 702 Calhoun, L. L., 582, 583, 589, 590 Calhoun, M. E., 618, 623 Call, T., 210, 225 Callaham, E. M., 721, 722, 726 Callan, R., 315, 325 Callender, M., 528, 543 Calles-Escandon, J., 888, 895 Callieco, R., 236, 247 Calne, D. B., 343, 485, 501 Calof, A. L., xxvii, xxxiv, xxxv, 76, 86, 96–98, 101–103, 106, 108, 109, 111, 112, 119, 121, 131–133 Calvert, G. M., 585, 588

Calvert, I. A., 526, 546 Calviño, A. M., 987, 993, 997 Calvo, P., 554, 563, 567 Camacho, F., 416, 418, 426, 437 Camara, C. G., 96, 101, 102, 106, 108, 616, 623 Camarino, G., 1001, 1017, 1022 Cambier, J., 488, 496 Cambillau, C., 19, 48 Cameron, A. T., 806, 820, 829, 841 Cameron, J., 36, 41 Camicioli, R., 564, 565 Camman, K., 582, 588 Cammarata, S., 505, 506, 523, 563, 572 Campagne, F., 713, 714, 717, 728 Campanacci, V., 19, 48 Campanella, G., 494, 496 Campbell, B. A., 310, 325, 390, 392, 397, 398 Campbell, C. B. G., 354, 377 Campbell, D. T., 788, 799 Campbell, I. M., 213, 214, 224, 490, 492, 496 Campbell, K., 77, 87, Campbell, K. H., xxxi, xli, 403, 408, 861, 734, 757, 873, 878 Campbell, N. A., 347, 374 Campbell, R. B., 920, 925 Campbell, R. G., 690, 704, 838, 841 Campbell, S. M., 941, 954 Campfield, L. A., 888, 902 Campioni, P., 647, 648 Cancalon, P. F., 558, 566 Candaras, M. M., 947, 957 Canessa, C., 719, 725 Canetto, S., 827, 842, 906, 927 Canguilhem, B., 618, 625 Cannon, D. S., 920–921, 925 Cannon, L., 634, 637 Cannon, T. D., 516, 519 Cano, J., 887, 895 Canonaco, M., 63, 70 Cantino, D., 149, 150, 155, 157, 162 Canto, P., 887, 903 Cantor, R. M., 848, 860 Cantrell, S. A., 552, 571, 582, 590 Canty, J. S., 882, 895 Cao, J. Q., 61, 69 Cao, L., 64, 73 Capdevila, J. H., 61, 67, 71 Capeless, C. G., 709, 710, 725, 726 Capra, C., 424, 433 Capretta, P. J., 316, 322, 838, 841, 908, 925 Caprio, J., 723, 725 Capurso, S.A., 618, 623 Carakostas, M. C., 582, 591 Carani, C., 359, 374 Carbajal, R., 829, 841 Cardello, A. V., 784, 794, 803, 818, 820 Cardon, L. R., 331, 342 Caretta, A., 369, 379, 976, 978 Carleton, A., 152, 154, 155, 156

Author Index Carlone, H. B., 63, 68 Carlsen, J., 147, 148, 153, 164, 170, 180, 413, 438 Carlson, A. D., 359, 377 Carlson, D. M., 644, 649 Carlson, J. R., xxv, xxxv, xxxiv, 334, 335, 340, 341, 342 Carlson, N. R., 410, 427 Carmanchahi, P. D., 30, 42 Carmelli, D., 483, 501 Carmichael, S. T., 167, 169, 170, 173, 176, 192, 194, 199, 263, 268, 269, 680, 693, 700, 702 Carnahan, J., 620, 624 Carner, E., 512, 513, 522 Carnes, K. M., 170, 176, 192, 199 Caroff, S., 516, 520 Caron, A.L., 4, 15 Caron, M. G., 27, 33, 46, 84, 85, 87–90 Caroom, D., 353, 374, 404, 407 Carpenter, J. A., 732, 734, 754, 917, 925 Carpenter, M. B., 947, 954 Carr, V. M., 75, 86, 95, 96, 98, 108, 145, 155, 616, 623 Carr, V. McM., 26–28, 30–33, 38, 34, 41, 45, 118, 121–125, 131, 132 Carr, W. E. S., 56, 60, 72, 807, 821 Carr, W. J., xxx, xxxi, xxxiv, 352, 357, 362, 374, 403, 404, 407, 422, 427, 867, 870, 876 Carraro, S., 890, 895 Carrasco, M., 212, 224 Carregal, E. J. A., 982, 999 Carrell, R. W., 509, 519 Carroll, B., 494, 496 Carrozzo, R., 127, 133, 1007, 1017, 1022 Carruth, B. R., 833, 845 Carson, C. A., 865, 876 Carson, J. S., 890, 891, 895 Carson, K. A., 146, 147, 148, 153, 155, 171, 176 Carstensen, K., 1004, 1022 Carter, B. L., 596, 609 Carter, C. S., xxx, xxxv, 352, 356, 374, 404, 405, 407, 422, 427, 432, 437 Carter, J. A., 147, 153, 157, 170, 177 Carter, J. L., 951, 954 Carter, J. M., 517, 521 Carter, J. N., 471, 476, 892, 899 Carter, R., 812, 813, 821 Carterette, E. C., xv, xxxiii, 799 Cartford, C., 723, 725 Carus, C. G., 658, 671 Carvalho, P. A., 593, 594, 610 Casanova, M. F., 603, 612 Casanova-Schmitz, M., 56, 67 Casarosa, S., 119, 121, 132 Cascio, C., 422, 430, 437 Casella, V. xxviii, xli Casper, R. C., 921, 925 Cass, M., 240, 244 Cass, S.P., 945, 954 Cassell, M. D., 551, 557, 565

1037 Casserius, J., xx, xxxiv, 652, 671 Cassidy, A. J., 58, 69 Cassidy, K. L., 831, 840 Casson, I. F., 341 Castano, D., 362, 373 Castillo, P. E., 154, 155 Castle, P. C., 329, 340 Castonguay, W., 864, 878 Castro, B. M., 368, 374 Castro, J. L., 509, 521 Casuto, D., 620, 624 Catalanotto, F. A., xxxi, xxxviii, xlv, 212, 224, 463, 475, 629, 637, 784, 787, 788, 790, 791, 793, 799, 800, 802, 831, 841, 889, 890, 896, 897, 938, 949, 954, 957 Caterina, D., 127, 134 Caterina, M. J., 983, 996, 1017, 1023 Catt, K. J., 1004, 1023 Cattarelli, M., 55, 69, 170, 176, 188, 190–192, 195, 198, 199, 255, 259, 271, 515, 520 Cau, E., 119, 121, 132 Caul, W. F., xxx, xxxiv Cauna, D., 11, 12, 15 Cauna, N., 11, 12, 15, 982, 996 Cavada, C., 170, 176, 192, 195 Cavaggioni, A., 359, 369, 374, 379, 970, 976, 976, 978 Cavaliere, F., 444, 456 Cavallaro, M., 621, 625 Cavanagh, D. G., 54, 71 Caza, P. A., 319, 322 Cazzato, G., 489, 501 Cecchi, C., 116, 132 Cecchini, A. P., 236, 247 Cecil, J. E., 887, 895 Cecyre, D., 619, 626 Cedaro, L., 890, 895 Cederberg, C. G., 553, 566 Cekic, M., 190, 196, 413, 430 Celebi, G., xxvii, xl, 93–95, 98, 111 Celio, M. R., 83, 85 Cendes, F., 268, 270, 424, 431 Centerwall, W. R., 833, 843 Cerf, B., 797, 800, 948, 954, 955, 960, 962, 965 Cerf-Ducastel, B., 256, 269 Cermak, L. S., 495, 497, 886, 896 Cernoch, J. M., 318, 319, 321, 322, 325, 338, 339, 340, 343, 834, 841 Cesare, P., 983, 996 Chaban, R., 442, 453 Chabaud, P., 174, 175, 176, 187, 188, 190, 191, 195, 198 Chabert, M., 888, 894 Chacon, R., 890, 895 Chadwick, D., 645, 649 Chadwick, P. R., 305, 306 Chaen, T., 54, 67 Chahl, L. A., 907, 932 Chaillan, F. A., 411, 412, 425, 427, 435 Chakrabortty, S., 621, 625

Chakravorty, S., 442, 458 Chalansonnet, M., 185, 195, 418, 427 Chalke, H. D., 216, 224 Chamberlain, M. P., 65, 67 Chambers, K. C., 907, 911, 915–916, 918–921, 925, 926, 932 Chambon, P., 62, 67 Champigny, G., 721, 730 Champlin, A. K., 348, 350, 352, 382, 383 Chan, G. C., xxvi, xliv, 24, 26, 27, 37, 49, 76, 80, 90, 397, 401 Chan, S., 516, 521, 882, 885, 895 Chandiok, S., 305, 306 Chandra, K. R., 909, 926 Chandrasekharappa, S., 514, 522 Chandrashekar, J., xxv, xl, 709–711, 714, 716, 717, 723, 724, 725, 728, 798, 798, 848, 858 Chang, C.Y., 945, 954 Chang, F. C. T., 683, 684, 700, 736, 747, 748, 754, 756, 918, 926 Chang, I. Y., 64, 71 Chang, L. W. 588 Chang, P. Y., 581, 591 Chao, S. K., 28, 48, 78, 89, 99, 113, 126, 130, 135, 137, 146, 160, 164, 296, 307, 354, 379, 560, 572 Chapelot, D., 882, 896 Chapman, C., 423, 431 Chapman, C. A., 191, 195 Chapman, P. R., 593, 594, 610 Chapman, V. M., 366, 374 Chapnik, J., 442, 444, 452, 457, 458 Chaput, M. A., 182, 184–186, 194, 195, 287, 293, 418, 421, 427 Char, H., 553, 563, 566 Charalambous, G. 843 Charles, P. C., 550, 551, 557, 566 Charpak, S., 151, 162 Charpentier, J., 289, 294 Chase, T. N., 510, 519, 602, 612 Chastrette, M., 991, 996 Chatterjee, A., 717, 724 Chaudhari, N., 664, 673, 690, 700, 723, 725 Chaudhry, M. R., 444, 453 Chauvet, X., 829, 841 Chavez, A. F., 421, 427 Chavez, M., 915, 924 Chawluk, J. B., 516, 520 Chazal, G., xxviii, xxxvi, 143, 156, 517, 519 Cheal, M., 768, 773, 778, 779 Checke, S., 914, 927 Checkoway, H., 564, 565 Chedester, A. L., 915, 931 Cheeseman, G. H., 204, 224 Chegini, N., 643, 649 Chen, C., 103, 108 Chen, C. C., 722, 725 Chen, D. Y., 917, 928 Chen, E. H., 942, 956 Chen, J., 26, 27, 48, 359, 376

1038 Chen, K., 291, 293 Chen, K. M., 480, 484, 488, 497, 563, 567 Chen, N., 789, 804, 953, 957 Chen, P., 970, 979 Chen, S., xxvii, xxxiv, 80, 130, 131 Chen, S. H., 480, 482, 498 Chen, S. R., 511, 521 Chen, T.-Y., 27, 41, 83, 86 Chen, W. R., 151, 155, 185, 195, 396, 399 Chen, X., 553, 567 Chen, Y., 54, 56–58, 67, 69, 969, 979 Chen, Z., 717, 724, 760, 780 Chencharick, M. S., 891, 900 Cheng, F. Y., 915, 929 Cheng, J., 76, 88 Cheng, L. H., 656, 658, 671 Cheng, S. W. T., 910, 930 Cheng, Y. S., 583, 588 Cheng-Mo, L., 580, 591 Cherniack, N. S., 450, 455, 999 Cherry, J. A., 27, 41, 972, 975, 977 Chesne, P., 318, 325, 343 Chess, A., 296, 306 Chester, A. C., 534, 544 Chhabra, R., 887, 898 Chi, H. Y., 581, 591 Chiaia, N. L., 760, 782 Chiang, C., 116, 132 Chiang, G., 417, 431 Chiarotti, F., 362, 374, 422, 428 Chiasson, B. J., 620, 623 Chiba, A. A., 173–175, 179, 182, 199, 421, 425, 435, 916, 927 Chiba, N., 709, 711, 727 Chickering, T. W., 1013, 1025 Chien, C. B., 670, 671, 767, 778 Chikaraishi, D. M., xxvii, xxxiv, 76, 86, 106, 108, 118, 119, 131, 137, 560, 573, 616, 626 Chilla, R., 789, 799 Chinn, H., 952, 955 Chinn, H. I., 915, 926 Chinnock, R. F., 833, 843 Chintanadilok, J., 554, 563, 569 Chiodo, L. K., 483, 500 Chipman, R. K., 365, 366, 374 Chirino, R., 370, 376 Chirurgi, R. J., 490, 500 Chisholm, C. A., 330, 340 Chisholm, D. J., 888, 895, 898 Chistoffel, K. K., 942, 956 Chitanondh, H., 261, 269 Chitayat, D., 311, 317, 323, 833, 842 Chkheidze, D. D., 148, 155 Chlebowski, R. T., 909–910, 928 Cho, E-S., 605, 611 Cho, H., 76, 80–81, 90 Cho, J. H., 443, 453 Cho, J. Y., 151, 155, 564, 566 Cho, Y. S., 468, 476 Chobor, K. L., xvi, xxxvii, 121, 137, 216, 228, 424, 436, 996 Chodos, E., 856, 857, 859

Author Index Chohan, K. S., 1004, 1025 Choi, E. J., 80, 86 Choi, J. O., 466, 476 Choiset, Y., 77, 86 Chorover, S. L., 185, 187, 198, 199 Chorsky, R., 1003, 1024 Chotro, M. G., 315, 322, 885, 896 Chou, B. J., 583, 590 Chou, J. H., 78, 90 Choudhary, K., 621, 623 Chouvet, G., 154, 162 Chowdhury, J. R., 58, 70 Chrislip, D. W., 585, 588 Chrisp, R. B., 807, 822 Christakos, S., 511, 522 Christensen, C. M., 644, 648, 887, 891, 895, 899, 950, 954 Christensen, E., 893, 903 Christensen, K. M., 313, 317, 325 Christensen, M. B., 444, 457 Christensen, N. J., 888, 894 Christensen, T. A., 181, 185, 186, 193, 195, 200 Christensson, E., 314, 322 Christensson, K., 313, 314, 322, 326, 327 Christian, J. J., 366, 367, 374 Christiansen, H., xxiv, xlii, 644, 649, 658, 664, 676 Christiansen, R. L., 793, 794, 801 Christie, J. M., 151, 155, 185, 195 Christman, S., 268, 270, 495, 497 Christopher, D. E., 797, 802 Christy, R. C., 664, 670, 676, 710, 725 Chu, C. C., 513, 519 Chuah, M. I., xxvii, xxxv, xxxix, 24, 35, 36, 41, 44, 93, 97, 101, 102, 104, 107, 108, 110, 113, 119, 120, 122–126, 128, 132, 134, 135, 137, 138, 140, 159, 511, 519, 825, 841, 1011, 1022 Chuang, H. Y., 581, 588 Chuck, B., 837, 843 Chui, H., 480, 496 Chui, H. C., 481, 485, 489, 491, 496, 497, 498 Chun, H. S., 511, 515, 519 Chung, C. K., 891, 895 Chung, S. K., xxxii, xxxv, 392, 398, 480, 497 Chuwang, I.-W., 124, 136 Cianfrone, G., 230, 232, 244 Ciaranello, R. D., 152, 160 Ciechanover, M., 950, 954 Cinelli, A. R., 145, 155, 158, 192, 193, 195, 268, 269 Cinotti, L., 258, 264–266, 272, 424, 435 Cinquanta, L., 54, 68, 101–103, 109 Cintron, N. M., 554, 563, 571 Ciombor, K. J., 149, 153, 156, 187, 196 Cisternino, M., 450, 454 Citron, J.H., 364, 381 Citti, L., 63, 64, 70

Civelli, O., 560, 570 Claas, B., 1001, 1003, 1023 Clair, A. L., 492, 501 Clancy, A. N., xxx, xxxv, 141, 142, 160, 162, 185, 198, 369, 381, 423, 437 Clapp, T. R., 709, 725 Clare, S., 698, 699, 700 Clark, A. B., 357, 374 Clark, A. W., 513, 523 Clark, C., 481, 491–493, 496, 498, 499 Clark, C. C., 795, 799 Clark, C. M., 480, 500, 514, 521 Clark, H. B., 512, 523 Clark, J. C., 267, 269 Clark, J. T., 922, 926 Clark, M. M., 838, 842, 908, 927 Clark, T. B. III., 793, 803 Clark, W., 643, 649 Clark, W. E. L., 145, 156, 556, 566 Clarke, D. L., 620, 621, 623, 624 Clarke, J. E., 365, 374 Clarke, R. W., 452, 453 Clarke, S. W., 440, 455 Clarke, J. L., xxii, xxxv Claude, P., 1013, 1025 Clausen-Schaumann, H., 305, 306 Claverie, J. M., 127, 134, 1017, 1023 Clavet, G., 466, 468, 476–477 Clearman, D. R., 886, 896 Clee, M. D., 891, 895 Clegg, F., 360, 374 Clegg, R. A., 411, 425, 428 Clegg, R. T., 452, 458 Cleland, J., 336, 338, 341 Clemens, L. G., xxx, xxxv, 404, 407 Clemens, M. G., 82, 85 Clement, P. A., 443, 458 Clemente, D. D., 9, 10, 15 Clemente, F. R., 3, 16, 969, 979 Clements, S., 644, 649 Cleveland, C., 538, 545 Cleveland, C. T., 715, 725, 793, 799, 807, 820 Cliffer, K. D., 773, 782 Clifton, P. G., 919, 926 Clinton, D. K., 923, 932 Cloquet, H., xv, xxxv Clos, J., 620, 623 Clough, I., 552, 567 Clugnet, M. C., 167, 169, 170, 173, 176, 192, 199, 263, 268, 269, 680, 693, 700 Clulow, F. V., 365, 374, 382 Clyne, P., xxv, xxxv Coates, E. L., 27, 41 Coblentz, J., 420, 432 Coburn, K. L., 421, 427 Coccia, V. J., 81, 86 Codina, J., 84, 88 Cogen, R. B., 941, 956 Coghlan, J. P., 124, 133, 742, 757 Cohan, F. M., 334, 342 Cohen, D., 127, 134, 241, 244, 1017, 1023

Author Index Cohen, E., 829, 843 Cohen, J., 116, 134, 856, 859, 923, 928 Cohen, J. A., 489, 496 Cohen, L. B., 190, 195 Cohen, M. H., 890, 903 Cohen, N. J., 411, 412, 414, 418, 419, 425, 427–429 Cohen, R.Y., 210, 227 Cohen, W., 604, 609 Cohen, Z., 856, 857, 859 Cohn, H., 714, 729 Coil, J. D., 906, 927 Coines, A., 98, 108 Colby, D. R., 368, 374 Cole, P., 4, 5, 11, 15, 441–444, 450, 452, 453–455, 457, 458, 467, 474 Colebatch, J. G., 267, 269 Coleman, E., 262, 265, 266, 271, 493, 499 Coleman, J. R., 774, 775, 779 Coleman, K. J., 887, 896 Coleridge, G., 403, 407 Coles, M. G. H., 240, 245 Coletti, L. M., 33, 34, 41 Colgan, P., 309, 317, 323 Colingo, K. A., 945, 957 Colletti, L. M., 118, 121–123, 132 Collingridge, G. L., 413, 420, 421, 433 Collings, V. B., 685, 700, 784, 790, 794, 799 Collins, G. E., 240, 248 Collins, L., 641, 648 Collins, M., 440, 456 Colliver, J. A., 209, 225, 872, 877 Colquhoun, D., 290, 293 Colton, T., 923, 928 Comar, D., 258, 264–266, 272, 424, 435 Combarros, O., 960, 965 Cometto-Muñiz, J. E., 217, 219, 224, 227, 256, 269, 576, 588, 981–983, 987, 988, 990–995, 995–997 Comiter, H., 268, 270 Compain, S., 127, 134, 1017, 1023 Company, T., 170, 176, 192, 195 Comstock, J. M., 554, 563, 571 Condon, C. D., 191, 195 Cone, J. E., 526, 538, 544, 634, 637 Cone, T. E., Jr., 319, 323 Conger, A. D., 891, 895, 939, 954 Conier, J. 845 Conn, F. W., 470, 474 Connell, D. W., 991, 997, 998 Connes, R., 660, 671 Connolly, M. E., 3, 15 Connors, B. W., 347, 372 Conover, J. C., 762, 779 Conrad, P., 480, 481, 485, 500, 501, 893, 895 Conrad, W. A., 441, 454 Conran, R. M., 3, 16 Constantinescu, C. S., 489, 496 Constanzo, R. M., 75, 86 Contarini, G., xx, xxxv Conti, M., 82, 85

1039 Conti-Fine, B. M., 553, 569 Continella, G., 367, 375 Contreras, R. J., 470, 476, 793, 684, 700, 747, 754, 799, 865, 876, 917–918, 929 Conzelmann, S., 36, 41, 48, 126, 137 Cools, A. R., 416, 434 Coombes, S., 909, 929 Coome, D., 93, 112 Coon, H., xxvii, xliv, 98, 113 Coon, M. J., 51, 53, 56–58, 61, 62, 64, 68, 71–73 Cooper, A. J., 310, 323 Cooper, D., 670, 674, 771, 781 Cooper, D. M. F., 80, 86, 89 Cooper, G. P., 891, 895 Cooper, K. J., 368, 374 Cooper, P.W., 452, 455 Cooper, R., 233, 249 Cooper, J. B., xxx, xxxviii Coopersmith, C. B., 422, 431 Coopersmith, R., 184, 187, 195, 355, 360, 374 Copeland, N. G., 116, 133, 136 Coplan, J., 839, 844 Copolov, D. L., 492, 493, 496 Coppola, D. M., 312, 323 Coquelin, A., xxx, xxxv, 364, 373 Coraboeuf, E., xxix, xxxiv Corbett, A., 471, 476, 892, 899 Corbett, D., 557, 570 Corbey, R., 554, 563, 571 Corbin, A., xv, xix, xxxv Corbit, J. D., 862, 878 Corcoran, C., 485, 493, 499, 501 Corcoran, P., 305, 307, 484, 501 Cordell, B., 513, 522 Corden, J. L., 116, 132 Cordero, M., xxvii, xli, 105, 107, 111 Cordick, N., 915, 926 Cordone, G., 505, 506, 523, 563, 572 Corey, D. P., 971, 977 Corey, J. P., 441, 442, 444, 450, 452, 454, 455, 457 Corey, L. A., 848, 860 Corey, P., 539, 546 Corfman, T. P., 919, 928 Corkin, C., 208, 209, 225 Corkin, S., 421, 424, 428, 481, 498 Cormier, Y., 440, 457, 458 Cornsweet, T. N., 206, 224 Cornwell, C. A. 374, 356 Cornwell-Jones, C. A., 405, 407 Coronas, V., 101, 103, 108, 151, 154, 156, 183, 184, 195 Corotto, F. S., 616, 617, 620, 622, 623 Correia, M. J., 749, 751, 757 Correig, X., 305, 306 Corridi, P., 362, 374, 422, 428 Corso, J. F., 216, 224 Corvol, J-C., xxv, xxxvii Corwin, J., 480, 481, 483, 485, 496, 500, 501, 893, 895

Coryell, C. D., xxviii, xli, 254, 271 Coslett, D. S., 62, 63, 67 Cossins, J.-M., 104, 108, 126, 132 Costa, E., 153, 162 Costa, L. G., 539, 544 Costanzo, D. J., 403, 404, 407, 422, 427 Costanzo, R. M., 18, 23, 24, 27, 30–32, 34, 36, 37, 42, 44, 46, 96, 108, 119, 125, 132, 215, 225, 395, 396, 400, 473, 474, 580, 584, 590, 604, 609, 616, 623, 627, 629, 631, 632, 633, 634, 635, 637, 638, 789, 799, 959, 960, 965 Coste, A., 444, 457 Costes, N., 174, 179, 193, 199, 258, 261, 264–266, 272, 424, 435 Cote, R. H., 81, 86 Cotman, C., 661, 676 Cotman, C. W., 151, 156, 320, 326, 481, 485, 489, 498, 601, 610 Cotter, M., 664, 677 Cottle, M. H., 4, 15 Cottler Fox, M., 661, 671 Couderc, S., 829, 841 Coudert, P., 317, 323, 369, 370, 374 Coughlin, M. D., 124, 132 Coulon, P., 551, 557, 565, 569 Coulson, A. H., 750, 751, 755 Coulter, D. M., 891, 895 Couly, G. F., 115, 132 Counsell, C., 893, 901, 950, 956 Coureaud, G., 310, 315, 317, 323, 369, 370, 374, 375, 381 Courtney, L., 920–921, 924 Coury, A., 422, 429 Cousens, G., 413, 414, 425, 428, 434 Cousineau, D., 828, 840 Coussen, F., 24, 44 Cova, L., 621, 625 Covey, E., 751, 754, 816, 821 Covington, C., 313, 324 Covington, J. A., 299, 307 Covington, J. W., 234–237, 240, 244, 245, 247, 342, 462, 476 Cowan, W. M., 680, 701 Cowart, B. J., xxxiii, 210, 221, 225, 227, 243, 248, 317, 321, 322, 466–467, 474, 599, 609, 783, 786–788, 790–792, 799, 801, 804, 825, 828–833, 853, 854, 859, 840, 841, 843, 845, 886, 889, 890, 894, 899, 902, 936, 950, 952, 954, 955, 957 Cowen, M. A., 491, 496 Cowey, A., 908, 933 Cowley, J. J., 310, 323, 363, 368, 375 Cox, D. J., 538, 539, 542, 544 Cox, D. R., 961, 965 Cox, G., 539, 544 Cox, K. A., 971, 978 Cox, N., 342 Coyle, J. T., 513, 523 Crabb, J. W., 83, 89 Craft, H. D., 359, 380

1040 Cragoe, E. J., 76, 89 Cragoe E. J. Jr., 718, 727 Craig, C. G., 620, 623, 625 Craig, U. K., 485, 488, 495 Craig, W., 865, 876 Crain, B., 603, 611 Cramer, C. P., 310, 322 Cramer, R. E., 913, 927 Crandall, J. E., 972, 978, 1016, 1025 Crandall, P. H., 424, 435, 603, 612 Cranny, A. W. J., 300, 306 Crapper McLachlan, D. R., 554, 555, 563, 566 Craven, M., 305, 307 Crawford, C. 343 Crawford, D., 5, 15, 37, 44, 484, 485, 486, 497, 501 Crawford, J., 1017, 1021 Crawley, B. A., 305, 306 Crebolder, J., 736, 756 Creelman, C., 209, 227 Cremer, H., xxviii, xxxvi, 143, 156, 517, 519 Crescimanno, C., 659, 661, 675 Crespo, C., 154, 156 Crespo-Facorro, B., 269, 269, 491, 496 Crews, M. L., 62, 72 Crick, F. H. C., 740, 749, 754 Crino, P. B., 507, 509, 514, 519 Crisp, A. H., 921, 926 Crist, R. E., 190, 196 Critchley, H. D., 192, 193, 195, 199, 263, 264, 269, 272, 421, 428, 435, 688, 692, 694, 695, 697, 698, 700, 704, 829, 844, 948, 952, 954, 956, 959, 966 Crites, S. L., 882, 899 Crofton, K. M., 60, 63, 68, 553, 566, 567, 583, 589 Croisile, B., 482, 499 Cromarty, S. I., 183, 193, 195 Cronquist, B., xxviii, xxxviii Crook, C. K., 824, 828, 829, 841, 883, 896 Cropp, C. B., 551, 557, 561, 570 Crosby, E. C., 128, 134, 468, 475, 605, 609 Crosher, R., 452, 455 Cross, B. A., 349, 375 Cross, C. E., 54, 67 Crossin, K., 1003, 1012–1016, 1018, 1024, 1025 Cross-Mellor, S. K., 921, 926 Crouch, C. N., 583, 590 Croul-Ottman, C. E., 617, 623 Crovetti, R., 882, 896 Crow, T. J., 76, 85 Crowley, W. R., 922, 926 Crowther, R. A., 510, 513, 514, 523 Cruickshanks, K. J., 461, 477 Crumbliss, A. L., 793, 803 Crump, B. L., 53, 68 Crusio, W. E., 336, 343

Author Index Cruveilhier, J., 981, 997 Cruz, A., 670, 671, 740, 745, 754 Cruz, L. A., 789, 803 Cruz-Rizzolo, R. J., 170, 176, 192, 195 Crysdale, W. S., 467, 474, 599, 609 Crystal, S. R., 830, 831, 833, 841 Cseh, C. L., 906, 930 Csernansky, J. G., 492, 501 Cubile, L., 370, 376 Cuénod, M., 151, 159 Cui, L., 236, 238, 245 Cui-Juan, Z., 580, 591 Cullen, M. R., 527, 528, 538, 542, 544, 546 Cullinan, W. E., 617, 623 Cummings, C. W., 14, 15, 942, 955 Cummings, D. M., 616, 617, 623 Cummings, T. A., xxv, xxxviii, 660, 671, 714, 715, 721, 725, 737, 754 Cummins, K. A., 471, 475 Cunnick, J., 81, 90 Cunningham, A. M., 24–28, 33, 41–43, 45, 78, 81–82, 85, 86, 89, 101, 102, 107, 619, 623 Cunningham, C. L., 910, 917, 933 Curbow, B., 528, 545 Curran, Jr.,W. J., 910, 929 Curry, S., 280, 293 Curtin, H. D., 598, 612 Curtis, K. S., 915, 926 Curtis, O. F., 788, 802 Curtis, R., 773, 782 Curtis, R. F., 346, 375 Cuschieri, A., 35, 36, 42, 104, 108, 118, 119, 123, 124, 132 Cutforth, T., xxvii, xxxiv, 130, 131 Cybulska, I., 891, 901 Czaja, J. A., 919–920, 925, 926 Czeizel, E., 341 Czerniawska, A., 551, 555, 566 Da Pos, O., 31, 42 Dabbs, J. M. Jr., 359, 373, 379 Dabdoub, G., 392, 399 D’Abundo, S., 645, 650 Dackis, C., 795, 803, 884, 893, 901 DaCosta, A. P. C., 424, 428 Daddona, P. E., 123, 137 Dade, L. A., 258, 259, 264–266, 269, 270 Dager, S. R., 538, 546 Dahan, E., 444, 457 Daher, M., 826–828, 842 Dahl, A. R., 51, 54–57, 59–64, 67, 69–73, 550, 558–566, 569, 572, 582, 590 Dahl, K. A., 528, 543 Dahl, R., 444, 456, 457 Dahlof, C. G., 553, 566 Daikoku, S., 1003, 1007, 1018, 022 Daikoku-Ishido, H., 1003, 1007, 1018, 1022 Dal Monte, M., 78, 86, 560, 566 Dale, A. M. 269 Dalhouse, A., 368, 382

Dalseg, R., 554, 568 Dalton, J., 910, 914, 929 Dalton, P., 210, 225, 234, 239, 245, 584, 588, 788, 801, 853, 854, 859, 982, 997 Dalvit, S. P., 919, 926 Dalvit-McPhillips, S. P., 919, 926 Daly, B. D., 600, 609 Daly, J., 602, 610 Daly, M., 422, 428 Daly, M. de B., 982, 998 Daly, R. L., 470, 475 Damadzic, R., 515, 520 Damak, S., xxvi, xlii, 708, 710, 713, 714, 717–719, 726, 728 Damasio, A. R., 268, 270, 512, 513, 515–517, 519, 521, 600, 610 D’Amato, F. R., 359, 375 D’Amico, A., 299, 306 Damrath, V., 284, 294 Dandy, W. E., 667, 673 Danger, J. M., 76, 86 Dangoria, N., 101, 103, 111, 619, 626 Danho, W., 24, 27, 33, 35, 45, 75, 80, 88, 710, 716, 728 Dani, R., 887, 901 Daniel, P. C., 183, 193, 195 Daniel, R. K., 4, 15 Daniel, S. E., 238, 248, 479, 485, 498, 514, 519, 520, 522 Daniell, W., 533, 539, 541, 546 Danielli, J. F., 347, 352, 378 Daniels, C., 469, 474, 715, 725, 737, 754 Daniels, S. B., 364, 381 Danielsson, B. R. G., 62, 69, 552, 559, 567 Dann, J. F., 311, 325 Dann, M., xxxi, xxxv, 204, 212, 213, 215–217, 225, 489, 497, 633, 637, 783, 791, 800 Dann, R., 516, 520 Dannan, G. A., 64, 69 Danowski, S., 885, 901 Dantas, Z. N., 554, 566 Dantzer, R., 410, 415, 416, 426, 427, 428, 430, 432, 434, 909, 929 Danz, C., 444, 458 Darby, Y., 467, 475 Darby-King, A., 152, 160 Dardzinski, B. J., 256, 257, 273 d’Argy, R., 556, 572 Darien-Smith, I., 679, 700, 702, 802, 913, 930 Dark, G., 332, 340 Darling, F. M., 392, 397, 398, 420, 428, 436 Darmer, D., 1004, 1022 Darras, J., 440, 456 Darrelmann, C., 94–96, 107, 1015, 1022 Darrow, B., xxvi, xxxvi Darwin, C., 312, 323 Das, P., 484, 497 Das, S. R., 849, 848

Author Index Das, G.D., xxviii, xxxiii Dasso, M., 710, 711, 729, 730 Dassule, H. R., 765, 779 Dastidar, P., 442, 454 Dateo, G. P., 713, 725 Datiche, F., 170, 176, 188, 190–192, 198, 199, 255, 259, 271, 515, 520 Dau, B., 24, 27, 33, 45, 75, 80, 88 Dauger, S., 390, 392, 397, 399 Daum, R. F., 485, 496 Daval, G., 172, 174, 176 DaVanzo, J. P., xxx, xxxv David, A. S., 687, 702 David, G. F. X., 61, 66 David, R. M., 56, 67 David, S., 125, 126, 132 Davidoff, A. L., 527, 528, 530, 532, 535, 538, 539, 543, 544, 545 Davidson, A., 553, 563, 568 Davidson, B., 492, 493, 501 Davidson, H. I. M., 936, 950, 954 Davidson, J., 603, 608 Davidson, M. J., 600, 609 Davidson, N., 80, 86 Davidson, R. J., 254, 270, 828, 842 Davidson, T. M., 204, 224, 462–463, 472–473, 474, 476, 477, 889, 896 Davies, A., 762, 779, 781 Davies, A. M., 450, 452, 454, 760–762, 764, 779 Davies, C.W., 329, 340 Davies, D. C., 512, 520 Davies, K., 621, 625 Davies, M., 209, 211, 227 Davies, R. H., 280, 293 Davies, S., 329, 340 Davis, B. J., 103, 108, 131, 134, 139, 140, 146–149, 151, 152, 155, 156, 160, 165, 167, 169, 171, 173, 176, 177, 552, 558, 564, 566, 569, 736, 754, 757 Davis, B. M., 152, 164, 772, 773, 780 Davis, C., 658, 671 Davis, D. E., 366, 374 Davis, H., 419, 428 Davis, J. C. 341 Davis, J., 296, 307 Davis, J. D., 862, 864, 865, 876, 878 Davis, J. L., 410, 428 Davis, K. L., 483, 501 Davis, L. B., 313, 317, 320, 321, 323, 325 Davis, L. M., 27, 41, 123, 131, 150, 155, 289, 294, 396, 397 Davis, M., 413, 420, 421, 433 Davis, R. A., 121, 133 Davis, R. C., 619, 624 Davis, R. G., 387, 392, 398 Davis, R. L., 27, 41 Davis, S. H., xxxi, xlv, 789, 804 Davis, T. H., 537, 541, 545 Daw, N. W., 184, 195 Dawes, C., 641, 648, 887, 897 Dawkins, R., 336, 340

1041 Dawley, E. M., 969, 977 Dawood, M. Y., 370, 376 Dawson, Jr., R., 920, 932 Dawson, T. M., 27, 42, 84, 87, 514, 520, 619, 626 Dawson, V. L., 514, 520 De, B. G., 947, 954 De, M. G., 494, 496 De Beun, R., 921, 923, 926 De Bias, S., 149, 150, 162 De Bilbao, F., 98, 109 De Boer, S. F., 416, 422, 432, 433 De Brabander, H., 416, 434 De Bruyn, A., 559, 568 De Cabo, R., 891, 900 De Carlos, J. A., 166, 169, 171, 177, 180 De Catanzaro, D., 365–367, 375, 378, 382, 383 De Conno, F., 891, 901 De Cristofaro, M., 103, 108 De Graaf, C., 340, 805, 807–812, 814, 817, 820, 821, 822, 832, 841 De Jong, N., 329, 340 De Jong, P. J., 717, 724 De la Pompa, J. L., 760, 780 De la Riva, C., 413, 427, 1003, 1023 De la Torre, J., 148, 163 De Leon, M. J., 600, 601, 609, 610 De Lorenzo, A. J., xxv, xxxv, 551, 555, 560, 561, 566 De los Santos, H. C., 795, 800 De Matteis, F., 57, 72, 552, 571 De Morsier, G., 603, 609, 1016, 1022 De Olmos, J., 165, 169, 171–173, 177, 420, 426 De Perceval, G. D., 314, 323 De Robertis, E. M., 116, 136 De Rosa, E., 170, 177, 413, 428 De Rossi, G., 647, 648 De Ruiter, A. J., 416, 432 De Slegte, R. G., 597, 610 De Toledo, L., 911, 926 De Wijk, R. A., 217, 218, 224, 226, 234, 236, 247, 256, 271, 494, 499, 991, 996 De Winter, F., 616, 625 De Zoysa, P. A., 472, 477 Deacarries, L., 515, 520 Deakin, J. F., 517, 522 Deal, R. J., Jr., 450, 455 Deamer, N. J., 56, 64, 67, 68, 552, 559, 566, 567 Dear, T. N., 77, 87 Deardon 276, 293 DeBlasi, A., 84, 85 DeBorde, D. C., 554, 563, 570 DeCasper, A. J., 833, 841 Decastro, M., 300, 308 DeChiara, T. M., 762, 779 Deckner, M.-L., 96, 108 DeCou, D. F., 285, 293 Deecke, L., 462, 476, 482, 499

Deems, D. A., xxxi, xxxv, 39, 47, 204, 216, 224, 225, 238, 245, 440, 450, 454, 463, 471, 474, 480, 484, 495, 496, 497, 513, 520, 525, 537, 544 563, 567, 587, 588, 595, 601, 604, 605, 609, 629, 635, 637, 787, 788, 790, 791, 795, 799, 890, 894, 896, 935, 938, 945, 950, 951, 953, 954, 959, 961, 963, 965 Deems, R. O., 683, 684, 691, 701, 889, 892, 893, 896 Defer, N., 80, 87 DeFries, J. C., 331, 340, 343, 366, 382 Defries, R. D., xxvi, xliv, 557, 572 Deger, K., 443, 456 Degueurce, A., 148, 155 DeHaas, C. J. 999 DeHamer, M. K., xxvii, xxxv, 101, 102, 108, 119, 132 DeHan, R. S., 660, 672 Dehejia, A., 514, 522 Deiss, V. C., 335, 340, 392, 398 Deitmer, T., 12, 13, 15 Del, R. G., 359, 374 Del Punta, K., xxv, xxxv, 972, 978 Delaleu, J. C., 183, 195, 388, 397, 982, 996 Delaney, S., 828, 846 Delank, K. W., 467–468, 474 Delapenha, R., 647, 648 Delay, E., 723, 725 Delay, R., 1005, 1022 Delay, R. J., 660–664, 671, 673 Delemarre-Van de Waal, H., 1017, 1023 Delgadillo, I., 305, 306 Dell, R. B., 893, 902 DellaCorte, C., 26, 33, 42, 43 Della-Fera, M. A., 788, 789, 790, 792, 798, 800, 801, 953, 955 Dellovade, T. L., 127, 132, 1012, 1022 Delon, M. R., 513, 523 DeLong, R. E., 982, 997 DeLucia, J., 529, 538, 544 Delwiche, J., 644, 648 Delwiche, J. F., 789, 799, 800 DeMarco, J., 244, 247 DeMartino, A., 244, 247 Demas, G. E., 422, 428 Dember, W. N., 348, 375 DeMesquita-Wander, M., 422, 427 Demets, D. L., 579, 589, 645, 648, 894, 897 Demski, L. S., 1001–1003, 1005, 1022, 1025 Dence, C., 253, 270 Dencker, L., 62, 69, 123, 133, 552, 559, 567 Denenberg, V. H., 310, 327, 367, 369, 375, 379 Deni, R., 320, 325 Denlea, N., 491, 492, 493, 501 Dennis, J. C., 26, 27, 48 Denny, M. R., xxix, xxxv

1042 Denson, M. A., 484, 499 Denton, D. A., 124, 133, 742, 757 DeOlmos, J., 147, 148, 156 Deonna, T., 553, 562, 569 Deragon, K. L., 1003, 1022 Derby, C. D., 183, 193, 195, 220, 228 Dermietzel, R., 105, 111 DeRosa, E., 191, 195 Derouiche, A., 26, 49 Derzie, A., 417, 431 Desage, M., 315, 326 Desai, P. B., 57, 67 Desantis, D., 367, 375 Desgranges, J. C., 660, 671 Deshpande, V. S., 57, 67 DeSimone, J. A., xxv, xxxvii, 643, 648, 713–716, 718, 720, 722, 725, 726, 728–730, 759, 775–777, 778, 779, 781, 782 DeSimone, S. K., 715, 718, 725, 726, 728 Desjardins, C., 358, 359, 366, 373, 374, 376, 379, 423, 430 Desmeules, M., 440, 458 Desmond, J. E., xxix, xliii, 174, 180, 255, 259–267, 269, 270–273, 424, 436 DeSnoo, K., 883, 896 Desor, J. A., 217, 224, 828–830, 832, 841, 843, 883, 886, 896 DeSouza, I., 417, 425, 434 D’Esposito, M., 258, 269, 273 DesRosiers, M. H., xxviii, xxxviii, xliii Dessauer, C. W., 26, 27, 48 Dessi-Fulgheri, F., 63, 70 Dethier, V. G., xxix, xxxv, 185, 195 Detwiler, P. B., 83, 89 Deurenberg, P., 888, 903 Deutch, A. Y., 516, 520 Deutsch, J. A., 884, 896, 917, 928 Deutsch, S., 296, 306 Devanand, D. P., xxxii, xxxv, 483, 497 Devata, S., 621, 624 Devine, J., 888, 902 Devitt, H., 554, 563, 566 Devon, R., 105, 108 DeVries, A. C., 422, 427 DeVries, E., 1002, 1006, 1022 DeWaard, R., 480, 486, 501, 514, 523 Dewes, W., 472, 476, 593, 594, 603, 610 Dewhurst, J. R., 216, 224 DeWit, H., 886, 896, 914, 933 Dewulf, N., 101, 113 DeWys, W. D., 890, 896, 909–910, 926 Deysher M., 836, 841 Dezell, H. E., 365, 373 Dhallan, R. S., 24, 42, 80, 82, 87 Dhariwal, A., 482, 498, 513, 520 Dhong, H. J., xxxii, xxxv, 392, 398, 451, 456, 480, 497 Dhooper, N., 58, 70, 505, 507, 510, 520 Di Benedetto, M., 233, 234, 246 Di Costanzo, A., 553, 563, 567 Di Ilio, C., 58, 66 Di Iorio, G., 514, 522

Author Index Di Magno, E. P., 887, 898 Di Natale, C., 299, 306 Di Prisco, G. V., 411, 426, 428 Di Rienzo, L., 599, 609 Di Scala, G., 174, 175, 177, 180, 420, 421, 429 Diakow, C. A., 362, 378 Diamant, H., 667, 671, 885, 896 Diamond, L., 645, 649 Diamond, P., 888, 896 Diaz, A. F., 300, 306 Diaz, C., 716, 729 Diaz, J., 839, 840, 843 Diaz, J. P., 660, 671 Díaz, M. L., 992, 995 DiBattista, D., 908, 926 Dibner, M. D., 124, 132 DiCara, L. V., 684, 705 DiCarlo, S. T., 856, 858 Dichter, M. A., 124, 133 Dick, R. B., 585, 588 Dickinson, T. A., 299, 306–308 Dickinson, K., xxvi, xlii Dickman, J. D., 746, 754 Didier, A., 149, 152, 156 Diedrich, O., 261, 269 Dien, J., 233, 240, 248 Dierich, A., 760, 779 Dierking, J., 512, 523 Dieter, M. P., 62, 72 Dietz, R., 983, 999 Dietz, V., 883, 900 Dijk, D. J., 422, 427 Dillon, W. P., 598, 612 DiLorenzo, M., 412, 436 DiLorenzo, P. M., 736, 748, 754 DiLoreto, D. A., 600, 611 Dimoglo, A. S., 991, 999 Dimov, D., 494, 496 Dimov, D. S., 952, 957 DiMuzio-Mower, J., 509, 521 DiNardo, L. A., 737, 754 DiNardo, L. J., 473, 474, 634, 638 Ding, X., 51, 53–58, 61–65, 67–73, 552, 559, 567, 568, 582, 589 Ding, Z. Q., 557, 565 Dingeldein, M. W., 552, 567 Dinh, L., 440, 457 DiNicolantonio, R., 886, 896 Dinis, P.B., 443, 454 Dionne, T. J., 917, 921, 925 Dionne, V. E., xxiv, xxxvii, 721, 727, 737, 755, 1005, 1022 Dippel, C., 887, 898 Disse, J., 1006, 1014, 1022 Dissinger, M. E., xxx, xxxiv, 352, 357, 374, 403, 404, 407 DiStefano, P. S., 773, 782 Distel, H., 218, 219, 226, 310, 315, 324, 355, 369, 370, 375, 377, 381, 830, 842 DiTraglia, G. M., 495, 497, 886, 896 Dittert, L. W., 553, 562, 568

Dittman, A. H., 80, 86, 426, 428, 433, 969, 979 Dittmann, B., 299, 307 Diu, C.K., 449, 459 Divecha, N., 644, 648 Dixon, A. K., 361, 375 Dixon, C. M., 553, 562, 565 Dixon, L. K., 342 Dizinno, G., 423, 428, 434 Djabali, K., 124, 132 Djuric´, V. J., 538, 539, 544 Djurup, R., 554, 563, 568 Dluzen, D. E., 416, 418, 428, 430 Doan, K., 554, 571 Doane, D. D., 886, 903 Doane, H. M., 310, 320, 325 Dobelle, W. H., 750, 756 Dobo, E., 153, 158 Dobrazanska, M., 887, 898 Dodd, G. H., xxvi, xlii, 244, 249, 296, 307, 560, 572 Dodd, J., 709, 724, 737, 753 Dodds, W. J., 644, 648 Dodo, H., 584, 589 Dodson, H. C., xxxi, xxxvi, 5, 15, 23, 30, 34, 38, 41, 42, 463, 475, 560, 565, 616, 623 Dodson, J. J., 426, 433 Doerr, E., 19, 23, 24, 30, 47 Doesburg, W., 554, 563, 571 Doetsch, F., 143, 156, 620, 624 Doetsch, G. S., 747, 748, 754 Doherty, A. E., 645, 650 Doherty, R. M., 992, 995 Doi, T., 942, 956 Dokumov, S. I., 952, 957 Dol, T., 942, 956 Dolack, M. K., 34, 47 Dolan, R. J., 516, 520 Dolan, T. F. Jr., 784, 801 Dolce, C., 642, 649, 670, 674, 939, 954 Dolev, Z., 450, 457 Dollken, A., 1002, 1006, 1022 Dominguez, H. D., 355, 366, 375, 885, 896 Dominguez, M. I., 154, 156 Dominguez, R. A., 472, 474 Dominic, C. J., 365, 366, 367, 375, 381 Domjan, M., 905, 908, 911, 913–914, 920, 924–927, 930 Domschke, W., 887, 898 Donahew, P. S., 923, 930 Donahue, J. L., 554, 563, 569 Donald, P. J., 598, 611 Donaldson, J. O., 889, 896, 949, 954 Donati, F., 946, 954 Donato, R., 32, 47 Donchin, E., 233, 240, 245 Donchin, E. A., 233, 240, 248 Donnal, J. F., 602, 609 Donnay, A., 536, 537, 545 Donndelinger, M. J., 264, 273 Donnelly, A., 561, 562, 567

Author Index Donoff, R. B., 946, 957 Donohoe, T. P., 920, 926 Dontchos, S., 837, 843 Doolin, R. E., 720, 726 Dopheide, T., 888, 903 Doraiswamy, P. M., 601, 611 Dore, F. Y., 426, 433 Dore-Duffy, P., 949, 954 Dorkin, H. L., 343 Dorman, D. C., 392, 399, 578, 588 Dorries, K. M., 61, 68, 217, 228, 469, 477 Dotti, M., 484, 485, 501 Doty, R. L., xv, xix, xxvi, xxvii, xxix–xxxii, xxxiv–xxxvi, xl, xliv, xlv, 5, 11, 15, 35, 37–39, 42, 44–46, 49, 61, 63, 64, 66, 68, 70, 103, 107, 108, 119, 127, 132, 138, 149, 159, 170, 177, 184, 195, 204, 206–213, 215–217, 224–228, 234, 238, 245, 255, 256, 259, 264–268, 269–273, 311, 312, 317, 323, 332, 334, 339, 340–344, 345, 353, 354, 356, 357, 359, 372, 373, 375, 376, 378, 379, 389, 392, 396, 397–400, 404, 405, 407, 413, 424, 428, 433, 440, 443, 445, 450, 451, 454–457, 459, 461–463, 466–467, 469–473, 474, 476–478, 479–495, 496–501, 503, 505, 512–516, 520, 522, 523, 525, 528, 530, 536, 537, 544, 545, 556, 563, 565, 567, 572, 576, 577, 580, 584, 585, 587, 588, 590, 593–596, 600–608, 609, 611–613, 616, 619, 620, 623, 624, 626, 629–631, 633–635, 637, 638, 639, 648, 656, 660, 674, 709, 721, 729, 732, 740, 742, 745, 755, 759, 764, 765, 768, 769, 773, 774, 778, 780, 783, 785–792, 795, 799–804, 823, 840, 842, 867, 872, 876, 889–892, 894, 895, 896, 918, 920–921, 924, 929, 935–938, 945, 950–953, 954–957, 959, 961, 963, 965, 981, 982, 984–988, 991, 995, 997, 999 Doucette, J. R., xxvii, xli Douek, E., xv, xxxi, xxxv, xxxvi, 15, 616, 623 Douglas, R. J., 406, 407 Douglas, W., 642, 647, 648 Douglass, H. O., 890, 902 Døving, K. B., 17, 48, 209, 225, 417, 436, 967, 971, 977, 979 Dow, C., 305, 307 Dowd, F. J., 641, 649 Dowdeswell, R. M., 300, 307 Dowling, M. M., 296, 306 Downey, L. L., 466, 468, 475 Drachman, D., 479, 499 Dragani, B., 58, 66 Draghia, R., 551, 567 Drago, F., 367, 375 Drago, J., 394, 396, 400 Dragoin, W., 913, 933

1043 Dragomir, A., 539, 544 Drake, A. F., 256, 273 Drake, B., 209, 225 Drapkin, P. T., 122, 132 Dravnieks, A., 218, 226, 297, 783, 795, 800, 991, 997 Drayer, B. P., 602, 609 Drenckhahn, D., 664, 672 Dresel, S., 593, 594, 612 Drevets, W. C., 680, 702 Drew, J., 914, 929 Drewett, R. F., 885, 896, 903, 919, 926 Drewnowski, A., 788, 798, 800, 802, 847–853, 855–858, 858–860, 882, 892, 896, 923, 926 Drexler, M., 218, 224 Dreyer, V., 893, 903 Dreyer, W. J., 289, 293 Drickamer, L. C., 357, 368, 375, 376, 379, 404, 407 Driezen, S., 891, 900 Drinkamer, L. C., 404, 407 Drinkwater, D., 972, 978 Drinnan, S. L., 79, 87 Drogou, I., 444, 457 Droste, C., 554, 563, 571 Droungas, A., 420, 428 Drucker-Colin, R., 420, 429 Drukarch, B., 480, 486, 501, 514, 523 Drutman, J., 595, 609 Dryer, L., 24, 48 Dryzga, M. D., 54, 72 DSM-IV-TR., 921, 926 Du, Y., 670, 674, 767, 781 Dubauskaite, J., 710, 726 Dube, W. V., 392, 399 Dubey, S. P., 597, 611 DuBois, G. E., 714, 726 Dubois-Dalcq, M., 621, 623 Dubois-Dauphin, M., 98, 109 Duchamp, A., 148, 156, 182–184, 186, 188, 195, 287, 293 Duchamp-Viret, P., 148, 156, 182–184, 186, 188, 195, 287, 293 Ducheny, K., 795, 800 Duclaux, R., 696, 700 Duclohier, H., 26, 41 Duda, J. E., 508, 510, 514, 520 Dudai, Y., 421, 435 Dudchenko, P. A., 414, 415, 425, 428, 429 D’Udine, B., 356, 357, 372 Dudley, C., 57, 61, 69, 971, 978 Duenholter, J. H., 311, 323 Duester, G., 62, 68 Duff, K., xxxii, xliii, 480, 482, 499 Duffy, V. B., 789, 798, 799, 803, 849–854, 855, 857, 858–860, 952, 955, 961, 965 Duffy, B., xxvii, xliv, 98, 113 Dugas du Villard, X., 875, 877 Duhra, P., 939, 955 Duke, V. M., 472, 477

Dulac, C., xxv, xxxvii, 78, 89, 126, 130, 135, 146, 160, 296, 307, 350, 351, 354, 373, 376, 379, 971, 972, 976, 977, 979 Dulay, M. F., 238, 245 Dulka, J. G., 389, 399, 1005, 1023 Dunbar, D., 61, 64, 73 Dunbar, I. A., 357, 375, 404, 407 Duncan, H. J., 14, 16, 37, 46, 394, 395, 398, 404, 407, 398, 463, 472, 475, 477, 630, 637, 788, 790, 803 Duncan, R. B., 579, 588 Dunér-Engström, M., 784, 800 Dunn, K. A., 530, 532, 538, 545 Dunn, L. M., 484, 497 Dunn, L. T., 916, 926 Dunn, T. P., 492, 496 Dunnick, J. K., 583, 588 Dunteman, G., 532, 546 Duong, H., 187, 197 Duquette, M., 698, 701 Durand, M., 154, 156 Durand-Lagarde, M., 236, 239, 245 Durbec, P., 517, 519 Durham, L. H., 12, 15, 451, 452, 454, 455 Durick, K., 723, 727 Durkin, T. P., 423, 429 Durlach, P. J., 420, 428 Dusek, J. A., 419, 428 Dussault, J. H., 620, 623 Dutra, A., 514, 522 Dutta, A. K., 367, 380 Duvall, D., xvi, xl, 368, 376 Duveau, A., 145, 158, 182, 197 Duvoisin, R. C., 514, 522 Duxbury, A. J., 951, 955 Dwalsaint, F., 444, 453 Dwyer, N. D., 76, 78, 87 Dye, L., 345, 372 Dyer, R. S. 588 Dynes, J. L., 126, 132 Dyson, G. M., 280, 293 Dyson, M., 665, 677 Dytch, H., 342 Earley, C. J., 919, 926 Eaton, D. C., 718, 727 Eaton, D. L., 58, 68 Eaton, J. R., 297, 308 Eaves, L. J., 342 Eayrs, J. T., xxxi, xxxiv, xxxvi, 386, 392, 397–399 Ebbell, B., 439, 454 Ebbesson, S. O. E., 551, 554, 555, 565 Ebendal, T., 101, 107, 111, 766, 773, 781 Eberling, J. L., 593, 594, 601, 610 Ebner, K., 416, 429 Ebner, V., 654, 658, 671 Eby, T. L., 960, 965 Eccles, R., 442, 444, 450, 452, 454, 456, 458, 981, 982, 984, 996, 997, 1005, 1022

1044 Echelard, Y., 551, 560, 568 Echeverri, F., 288, 291, 294 Echols, D. H., xxvi, xli, 557, 570 Eckel, L. A., 915, 917, 920–921, 926, 927, 930 Ecker, A., xxii, xxxvi, 17, 18, 42, 981, 997 Eckert, E., 923, 928 Eckhard, C., xxii, xxxvi, 42, 981, 997 Eckhert, S. J., 310, 326 Eckmann, K. W., 192, 195, 411, 421, 425, 428 Eckstein, A., 826, 828–830, 841 Eckstrom, P., 1004, 1022 Economos, D., 444, 458 Eddy, A., 218, 221, 226 Edelhoch, H., 645, 648 Edeline, J. M., 191, 195 Edelman, G. M., 1003, 1012–1016, 1018, 1024, 1025 Edelman, L., 563, 572 Edelstein, E. L., 951, 957 Edgar, W., 647, 649 Edison, A. F., 582, 583, 588, 590 Edmondson, J., 78, 89, 126, 130, 135, 146, 160, 296, 307, 354, 379 Edwards, D. A., 361, 381, 618, 624 Edwards, J., 491, 492, 496 Edwards, R. H., 153, 161 EEOC 542, 544 Effenberger, F., 2, 16, 99, 112 Efron, E. H., 910, 914, 932 Egawa, M., 581, 589 Eggerickx, D., xxv, xli Eggers, G., 467, 476 Eggert, F., 318–320, 323, 324, 337, 339, 341, 342 Eggum, B. O., 698, 704 Egid, K., 318, 324, 337, 341, 342 Egide, M. S., 644, 648 Eglitis, J., 656, 658, 673 Eglitis, J. A., 784, 802, 825, 843 Egolf, L. M., 991, 997 Eguchi, E., 26, 35, 48 Ehrenberg, C. G., 652, 671 Ehrlich, B. E., 511, 519 Ehrlich, P., xxiv, xxxvi, 654, 671 Ehrmann, S., 298, 307 Eibl-Eibesfeldt, I., 347, 376, 406, 407 Eichel, B. S., 468, 475 Eichenbaum, H. B., 174, 175, 178, 179, 188, 191–193, 195, 198, 199, 208, 209, 225, 296, 307, 410–412, 414, 415, 417–419, 421, 422, 424–426, 426–429, 433–435, 437, 438 Eidelman, A. I., 321, 324 Eidson, A. F., 62, 72 Einarson, T., 829, 845 Einav, S., 448, 454 Eindhoven, G. B., 945, 955 Eipper, B. A., 76, 87 Eischen, S. E., 240, 248 Eisele, S. G., 919, 929

Author Index Eisenberg, H. M., 604, 611, 631, 632, 637 Eisenbrandt, D. L., 583, 588 Eisenmenger, W., 517, 522 Eisler, I., 923–924, 931 Eisthen, H. L., 34, 35, 42, 1003, 1005, 1022 Ekloev, T., 305, 306 Eklund, A., 320, 322 Ekman, G., 204, 210, 223, 225, 988, 996 el Mously, M., 962, 965 el Naggar, M., 468, 475 Elaagouby, A., 191, 199, 415, 426, 435 Elad, D., 448, 454 Elad, Y., 450, 457 Elder, D. E., 599, 608 Eldridge, S. R., 583, 588 Eleftheriou, B. E., 365, 366, 373 El-Etri, M. M., 154, 156 Elgar, G. J., 124, 135 Elias, M., 359, 376, 489, 497 Elias, S., 342 Eliot, C., xxiv, xxxiv, 17, 48 Elizade, G., 906, 927 Elkabes, S., 1003, 1007, 1012, 1025 el-Kalliny, M., 551, 556, 558, 565 Elkes, J., xxviii, xliii Elkin, E., 359, 376 Elkins, R. L., 910, 927 Elkon, D., 596, 609 Ell, S. R., 451, 454 Eller, O. M., 825, 843 Eller, P. M., xxxi, xxxvii, xl, 27, 33, 38, 43, 45, 123, 134, 463, 466, 472, 476, 600, 608, 610, 631, 638, 662, 673 Ellermann, J. M., 254, 269, 271 Elliffe, D. M., 786, 802 Ellins, S. R., 913, 927 Elliot, E. J., 720, 726 Elliot, P. B., 209, 225 Ellis, L. B., 365, 382 Ellis, W. G., 513, 522 Ellison, D. W., 233, 247, 463, 473, 476 Ellison, J., 309, 327 Ellison, R. C., 886, 903 Ellman, C., 828, 842 Elmer, P. J., 886, 896, 897 Elmes, D. G., 204, 227, 255, 271 el-Nemr, F., 388, 398 Elovaara, I., 606, 609 Elsberg, C. A., 204, 225, 469, 475, 580, 588, 985, 997 ElShamy, W. M., 771, 772, 781 Elwany, S., 445, 454 Elwell, M. R., 583, 588 Emery, P., 467, 474 Emge, D. K., 616, 623 Emile, J., 505, 519 Emile, P., 38, 39, 41, 47, 127, 137, 472, 477 Emko, P., 38, 47, 472, 477 Emmers, R., xxiv, xxxvi

Emmett, E. A., 579, 588 Emori, Y., 663, 673, 709, 711, 715, 716, 723, 727, 728 Emory, C. R., 551, 556, 572 Emson, P. C., 26, 44, 120, 123, 136 Endo, H., 235, 244, 783, 797, 801 Endo, S., 963, 966 Endröczi, E., 359, 367, 376, 380 Enerdal, J., 454, 454 Eng, D. L., 28, 42, 140, 156 Engel, D., 941, 956 Engel, L. S., 564, 565 Engelen, C., 299, 307 Engelender, S., 511, 514, 520, 523 Engelhardt, H., 299, 308 Engelhart, A., 552, 564 Engelman, K., 735, 754, 886, 888, 891, 894, 895, 899, 902 Engelmann, M., 416, 429, 432 Engelmann, T. W., 660, 672 Engen, T., 210, 217, 223, 225, 314, 324, 424, 430, 433, 542, 543, 828, 838, 839, 841, 842, 988, 996 England, S., 722, 725 Englander, H. R., 643, 648 Engle, J. Jr., 686, 704 Englehardt, R. A., xxxi, xlv, 789, 804 Engleman, K., 831, 840 English, R. J., 600, 612 Englund, U., 515, 520 Engström, H., xxv, xxxv, 19, 42, 661, 672 Enko, P., 1017, 1025 Enna, S. J. 999 Ennis, D. M., 807, 808, 813, 821 Ennis, M., 100, 105, 109, 139, 143, 146, 147, 149, 151, 153, 154, 155–158, 163, 181, 183–185, 187, 191, 192, 194–197, 200, 201, 409, 413, 424, 431, 436 Enns, J. T., 220, 225 Enns, M. P., 832, 842 Ensoli, B., 103, 108 Ensoli, F., 103, 108 Epplen, J. T., 509, 514, 521 Epstein, A. N., 747, 756, 833, 845, 886, 902 Epstein, L. H., 887, 896, 900, 903 Erb, M., 261, 269 Erhardt, H., 33, 42 Erick, M., 950, 955 Erickson, J. C., 76, 85 Erickson, J. T., 762, 779 Erickson, R. P., xxiv, xli, 216, 228, 681, 684, 691, 700, 701, 704, 732, 734, 736, 740, 747–751, 754, 755, 757, 758, 816, 817, 821, 822, 825, 842, 864, 877, 884, 902 Erikssen, J., 291, 293 Eriksson, C., 64, 65, 68, 552, 566, 582, 588 Eriksson, P., 93, 108 Eriksson, R. K., 305, 306 Eriksson, U., 123, 133

Author Index Erlanger, J., xxiv, xxxvi Ernfors, H., 762, 767, 780 Ernfors, P., 620, 625, 761, 762, 771, 772, 779, 781 Erras, A., 791, 798, 801 Ervin, F. R., 396, 398 Ervin, R. R., 910–911, 927 Erzurumlu, R. S., 764, 782 Esaki, Y., 585, 590 Eschricht, D. F., 981, 997 Escurat, M., 124, 132 Esiri, M. M., 60, 72, 482, 499, 512, 513, 520, 522, 551, 557, 563, 567, 600, 609 Eskandar, E. N. 223, 225 Eskenazi, B., 268, 270, 494, 496, 949, 954 Eslinger, P. J., 256, 257, 268, 270, 273, 949, 956 Esser, P. D., 262, 265, 266, 271, 491, 492, 499 Esses, B. A., xxxi, xxxvii, 38, 43 Esses, V. M., 882, 896 Essock-Vitale, S. M., 363, 376 Estabrook, R. W., 57, 61, 67, 71 Esteban-Santillan, C. E., 485, 487, 488, 495, 501 Etienne, N., 495, 496, 605, 608, 891, 895 Etscorn, F., 319, 325 Etter, L., 853, 854, 855, 860 Etteri, S., 621, 625 Euker, J. S., 366, 376 Eura, Y. S. K., 232, 245 Evans, A. C., xxix, xliii, xlv, 190, 201, 258, 259, 262, 264–267, 270, 272, 273, 699, 705, 797, 803, 948, 957 Evans, C., 17, 42 Evans, C. M., 362, 376 Evans, D. J., 829, 844 Evans, G., 551, 557, 571 Evans, J., 551, 555, 567, 568 Evans, J. E., 33, 45, 388, 394, 396, 398, 578, 581, 583, 588, 589 Evans, M., 621, 623, 624 Evans, S. M., 535, 546 Evans, W. J., 232, 234, 236–238, 245 Evans J. E., 556, 567 Evdokimov, V. J., 54, 71 Everall, I. P., 605, 609 Everitt, B. J., 423, 435, 916, 926 Evers, K. A., 945, 955 Evershed, R. P., 971, 978 Everts, H. G., 416, 429, 432 Ewbank, D., 514, 521 Ewer, R. F., 406, 407 Ewing, J. F., 82, 87 Exner, S., xxii, xxxvi Exworthy, T., 510, 520 Eyman, R. K., 210, 225 Fabre-Nys, C., 423, 434 Fabrizio, P. A., 3, 16, 969, 979 Fabsitz, R. R., 342

1045 Facchinetti, F., 359, 374 Fadem, B. H., 1003, 1024 Fadool, D. A., 81, 87 Fagan, A., 411, 412, 414, 425, 428 Fagel, P. L., 512, 513, 522 Fagius, J., 888, 896 Fahrenkrug, J., 450, 453 Fahrion, N., 342 Fährmann, W., 660, 672 Fahy, C., 946, 954 Fairchild, G. A., 583, 584, 589 Fairley, J.W., 451, 454 Fajen, J. M., 585, 588 Falck, J. R., 61, 67, 71 Falconer, D. S., 367, 374 Falkai, P., 517, 520, 603, 609 Falkner, B., 891, 900 Fallon, J. H., 170, 177 Faludi, B., 689, 701 Fang, H., 481, 485, 489, 498, 621, 624 Fang, H. S., 62, 69 Fanselow, M. S., 907, 926 Fantino, M., 696, 700, 907, 927 Faraday, R., 410, 436 Faraone, S. V., 491, 492, 501 Farb, P., xv, xxxvi Farber, N. B., 124, 137 Farbman, A. I., xv, xxvii, xxxv, xxxvi, 22, 24, 26–28, 30–36, 38, 41, 42, 44, 45, 47–49, 75, 81, 85–87, 95–97, 98, 101–104, 107, 108, 110, 112, 113, 118–126, 130, 131, 131–133, 135, 136, 138, 145, 150, 155, 396, 397, 616, 623, 660–663, 665, 670, 672, 674, 713, 726, 768, 762–766, 768, 769, 775, 779, 780, 968, 979 Farde, L., 252, 273 Farget, V., 174, 175, 176, 187, 188, 190, 191, 195, 258, 264–266, 272, 424, 435 Fariello, R. G., 512, 513, 522 Farinas, I., 670, 674, 761, 762, 767, 771–773, 779–781 Farkas, B. K., 883, 902 Farleigh, C. A., 893, 902 Farlow, M. R., 480, 482, 498 Farmer, P. V., 920, 930 Farnsworth, D., 891, 898 Farrior, B., 468, 475, 605, 609 Farrior, R. T., 3, 15 Farrow, J. A., 923, 927 Fasano, W. J., 553, 566 Fasolo, A., 35, 48, 76, 86, 101, 111, 124, 131, 149, 150, 157, 162 Fast, K., 784, 787, 799, 853–857, 860, 961, 965 Fattori, B., 54, 68, 101–103, 109 Faure, N., 554, 569, 572 Faurion, A., 714, 723, 726, 797, 800, 813, 821, 948, 954, 955, 960, 962, 965 Favarato, M., 551, 555, 573 Favor, J., 116, 133 Fawcett, J. R., 553, 567

Fazakerley, J. K., 550, 551, 557, 564, 570 Fazekas, A. T., 554, 569, 572 Fechner, G. T., 203, 225, 784, 787, 800, 806, 821 Federico, G., 54, 68, 101–103, 109 Federoff, I. C., 923, 931 Fedio, P., 268, 270 Fedoroff, I. C., 472, 475 Feeley, A., 411, 425, 428 Fehm, H. L., 240, 247, 554, 563, 571 Fehm Wolfsdorf, G., 236, 248 Feigin, M. B., 794, 800 Feijen, A., 101, 113 Feil, P., 366, 379 Feil, V. J., 63, 67, 550, 552, 559, 566, 577, 582, 588 Feinleib, M., 342 Feinstein, P., 126, 137, 138, 972, 973, 978 Feinstein, P. G., xxiv, xli, 24, 44 Feistel, H., 241, 248 Feldman, G. M., 722, 728, 729 Feldman, J., 96, 110, 618, 625 Feldman, M., 887, 896, 901, 902 Feldman, R. S., 783, 788, 790, 795, 799 Feldman, S., xxxiii, 173, 179 Feldon, S. E., 605, 610 Fellows, D. W., 596, 609, 613 Feneis, H., 669, 672 Feng, L., 709, 711, 723, 725, 728, 848, 858 Feng, P. C., 550, 567 Feng, W.-H., 22, 37, 42, 48, 504, 523 Fenner, D. P., 839, 840 Fenster, L., 359, 376 Fentress, J. C., 670, 671 Fenwick, G. R., 857, 859 Ferguson, D., 445, 455 Ferguson, K., 27, 49, 82, 90 Ferguson-Segall, M., xxx, xxxiv, 392, 398, 469, 474, 493, 494, 497, 498 Fernandez, A. D., 341, 471, 474 Fernandez, J., 421, 427 Fernandez, M., 538, 544 Fernandez-Fewell, G. D., 975, 977 Fernandez-Ruiz, J., 420, 429 Fernandez-Salguero, P., 57, 70 Fernandez-Urrusuno, R., 554, 563, 567 Ferner, H., 662, 674 Fernstrom, A., 893, 896 Féron, F., xxvii, xxxvi, xxxix, xl, 13, 14, 15, 36, 46, 101–103, 107–109, 111, 466, 475, 516, 520 Feron, V. J., 582, 588, 591 Ferrand, J., 17, 48 Ferrans, V. J., 620, 624 Ferrante, M. A., 470, 475 Ferrari, C. C., 30, 42 Ferreira, G., 172, 178, 423, 429 Ferrell, F., 777, 778 Ferrell, M. F., 775, 777, 779, 886, 895 Ferreyra-Moyano, H., 192, 193, 195, 268, 269, 479, 497, 563, 567

1046 Ferri, T., 656, 671 Ferriero, D., 96, 110, 618, 625, 629, 637, 959, 960, 963, 965 Ferris, A. M., 831, 841, 858, 858, 889, 890, 896 Ferris, S.H., 600, 609 Ferry, B., 174, 175, 177, 180, 420, 421, 429 Ferstl, R., 233, 234, 236, 239–241, 247, 248, 319, 320, 323, 341, 350, 380, 469, 474, 476 Fesenko, E. E., 54, 71 Festa, D., 581, 589 Feuz, A., 910, 927 Feyereisen, R., 57, 71 Fiedler, N., 526, 527, 529, 530, 535, 538, 544 Field, F. H., 364, 381 Field, P., xxvii, xxxix, 105, 110 Field, P. M., 154, 162 Field, R. B., 645, 650 Fielden, J. A., 359, 373 Fielder, C. P., 467, 475 Fields, R. D., 1001, 1022 Fienberg, A. A., xxv, xxxvii Fife, D., 629, 637 Fifer, W. P., 833, 841, 842, 845 Figarella, C., 887, 901 Fijimoto, T., 81, 87 Fildes, V. A., 312, 323, 837, 842 Filer, L. J., 689, 705 Filiaci, F., 444, 456 Filla, A., 494, 496 Fillion, T. J., 320, 323, 356, 376, 828, 829, 841, 845 Finch, G. L., 64, 73 Fincher, C. E., 539, 546 Findlay, J. B. C., 970, 976 Findlay, M. W. Jr. 306, 308 Finelli, P. F., 469, 475 Finger, T. E., xxix, xxxvii, 24, 28, 34, 37, 38, 40, 44, 47, 49, 148, 161, 165, 170, 179, 351, 383, 659, 660, 672, 676, 719, 727, 746, 757, 765, 769, 779, 782, 802, 983, 996–999 Fink, R. P., xxvii, xl, 93–95, 98, 111 Finkel, D., 331, 332, 341 Finkenzellar, P., 230, 245 Finley, A., 257, 269, 271 Finley, M. F., 621, 624 Fiol, M. E., 686, 700 Fiorelli, V., 103, 108 Fiorino, D. F., 422, 429 Firestein, S., xxiv, xxv, xxvi, xxxvi, xlv, 27, 42, 76, 78–79, 84, 87, 91, 145, 149, 155, 164, 221, 223, 287, 293, 294, 396, 400, 551, 573 Fisch, U., 960, 965 Fischer, M. E., 789, 803, 946, 957 Fischer, R., 848–850, 855–858, 859 Fischer. E., xxii, xxxvi Fischl, B. R., 269, 270 Fischman, A. J., 686, 702

Author Index Fisher, E. W., 450, 454 Fisher, G., 116, 134 Fisher, L. J., 621, 624 Fishman, M., 600, 608 Fishman, P. S., 551, 561, 569 Fiske, B. K., 143, 156 Fitzgerald, R. E., 422, 427 Fitzsimon, J. S., 469, 475 Fixsen, C., 652, 660, 672 Flaherty, C. F., 914, 927 Flamand, A., 551, 557, 565, 569 Flanagan, L. M., 910, 927, 932 Flanagan, P., 450, 454 Flatt, J. P., 919, 930 Flattery, J. J., 481, 501 Flaum, L. E., 310, 326 Flaum, M., 491, 492, 498 Flax, J. D., 621, 624 Fleischer, A., 472, 477 Fleming, A. S., 423, 429, 432 Fleming, L. E., 585, 588 Fleshman, A., 447, 456 Fletcher, D. J. C., 343 Fletcher, M. L., 190, 196 Fletcher, P., 516, 520 Flieger, S., 960, 962, 965, 966 Flipse, B. G., 891, 895 Flood, J., 444, 457 Flor, H., 261, 269 Flores, G., 619, 626 Flor-Henry, P., 492, 493, 500 Floriano, W. B., 276, 289, 293 Flory, J., 512, 516, 519 Flory, W., 60, 63, 72, 553, 572, 583, 591 Flower, D. R., 970, 976 Fluegel, K.W., 698, 705, 797, 804 Flumerfelt, B. A., 38, 42 Flynn, F. W., 683, 700, 862, 865, 866, 877, 907, 924 Flynn, R. E., 971, 978 Flynn-Rodden, K., 466–467, 474, 599, 609 Focacci, C., 647, 648 Fode, C., 121, 133, 760, 779 Fogarty, L., 535, 539, 544 Foley, J. F., 60, 62, 63, 66 Foley, P. 223, 228 Folio, C. J., 784–786, 792, 804 Folstein, M., 479, 481, 499 Folstein, S. E., 481, 489, 499, 602, 612 Fomon, S., 4, 15 Fong, K. J., 54, 62, 68 Fons, M., 786, 800 Font, C., 152, 156, 561, 565 Fontenot, D. T., 718, 719, 720, 726 Forbes, M. E., 773, 780 Forbes, W. B., 94, 96, 112, 141, 162, 412, 433, 616, 626 Ford, D. P., 581, 585, 588, 590 Ford, J., 488, 498 Ford, M., 104, 105, 110, 216, 224 Ford, M. D., 620, 624 Ford, M. J., 923, 928 Ford, R., 553, 573

Forehand, R. L., 369, 382 Foreman, J. C., 443, 453 Formaker, B. K., 717, 720, 726, 734, 751, 754, 755, 775, 779, 788, 793, 800, 952, 955 Fornasari, G., 450, 454 Forno, L. S., 513, 520, 522 Forrai, G., 331, 341, 858, 859 Fort, A., 174, 175, 179 Fortun-Lamothe, L., 369, 374 Foscolo, M., 356, 380 Fosdick, L. S., 643, 648 Fosmire, G. J., 891, 894, 895, 896 Foster, D. C., 82, 87 Foster, D. F., 864, 878 Foster, J. D., 30, 35, 42, 54, 62, 68, 72, 560, 567 Foulds, I. S., 940, 955 Fourcroy, A. F., xix, xxxvi Foureman, G. L., 583, 589 Fournel-Gigleux, S., 58, 70 Foville, T., xxiv, xxxvi Fowler, J. S., 594, 609 Fox, A. F., 847, 848, 859 Fox, A. L., 709, 726 Fox, D. A., 581, 589 Fox, E. A., 682, 702 Fox, G. D., 415, 438 Fox, K. A., 365, 366, 374 Fox, M. M., 923, 928 Fox, N. A., 828, 842 Fox, P., 647, 648 Fox, P. C., 646, 650 Fox, P. T., xxviii, xxxvi, 253, 270 Frackowiak, R. S., 253, 258, 270, 516, 520 Fraietta, L., 450, 454 Frame, S. R., 56, 67, 582, 588 Frampton, M., 539, 543, 587, 588 Franceschini, I., 104, 105, 108, 111 Franchi, C., 318, 325, 343 Francis, G. W., 219, 221, 226 Francis, J., 887, 895 Francis, S., 193, 198, 259, 264, 266, 271, 698, 699, 700, 702, 797, 802 Franciscus, R. G., 439, 454 Franco, B., 127, 133, 1001, 1017, 1021, 1022 Francoeur, R. T., 329, 342 Francom, S.F., 550, 453 Francot, C. M., 486, 501, 514, 519, 523 Frank, C., 619, 627 Frank, M. E., xv, xxiv, xxxvi, 466–467, 472, 473, 476, 644, 649, 684, 700, 714, 717, 721, 725, 726, 729, 732, 734, 738–742, 746–749, 751, 752, 754–757, 783–797, 799–801, 803, 864, 875, 876, 878, 946, 952, 953, 955, 957, 962, 963, 965 Frank, R. A., 795, 797, 798, 800, 812, 813, 817–819, 821, 856, 859 Franke, G., 439, 454 Frankland, P. W., 417, 420, 432, 436

Author Index Franklin, D., 888, 903 Franklin, R. J. M., 28, 29, 42 Frankmann, S. P., 740, 755 Franks, N. P., 280, 293, 988, 997 Franssen, H., 686, 704 Franz, B. R., 517, 519 Franzen, L., 27, 41, 123, 131, 150, 151, 155, 396, 397, 564, 566 Franzon, M., 554, 571 Fraser, D., 410, 411, 436 Fraser, N. W., 551, 557, 572 Frater, G., 276, 293 Fraùsto da Silva, J. J. R., 281, 293 Frazier, L. I., 564, 567 Frazier, L. L., 129, 131, 139, 155, 616, 617, 623 Fredholm, B. B., 784, 800 Fredman S. M., 555, 567 Fredrickson, J. M., 942, 955 Free, M. L., 212, 214, 225 Freed, W. J., 119, 121, 124, 137 Freedman, L. J., 515, 522 Freeman, F. N., 342 Freeman, S. K., 363, 376 Freeman, W. J., 124, 133, 152, 162, 163, 170, 174, 175, 177, 178 186, 187, 191, 193, 194, 196, 197, 200, 296, 307, 411, 413, 426, 428–430 Fregly, M. J., 862, 877 Frei, E., 64, 67 Freise, B., 909, 929 Freitag, J., 709, 710, 729 French, S. J., 882, 898 Frese, K., 551, 557, 570 Freund, M. S., 300, 307 Frey, L., 947, 955 Frey, P. W., 209, 225, 872, 877 Frey, S., 258, 266, 272, 698, 704, 797, 800, 803, 916, 930 Frey, W. H. I., 553, 567 Frey, W. H., 2nd 551, 556, 572 Freygang, W. H. J., xxviii, xxxvi, xxxviii, 253, 271 Friberg, L., 579, 589 Friberg, U., 658, 661, 670, 671, 825, 841 Fricker, J., 882, 896 Fricker-Gates, R. A., 515, 520 Fricton, J. R., 952, 957 Fridkin, M., 553, 563, 568 Friedewald, W. T., 645, 648, 894, 897 Friedland, R. P., 481, 495, 499 Friedman, B., 618, 624, 762, 779 Friedman, C., 17, 42 Friedman, D., 151, 156, 157, 185, 196 Friedman, D. P., 686, 704 Friedman, I., 18, 43 Friedman, J. H., 493, 499 Friedman, J. M., 603, 610 Friedman, M., 468, 475 Friedman, M. I., 682, 683, 701, 702, 732, 755 Friedman, M. P., xv, xxxiii, 799 Friedman, W. H., 9, 16

1047 Friedrich, R. W., 126, 133 Friedwald, W. T., 579, 589 Friefeld, G., 440, 456 Friend, K. B., 268, 270 Friendman, L. S., 889, 892, 893, 896 Friendman, M. I., 889, 892, 893, 896 Frier, B. M., 893, 901, 950, 956 Frierson, H. F., 38, 48 Frijters, J. E. R., 211, 218, 219, 225, 228, 806–815, 817, 820, 821, 822, 892, 897 Frings, S., xxv, xxxvi, 26, 27, 46, 47, 76, 80, 86, 87 Frisen, J., 620, 621, 623, 624 Frisen, J. T., 96, 108 Frisna, R. D., 255, 270 Friston, K. J., 258, 267, 269, 270, 516, 520 Frith, C. D., 516, 520 Fritsch, G., 1001, 1022 Fritschy, J.-M., 150, 156 Fritzsch, B., 767, 771, 779 Froment, J. C., 258, 264–266, 272, 424, 435 Frommer, G., xxiv, xli, Frommes, S. P., 30, 46 Froriep, R., 665, 672 Frosch, M. P., 124, 133 Frumkin, H., 581, 590 Fry, F. A., 449, 454 Frye, P., 873, 877 Frye, R. E., xxvi, xxvii, xxxv, xxxvi, 11, 15, 64, 68, 204, 215, 216, 217, 224, 225228, 445, 450, 451, 454, 463, 473, 475, 483, 484, 499, 500, 525, 537, 544, 580, 584, 585, 587, 588, 590, 595, 608, 609, 612, 823, 842 Fucetola, R., 495, 498 Fuchs, A. R., 370, 376 Fuchs, W., 718, 726 Fueshko, S. M., 1012, 1013, 1022, 1025 Fuji, M., 584, 589 Fujii, H., 62, 68, 507, 512, 521 Fujii-Kuriyama, Y., 62, 68 Fujikane, M., 789, 800, 960, 962, 965 Fujimoto, S., 661, 662, 664, 674, 672, 773, 779 Fujimoto, Y., 723, 730, 917–918, 933 Fujino, S., 887, 889, 990 Fujisawa, H., 145, 160 Fujita, H., 121, 123, 125, 138 Fujita, K., 662, 675 Fujita, S. C., 121, 135, 560, 570, 972, 978 Fujita, T., 28, 48, 124, 137, 662, 663, 672, 677 Fujiwara, H., 509, 522 Fujiwara, M., 26, 34, 49, 463, 477 Fujiwara, Y., 554, 570 Fujiyama, R., 720, 722, 728 Fukuchi, Y., 580, 591 Fukuda, H., 4, 16, 233, 249, 266, 267, 272 Fukukita, K., 37, 49

Fukunaga, I., 723, 730 Fukushi, H., 551, 570 Fukushima, H., 509, 523 Fukutake, T., 952, 955, 957 Fukuwatari, T., 884, 903 Fulbright, R. K., 167, 178, 259, 264–266, 270 Fulfaro, F., 891, 901 Fulker, D. W., 331, 340 Fülle, H.-J., 27, 43, 82–83, 87, 88 Fuller, C. M., 26, 29, 30, 31, 33, 45 Fuller, J., 914, 925 Fuller, T. A., 170, 176, 177, 179 Fulop, Z., 618, 624 Fulton, J. F., xxviii, xxxvi, 252, 270 Funakoshi, M., xxiv, xxxvi, 714, 720, 728, 730, 737, 747, 756, 758, 791, 802, 875, 877, 885, 896 Fung, K.-M., 98, 109 Funk, D., 172, 174, 177 Furi, S., 319, 327, 343 Furlong, C. E., 539, 544 Furlong, P. L., 686, 700 Furman, V., 262, 265, 266, 271 Furstenberg, A. C., 468, 475, 605, 609 Furudate, S., 367, 376 Furukawa, M., 101, 102, 111, 215, 225, 232, 246, 392, 397, 399, 634, 638 Furukawa, Y., 471, 476 Furukubo-Tokunaga, K., 116, 134 Furuta, S., 581, 589 Furuya, N., 243, 247 Fushiki, T., 884, 887, 889, 900, 903 Fuster, J. M., 411, 422, 429 Fuxe, K., 538, 546 Gaafar, H., 388, 398 Gabel, J., 535, 544 Gable, D., 357, 362, 374 Gabrieli, J. D., xxix, xliii, 174, 180, 452, 458 Gabrieli, J. D. E., 190, 200, 255, 256, 259–267, 269–273, 424, 436 Gabrielsson, J., 62, 69, 552, 559, 567 Gaburro, D., 642, 650 Gadoth, N., 952, 955 Gaffan, D., 414, 429, 692, 700 Gaffield, W., 765, 766, 781 Gage, F., 93, 108 Gage, F. H., 62, 72, 619–621, 625, 626 Gaggar, A., xxvi, xliv, 24, 26, 37, 49, 76, 80, 90, 397, 400 Gagliardo, A., 560, 565 Gagne, G.M., 442, 445, 455 Gagne, M., 359, 380 Gagnon, J., 795, 803, 884, 902 Gahide, I., 495, 500, 960, 961, 962, 966 Gainer, H., 122, 138, 1003, 1007, 1012, 1013, 1025 Galanter, E. H., 854, 860 Galef, B.G., Jr., 316, 323, 355–358, 360–362, 372, 376, 419, 429, 430, 838, 842, 908–909, 913, 927

1048 Galen, C., 651 Galioto, G., 450, 454 Galioto, P., 450, 454 Galizia, C.G., 182, 183, 193, 197, 220, 226 Gall, C., 102, 105, 109, 182, 196, 622, 624 Gall, C. M., 145, 151, 152, 156, 157, 183, 185, 196, 201, 412, 425, 431 Gall, J. A., 339, 341 Gallagher, M., 173–175, 179, 182, 199, 394, 396, 400, 414, 420, 421, 425, 430, 435, 438, 916, 927 Gallagher, P., 890, 897 Gallagher, R. M., 553, 562, 567 Gallego, J., 390, 392, 397, 399 Gallo, M., 915, 924, 927 Gallo, O., 62, 71 Gallousis, G., 165, 168, 171, 179 Gallucci, M., 602–603, 609 Galton, F., 330, 332, 338, 341 Galvin, J., 514, 520, 521 Gamble, J. F., 576, 589 Gambrieli, J. D. E., 486, 501 Gammage, R. B., 542, 545, 995 Gamzu, E., 914, 927 Ganchrow, D., 658, 659, 661, 664, 665, 670, 672, 677, 764, 769, 779, 824, 825, 842 Ganchrow, J. R., 467, 475, 658, 659, 661, 664, 665, 670, 672, 677, 747, 755, 764, 769, 779, 826–829, 841, 842, 843, 906, 927 Gandelman, R. J., 411, 437 Ganesan, R., 920, 927 Gangdev, P. S., 951, 955 Gangestad, S. W., 329, 341 Gannon, K. S., 664, 677, 709, 710, 711, 726, 730 Gans, R., 647, 648 Gansler, D. A., 495, 498 Ganzevles, P. G. J., 785, 800 Garattini, S., 689, 705 Garb, J.L., 905, 910, 918, 927 Garbers, D. L., 27, 42, 43, 82, 83, 88 Garbini, A., 314, 323 Garcea, M., 738, 741, 758, 864, 865, 874, 875, 879 Garcia, C., 166, 178 Garcia, G., 143, 157 Garcia, J., xxix, xxxi, xxxvi, 396, 398, 419, 420, 421, 427, 431, 434, 838, 843, 873, 877, 906, 910–911, 913–914, 917, 923, 925, 927, 928, 931 Garcia, J. R., 732, 755 Garcia, M. -S 1003, 1004, 1009, 1024 Garcia, P., 170, 178 Garcia-Diaz, D. E., 187, 196, 982, 997 García-Medina, M. R., 987, 993, 997 Garcia-Osuna, M., 564, 565 Garcia-Segura, L.-M., 27, 41, 56, 67 Garcia-Velasco, J. G., 3, 15 Garcia-Verdugo, J. M., xxviii, xxxix, 128, 134, 143, 156, 159, 620, 624

Author Index Gardell, S. J., 509, 521 Gardini, G. P., 300, 306 Gardner, E., 853–855, 860 Gardner, J. W., 297, 299–300, 305, 307 Gardner, L., 209, 211, 227 Gardner, L. E., 404, 408 Gardner, L. E., Jr., 320, 324, 349, 356, 379 Gardner, P., 81, 88 Garfinkel, L., 882, 902 Garibotti, M., 78, 87 Garlow, S. J., 152, 160 Garner, W. R., 817, 821 Garrett, J. R., 639, 648 Garrett-Laster, M., 893, 897 Garris, D. R., xxx, xxxv Garrity, D. L., 533, 534, 544 Garruto, D., 411, 434 Garten, S., xxiv, xxxvi, Gartside, P. S., 888, 897 Garty, H., 718, 726, 776, 777, 779 Gash, D. M., 130, 135 Gaskell, B. A., 62, 65, 67, 68, 583, 589 Gaskell, S. J., 971, 978 Gasser, B., 1003, 1012, 1021 Gasser, R. F., 982, 997 Gasser, H. S., xxiv, xxxvi Gasztonyi, Z., 332, 341 Gatch, M. B., 861, 863, 876 Gatenby, J. C., 797, 799 Gaulin-Kremer, E., 831, 841 Gaultier, C., 390, 392, 397, 399 Gauss, R., 722, 729 Gauthier, G., 603, 609 Gautschi, I., 719, 725 Gawel, M. J., 484, 497 Gawley, D. J., 914, 929 Gay, B., 55, 69 Gaykema, R. P., 170, 177 Gayner, S. M., 942, 955 Geary, R. S., 554, 563, 571 Gebhardt, L. P., xxvi, xlii, 2, 16, 60, 72, 556, 567, 571 Gebhardt, S. P., 615, 626 Gebruers, E. M., 889, 897 Geckle, R. J., xxix, xlv, 37, 49, 256, 259, 264–267, 269, 273, 344, 470, 478, 495, 497, 501, 512, 523, 593, 603, 613, 629, 630, 631, 633, 634, 637, 638 Gedda, L., 342 Geddes, J. F., 238, 245, 481, 490, 498 Gee, D. J., 318, 326 Geervani, P., 698, 704 Gehri, M., 553, 562, 569 Gehring, W. J., 116, 134 Geiger, L. E., 583, 590 Geiger, S. R., 913, 930 Geil, R. G., 583, 589 Geinisman, Y., 169, 180, 192, 200 Geisler, M. W., 232–238, 240, 244, 245, 247, 342, 462, 476 Geissler, A. H., 894, 897 Geisslinger, G., 238, 245

Gelb, A., 686, 702 Gelboin, H. V., 61, 73 Geller, A. M., 394, 396, 400 Geller-Bernstein, C., 450, 457 Gellman, R. L., 170, 177 Gemberling, G. A., 908, 927 Geneste, O., 64, 67 Genin, E., 339, 341 Genkova, P. I., 952, 957 Gennari, P. U., 656, 671 Gennings, J. N., 61, 68 Genow, A., 797, 800 Gent, J. F., xvi, xxxix, 211, 212, 215, 224, 227, 463, 466–467, 472, 473, 475, 476, 593, 610, 629, 633, 637, 709, 726, 734, 751, 752, 754, 756, 783–792, 795, 796, 798, 799–803, 816, 822, 857, 859, 889, 890, 896, 897, 946, 957 Genter, M. B., 56, 57, 60, 62–65, 67–69, 552, 553, 558, 564, 566, 567, 583, 589 Genton, M., 525, 544 Genzel, C., 319, 321, 326 George, A. E., 600–601, 609, 610 George, D. T., 922, 929 George-Hyslop, P., 509, 522 Geran, L. C., 867–871, 877 Gerard, C., xxv, xli, Gerber, S., 830, 842 Gerbert, B., 472, 477 Gerfen, C., 558, 560, 571, 572 Gerhardt, G. A., 153, 163 Gerin, M., 559, 568 Gerlach, J., 654, 672 Gerling, S. A., 361, 365, 377 German, D. C., 563, 564, 571 Gerrits, N., 557, 568 Gerritsen, G. J., 597, 610 Gertsberger, R., 416, 432 Gerull, G., 234, 244 Gervais, R., 148, 153, 154, 159, 162, 169, 174, 175, 176, 177, 179, 187, 188, 190, 191, 193, 195, 196, 198, 199, 261, 264, 272, 370, 377, 410, 413, 415, 418, 423, 426, 427, 429, 432, 433, 435 Gervasi, P. G., 55–61, 63–65, 66, 68, 70, 71, 561, 567 Gescheider, G. A., 204, 209, 225, 255, 270, 869, 871, 877 Gesteland, R. C., xxiv, xxxvi, 24, 28, 35, 42, 43, 84, 88, 123, 124, 132, 133 Getchell, M. L., 30, 35, 42, 53, 54, 56–58, 60, 67–71, 73, 75–76, 87, 119, 133, 145, 156, 221, 225, 233, 245, 507, 509, 510, 511, 519–523, 559, 560, 567, 570, 577, 589, 660, 672, 969, 979, 983, 997 Getchell, T. V., xxiv, xxxvi, 26, 27, 30–33, 35, 37, 42–44, 47, 53, 54, 56–58, 60, 67–71, 73, 75–76, 87, 96, 101, 102, 109, 111, 119, 132,

Author Index 133, 145, 156, 207, 208, 210–212, 216, 221, 224, 225, 233, 245, 341, 479, 480, 497, 505, 507, 508, 510, 511, 520–523, 552, 558–560, 567, 570, 573, 577, 589, 639, 648, 656, 660, 672, 674, 709, 732, 755, 759, 765, 768, 773, 780, 799–803, 840, 918, 924, 935, 938, 945, 950, 951, 954, 955, 969, 979, 982, 983, 997, 999, 1001–1004, 1025 Getschman, E., 661, 675 Getsy, J., 443, 451, 459 Getz, L. L., 422, 427 Geula, C., 680, 702 Gewirtz, G., 505, 510, 511, 516, 521 Ghantous, H., 62, 69, 552, 559, 567 Ghatei, M. A., 76, 85 Ghetti, B., 509, 522 Gheusi, G., xxviii, xxxvi, xli, 143, 156, 410, 415, 416, 427, 430 Ghiselli, E. E., 413, 427 Ghorbanian, S. N., 445, 455, 467, 473, 475 Ghosh, A., 620, 624 Giachetti, I., 187, 188, 196, 199, 421, 434, 682, 702 Giambanco, I., 32, 47 Giannetti, N., 388, 398 Gianutsos, G., 551, 567 Giasson, B., 509, 522 Gibber, J. R., 919, 929 Gibbs, J., 691, 701, 923, 928 Gibbs, R. B., 1012, 1026 Giberson, R., xxxi, xxxv, 107, 108, 213, 216, 225, 341, 461, 474 Giblen, G., 515, 520 Gibson, A. D., 27, 42 Gibson, E. L., 884, 897 Gibson, J. J., 371, 376 Gibson, M., 561, 562, 567 Gibson, R. S., 894, 897 Gibson, T. D., 305, 307 Gibson, W. P. R., 786, 789, 791, 801 Giddens, W. E., 583, 584, 589 Giduck, S. A., 936, 955 Gieroba, Z. J., 557, 565 Giese, J., 883, 897 Giesen, M., 230, 245 Gietzen, D. W., 865, 877, 884, 897, 900, 906, 927 Giger, R. J., 762, 763, 781 Gil, R., 482, 496 Giladi, E., 553, 563, 568 Gilbert, A. N., 318, 323 Gilbert, C., 919, 927 Gilbert, C. D., 190, 196 Gilbert, J. G., 4, 15 Gilbert, M., 642, 649 Gilbert, M. E., 538, 543 Gilbertson, D. M., 721, 726 Gilbertson, T. A., 697, 698, 701, 702, 708, 710, 711, 718–720, 721, 725–727, 737, 738, 752, 755, 829, 842, 881, 884, 897

1049 Gill, J. M., 748, 755 Gill, J. R. Jr., 784, 801 Gillett, N. A., 62, 72 Gillman, J., 919, 927 Gillmore, R., 884, 901 Gilman, A., 920, 927 Gilman, A. G., 24, 44, 80, 90, 915, 933 Gilmore, M., 832, 844 Gilmore, M. M., 480, 481, 500, 789, 795, 797, 800, 853, 854, 859, 864, 877 Gilmore, R. L., 950, 957 Gilmore, R. W., xxx, xxxiii, 403, 404, 407 Gingrich, B., 422, 430, 437 Giniès, C., 370, 381 Giombini, S., 621, 625 Giordano, T., 620, 624 Giorgi, D., 17, 42, 291, 294 Giorgi, P. P., 560, 569 Giovanetti, A., 38, 44 Giovanni, M. E., 787, 802, 884, 885, 901 Girard, P. R., xxxiii Girardin, M., 447, 454 Girardot, M. N., 183, 193, 195 Girault, J-A., xxv, xxxvii Girelli, M., 621, 625 Girgis, R., 388, 398 Girod, R., 154, 156 Girvin, J. P., 686, 702 Gispen, W. H., 125, 138, 417, 433 Gisselmann, G., xxv, xliv, 287, 293 Giugno, L., 915, 930 Giustetto, M., 149, 150, 157, 162 Giza, B. K., 680, 683, 684, 688, 691, 701, 704, 734, 736, 740, 744, 745, 747, 748, 751, 752, 755, 757, 758, 873, 878 Gizurarson, S., 553, 554, 564, 565 Gjuric, M., 243, 249 Gladue, B. A., 359, 379 Glanville, E. V., 857, 858, 859 Glasbrenner, B., 888, 897 Glaser, D., 713, 725 Glass, L., 828, 844 Glasscock, M. E., 946, 955 Glatt, C. E., 58, 73, 76, 81–82, 90 Gleason, K. K., 404, 407 Gleeson, R. A., 56, 60, 72, 81, 89, 220, 223, 807, 821 Glemarec, A., 219, 226 Glendinning, J., 644, 648, 738, 755, 865, 877, 881, 897 Glenn, J. F., 691, 701, 736, 755 Glickman, S. E., 404, 407 Glickstein, M., 266, 273 Globus, J. H., xxviii, xxxvi Glod, C. A., 538, 546 Glode, B. M., 174, 180, 194, 201, 394, 400, 411, 415, 426, 438 Gloor, P., xv, xxxvi, 948, 955 Glosser, G., 514, 521, 886, 897

Glover, G. H., xxix, xliii, 174, 180, 255, 257, 259–267, 269–273, 424, 436 Glover, P., 392, 394, 400 Glück, J., 839, 843 Glusman G., 17, 43, 145, 157 Goddard, W. A. III 276, 289, 293 Godde, M., 76, 87 Godfrey, D. A., xxx, xxxvi, 147, 153, 157, 170, 177 Godfrey, J., 403, 404, 407 Godinot, D., 174, 179, 264, 272 Godinot, F., 105, 107 Godo, M. N., 55, 70, 578, 589 Goedert, M., 505, 506, 509, 510, 513, 514, 521, 523 Goehler, L. E., 909–910, 919, 924 Goertzen, W., 468, 473, 475 Goetz, C. G., xxxii, xxxv, 588 Goetz, R. R., 493, 499 Goetzl, F. R., 882, 897 Goff, W. R., xxxi, xxxvi, xli, 230, 244, 248, 387, 398, 399, 404, 407 Gogola, G., 619, 626 Gogos, J. A., 146, 157 Gola, J. M. R., 987, 988, 991, 993, 994, 995, 997 Golbe, L. I., 486, 497, 514, 522 Gold, G. H., xxiii–xxvii, xxxiii, xxxiv, xxxix, xl, xliv, 24, 26, 35, 36, 37, 41, 43, 44, 46, 49, 75–76, 79–80, 81, 83–84, 85, 86, 88–90, 126, 130, 131, 134, 220, 226, 355, 382, 397, 400, 426, 437, 622, 626 Gold, M. S., 983, 998 Goldberg, H. I., 602, 608 Goldberg, S. J., 188, 198 Goldberg, T. E., 603, 612 Golden, G.T., 512, 513, 522 Golden, J. A., 116, 133 Goldfoot, D. A., 363, 376, 919, 927 Golding-Wood, D. G., 467, 475 Goldman, A., 894, 897 Goldman, A. S., 839, 842 Goldman-Rakic, P.S., 193, 196 Goldschmidt, H., 824, 842 Goldsmith, D., 786, 791, 803 Goldstein, B. J., 33, 35, 43, 53, 62, 69, 76, 87, 101, 102, 106, 109, 118, 119, 133, 617, 621, 624 Goldstein, I. J., 714, 727 Goldstein, J. A., 61, 70 Goldstein, J.M., 491, 492, 501 Goldstein, M., 103, 109, 505, 509, 514, 516, 520 Goldstein, R. B., 532, 544 Goldstein, R. S., 661, 672 Goldwasser, R. A., 557, 570 Goller, F., 216, 224 Gollomp, S. M., 215, 225, 484, 485, 497, 514, 520, 521 Golombowski, S., 508, 509, 521 Gomes, A., 443, 454 Gomez, G., 185, 196, 221, 227

1050 Gomez, J., 923, 928 Gomez-Carneros, C., 847, 857, 858, 858 Gong, Q., 100, 111, 109, 119, 120, 122, 123, 125, 127, 130, 133 Gonoi, T., 715, 728 Gonyou, H. W., 315, 325 Gonzales, E., 343 Gonzales-Estrada, M. T., 124, 133 Gonzalez, F., 57, 61, 70, 73, 124, 133 Gonzalez, J. S., 948, 955 Gonzalez, K., 81, 90 Gonzalez, M. F., 917, 928 González, M. de L., 130, 133 Gonzalez-Bono, E., 359, 376, 382 Gonzalez-Mariscal, G., 370, 376 Goo, C. F., 581, 591 Good, A., 321, 324 Good, K. P. 271, 342, 491–493, 495, 498, 499, 961, 965 Good, P. F., 551, 555, 567, 570 Good, J. M., xxii, xxiii, xxxvi Goodall, G., 410, 415, 416, 430 Goode, J., 645, 649 Goode, R. L., xxix, xliii, 174, 180, 259, 261, 264, 266, 271, 272, 424, 436, 441, 454 Goodhouse, J., 420, 435 Goodison, T., 366, 375 Goodlett, C. R. 843 Goodman, J.M., 491, 492, 501 Goodman, L. S., 920, 927 Goodman, M. 111, 109 Goodman, P. E., 530, 532, 538, 545 Goodman, S. I., 833, 843 Goodspeed, R. B., 204, 212, 224, 463, 475, 593, 606, 610, 629, 633, 637, 790, 791, 800, 889, 890, 896, 897 Goodwin, M., 346, 382 Goosens, K. A., 670, 674, 767, 771–773, 781 Gopel, W., 304, 308 Gorczyca, W. A., 83, 89 Gordon, A. S., 472, 476, 600, 610 Gordon, A. S. D., 451, 454 Gordon, C. R., 952, 955 Gordon, M. K., 121, 133 Gore, J. C., 259, 264–266, 270, 797, 799 Gorell, J. M., 556, 564, 568 Gorham, D., 124, 133 Goridis, C., 760, 779 Gorman, J. M., 262, 265, 266, 271, 493, 499, 516, 523 Gormazano, G., 914, 929 Gormican, A., 890, 891, 895 Gorski, R. A., xxx, xxxv, 919, 923, 929, 932 Gorsky, M., 952, 955 Goschnick, J., 298, 307 Gosepath, J., 444, 445, 454 Gossler, A., 621, 624 Göthe, C. J., 539, 544 Goto, N., 787, 793, 801 Goto, R., 266, 267, 272

Author Index Gotoh, O., 57, 62, 68, 71 Gots, R. E., 539, 544, 587, 589 Gottesman, I. I., 331, 332, 341 Gottlieb, D. I., 121, 124, 137, 145, 162, 621, 623 Gottofrey, J., 551, 555, 568 Gottwald, B., 469, 474 Goudie, A. J., 914, 920, 927 Gould, E., xxviii, xxxvii, xliii, 622, 624 Gould, K. L., 600, 610 Gould, V. E., 38, 48 Goumans, M.-J., 101, 113 Gourlay, S. G., 554, 563, 568 Goursaud, A. P., 320, 323 Gower, D. B., 61, 68 Goy, R. W., 363, 376, 919, 926, 927 Goyens, J., 357, 362, 376 Gozes, I., 553, 563, 568 Gozzo, S., 618, 624 Graamans, K., 598, 610 Grabauskiene, S., 765, 766, 781 Gråberg, J., 665, 672 Gradwohl, G., 119, 121, 132, 760, 779 Graeber, M., 509, 514, 521 Graf, P., 444, 451, 454, 454, 455 Graf, W., 557, 568 Graff-Radford, N. R., 469, 475 Graftnan, J., 216, 224, 680, 702 Graham, A., 760, 778, 779 Graham, B. G., 793, 803, 884, 893, 895, 902, 950, 951, 954, 956 Graham, C., 366, 375 Graham, C. H., 750, 751, 755 Graham, G., 104, 111 Graham, H. N., 698, 701 Graham, J. M., 359, 376, 423, 430 Graham, P. W., 420, 430 Graham, S., 145, 163, 392, 394, 400 Graillon, A., 828, 829, 842 Grajski, K. A., 411, 430 Granata, A. R., 359, 374 Grandes, P., 151, 159 Grandt, D., 94–96, 107, 1015, 1021 Granger, R., 188, 191, 198, 410, 412, 425, 426, 426, 431, 433 Granie-Prie, M., 660, 671 Granier-Deferre, C., 312, 326 Grannot-Reisfeld, N., 103, 111, 618, 625 Grant, E. C., 361, 378 Grant, M. M., 882, 900 Grant, P., 122, 138, 1003, 1007, 1012, 1025 Grant, R., 786, 791, 801 Grant, S., 923, 928 Grant, V. L., 905, 927 Gravel, R., 827–829, 840 Graves, A. B., xxxii, xxxvii, 334, 341, 483, 498 Gray, H., 5, 7–12, 15 Gray, A. J., 482, 498, 513, 520 Gray, C. M., 170, 177, 413, 430 Gray, E. G., 143, 157 Gray, J., 368, 382

Gray, J. A., 687, 702 Gray, J. M., 920, 930, 933 Gray-Keller, M. P., 83, 89 Graziadei, P. P. C., xxii, xxvii, xxxvii, 17, 18, 31, 36, 43, 45, 52, 62, 69, 75, 76, 86, 87, 93, 94, 96–98, 100, 106, 108–110, 118, 119, 121, 123, 125, 127, 129, 130, 132, 133, 135–137, 141, 157, 616, 625, 660, 672, 972, 979, 1015, 1022 Graziani, A., 891, 899 Grecksch, G., 619, 627 Green, B. G., 210, 225, 670, 671, 740, 745, 754, 755, 783, 788, 795, 797, 800, 801, 853, 854, 859, 864, 877, 982, 997, 999 Green, C. A., 29, 48 Green, D. M., 209, 225, 869, 871, 877 Green, J. E., 481, 498 Green, K. F., 732, 755 Green, M., 58, 70 Green, M. A., 318, 326 Green, P., 216, 224 Green, S. J., 795, 800 Greenamyre, J. T., 564, 565 Greenberg, B., 507, 509, 514, 519 Greenberg, L., 947, 957 Greenberg, M. E., 620, 624 Greenberg, M. J., 1004, 1024 Greenberg, M. M., 583, 589 Greenberg, R. A., xxxiii, 142, 155 Greenberg, R. M., 81, 88, 89 Greenberg, J., xxviii, xli Greene, D., 553, 562, 572 Greene, J. A., 893, 895 Greene, L. S., 832, 841, 883, 886, 896 Greengard, P., 84 Greenstein, J. A., xxix, xliv Greenwood, M. M., 828, 843 Greenwood, M. R. C., 892, 896 Greep, R. O., 919, 927 Greer, C. A., 104, 105, 109, 110, 129, 130, 133, 134, 137, 139–146, 148, 149, 151, 152, 155, 157–160, 163, 164, 181, 184, 185, 186, 200, 220, 228, 258, 273, 335, 341, 350, 355, 380, 511, 514, 523, 621, 624 Greer, G., 484, 486, 495, 496 Greff, G., 991, 998 Greger, J. L., 894, 897 Gregg, B., 417, 430 Gregler, L. L., 600, 610 Gregoire, A. T., 363, 376 Gregoire, M. C., 174, 179, 258, 264–266, 272, 424, 435 Gregor, T., 204, 206, 215, 225, 461, 462, 474, 480, 481, 497, 563, 567, 600, 609 Gregory, E., 182, 199 Gregory, E. H., 356, 376 Gregson, R. A. M., 212–214, 224, 225, 490, 492, 496, 817, 821 Greiner, R. S., 394, 398

Author Index Gresack, J., 865, 877 Gresham, L. S., 64, 69 Grewal, M. S., 470, 477 Grier, J. B., 209, 225 Griesbach, G. S., 412, 430 Griff, E. R., 153, 154, 156, 157, 187, 197, 413, 424, 431 Griff, I. C., 329, 334, 335, 341 Griffin, F., 848–850, 855–858, 859 Griffin, J. W., 445, 455 Griffin, K., 116, 137 Griffin, M. G., 416, 430 Griffin, P. M., 883, 900 Griffith, G., 28, 43 Griffith, G. C., 829, 844 Griffith, I. P., 216, 225 Griffith, J., 392, 399 Griffith, W. C., 56, 59, 60, 64, 70 Griffiths, D. H., 452, 454 Griffiths, G., 828, 829, 842 Griffiths, J., 365, 375 Griffiths, M. V., 962, 965 Griffiths, R. R., 535, 546 Griffo, W., 352, 361, 378 Grigaliunas, A., 762, 777, 779 Grigson, P. S., 682–684, 701, 703, 704, 914, 916, 918, 927, 928, 931 Grijalva, C. V., 420, 427 Grill, H. J., 682, 683, 700, 701, 732, 734–736, 755–758, 828, 842, 862, 863, 865–868, 870, 873–875, 876–878, 907–908, 913–914, 916–918, 924, 928, 931, 932 Grillo, M., 27, 41, 103, 111, 123–125, 131, 135, 138, 150, 155, 160, 396, 397, 552, 558, 564, 566, 570, 572, 618, 625, 626 Grimes-Hillman, M., 343 Grimm, R. H., 886, 896 Grimmelikhuijzen, C. J., 1004, 1022 Grimsby, S., 101, 113 Grimsley, D. L., 907, 928 Grinker, J. A., 828, 832, 842, 892, 897 Grinspan, J. B., 10, 133 Grinvald, A., 259, 270, 271 Grioni, E., 621, 625 Gritzmann, R., 888, 897 Grodd, W., 261, 269 Grolli, S., 19, 48 Groom, C. R., 970, 976 Grootegoed, A., xxv, xli Gropman-Rubin, J., 828, 842 Gros, F., 124, 132 Gross, E. A., 54, 55, 70, 71, 578, 582, 588, 589, 591 Gross, C. G., xxviii, xxxvii Gross, G. W., 552, 558, 568, 573 Grosser, K., 234, 238, 239, 245, 246, 444, 445, 455, 463, 476 Gross-Isseroff, R. G., 331, 341, 445, 457, 467, 473, 475, 477, 491–493, 498, 501, 769, 779, 891, 900 Grossman, E., 509, 517, 521

1051 Grossman, M. I., 882, 897 Grossman, R. I., 602, 608 Grossman, Y., 412, 425, 435 Grossmann, S., 923, 933 Grosvenor, M., 909–910, 928 Grosvenor, W., 660, 672 Grover Johnson, N., 660, 672 Groves, J., 786, 789, 791, 801 Groves, P., 347, 376 Grovum, W. L., 948, 955 Growdon, J. H., 481, 498 Gruen, J. R., 716, 730 Grumbach, M. M., 38, 48 Gruneberg, H., 1004, 1022, 1024 Grushka, M., 786, 788, 790, 791, 802, 952, 955 Gruss, L., 907, 931 Gruss, P., 116, 133, 136 Gruünbaum, L., 716, 728 Gryllus, L., xx, xxxvii Grymer, L. F., 443, 450, 455 Gu, J., 53, 56–58, 61–65, 68, 69, 72, 73, 552, 559, 567, 568, 582, 589, 917, 928 Guadagni, D. G., 204, 225 Guagliardo, N. A., 734, 751, 758, 867–869, 871, 874, 877–879 Guagliardo 766 Gualtieri, T., 961, 965 Guan, X., 418, 430 Gubernick, L. T., 908, 928 Gudermann, T., 76, 90, 972, 977 Gudziol, H., 990, 997 Guengerich. F. P., 57, 58, 63, 64, 66, 69, 73 Guerin, J., 482, 499 Guevara, J. L., xxvii, xxxv, 101, 102, 108, 119, 132 Guevara-Aguilar, R., 187, 196, 982, 997 Guevara-Guzman, R., 423, 431 Guggenheimer, J., 939, 954 Guglielmi, R. E., 538, 539, 542, 544 Guglielmotti, V., 165, 171, 178 Guild, R. S., 1001–1004, 1023 Guilford, J. P., 204, 210, 214, 225 Guillamón, A., 975, 978 Guillemot, F., 119, 121, 132, 133, 760, 779 Guillette, B. J., 441, 442, 455 Guinard, J. X., 832, 843, 887, 897 Guiney, S., 660, 673 Guioli, S., 127, 133, 1017, 1022 Guizzetti, G., 236, 247 Gulati, P., 472, 474 Gulick, W. L., 255, 270 Gulisano, M., 116, 137 Gulya, K., 154, 158, 512, 521 Gulyas, B., 174, 179, 258, 259, 262–264, 266–268, 272, 350, 381, 424, 435 Gumpel, M., 124, 132 Gundelfinger, E., 84, 86 Gundersen, J., 923, 930 Gungor, A., 450, 452, 455 Gunsalus, I. C., 57, 71

Güntherschulze, J., 22, 43 Guo, J., 55, 57, 58, 72, 73 Guo, S. W., 798, 801, 847, 848, 855, 858, 859 Guo, W., 709, 711, 721, 725, 730 Guo, X., 509, 514, 520 Gupta, B. N., 578, 583, 591 Gupta, K. C., 165, 168, 171, 180 Gur, R. C., 269, 271, 479, 491–493, 498, 499, 511, 515–517, 519, 520, 522 Gur, R. E., xxxii, xliv, 269, 271, 273, 479, 491–493, 498, 499, 512, 515–517, 519, 520, 522, 523 Gurkan, S., 644, 648, 658, 663, 672, 768, 769, 781 Gurney, N. L., 362, 378 Guroff, G., 620, 624 Gurski, M. R., 184, 196 Gurwitz, S. B., 828, 844 Gussack, G. S., 597, 610 Gustafson, A.-L., 123, 133, 559, 565 Gustafson, R. O., 596, 612 Gustafsson, J. A., 538, 546 Gustavson, C. R., 920–923, 928, 930 Guterman, A., 891, 900 Guth, L., 773, 779 Guthrie, J. F., 923, 931 Guthrie, K. 111, 109 Guthrie, K. M., 145, 157, 182, 184, 185, 187, 196, 201, 558, 560, 568, 617, 622, 624 Gutierrez, J., 81, 89 Gutman, L. A., 391, 393, 400 Gutman, N. J., 807, 821 Gutnick, D. N., 490, 500 Guttman, N., 866, 877 Gutzke, W. H. N., 970, 978 Gutzwiller, F., 586, 589 Guz, A., 267, 269 Gwinn, K. A., 489, 495, 495, 498 Gwirtsman, H., 922, 929 Gyo, K., 942, 957 Gysling, K., 152, 153, 154, 155 Haaheim, L. R., 554, 568 Haberkorn, K., 319, 323, 341 Haberly, L. B., xxxi, xxxiv, 146, 147, 157, 165, 167–173, 176, 177, 180, 181, 187, 188, 190, 191–193, 196, 197, 200, 268, 270, 409, 410, 425, 426, 430, 431, 974, 977 Habermehl, K. H., 655, 666, 672 Hachinski, V. C., 600, 608, 686, 702 Hackenberg, T. D., 734, 758, 868, 874, 879 Hacker, B., xxvi, xliv, 24, 26, 37, 49, 76, 80, 90, 397, 400, 426, 428 Hackett, J. B., 342 Hackney, D. B., 604, 613 Hadden, R., 315, 326 Hadizath Taimagari, G., 832, 844 Hadjigeorgiou, G. M., 514, 522 Hadley, T., 516, 519

1052 Hadley, W. M., 51, 55–57, 59, 60, 62–64, 67, 69, 558, 559, 560, 561, 566 Haerer, A. F., 947, 955 Haertle, T., 77, 86 Haga, T., xxv, xliv, 287, 294 Hagan, P. J., 604, 610 Hagbarth, K. E., 146, 157 Hagen, R. L., 882, 895 Hagenmaier, H., 552, 564 Hahn, C. G., 511, 516, 519, 523 Hahn, F. F., 60, 67, 70, 583, 588 Hahn, H., 785, 801, 806, 812, 818, 821 Hahn, I., 55, 56, 69, 266, 270, 448–450, 455, 457 Haider, H., 443, 454 Haight, J. S. J., 5, 15, 441, 450, 452, 453, 455 Haines, R. F., 321, 322 Haines, S. J., 469, 476 Hainsworth, F. R., 643, 648 Hajnal, A., 691, 701, 736, 755 Hakanson, R., 907, 932 Hakkola, J., 57, 70 Halabisky, B., 151, 157 Halaris, A. E., 170, 178, 179 Halasz, N., 103, 109, 130, 133, 139, 140, 143, 146, 148–153, 157, 165, 166, 167, 171, 177, 184, 198, 514, 520 Haldane, E. S., xxi, xxxvii Halem, H. A., 972, 975, 977 Haley, P. J., 582, 590 Haley, R. W., 538, 539, 544 Halfter, W. 111, 112 Halgren, E., 269, 270 Hall, H., 646, 648 Hall, J. M., 765, 779 Hall, M., 62, 73, 646, 648 Hall, M. J., 709, 726, 856, 859 Hall, R. D., 618, 624 Hall, W. C., 165, 180 Hall, W. G., 171, 178, 190, 197, 310, 322 Hall, W. J., 889, 897 Hallen, H., 444, 451, 454, 454, 455 Hallett, M., 441, 458 Halliwell, B., 54, 67 Hallonet, M., 116, 133 Halloran, F. J., 148, 163 Hallsworth, P. G., 557, 565 Halmi, K. A., 922–923, 928, 930 Halpern, B. P., xxiv, xli, 643, 649, 746, 751, 755, 785–787, 789, 793, 801, 864, 873, 875, 877, 879, 891, 899 Halpern, M., 148, 159, 165, 168, 177, 178, 351, 376, 970–973, 977–979 Halpern-Sebold, L., 1003, 1022 Halpin, Z. T., 317, 323, 417, 422, 430, 433 Haly, P. J., 583, 588 Ham, M., 365, 375 Hama, K., 151, 152, 158, 183, 184, 197, 618, 624 Hamada, H., 62, 68 Hämäläinen, M. S., 241, 245, 245

Author Index Hamasaki, K., 664, 672 Hamazu, Y., 233, 249 Hamberger, A., 185, 197 Hambloch, H., 815, 816, 821 Hamburg, R., 443, 458 Hamburger, R., 481, 500 Hamedi, S., 166, 178 Hamilton, G. van T., 386, 398, 403, 407 Hamilton, J. M., 489, 498 Hamilton, K. A., 145, 151, 154, 155, 158, 184, 185, 194, 196 Hamilton, L. W., 411, 437 Hamilton, R. B., 679, 680, 684–686, 702, 913, 928, 948, 956 Hamilton, W. D., 336, 341 Hamilton 774, 779 Hamm, T. E. J., 582, 588 Hammarlund, E., 661, 671 Hammer, M., 991, 997 Hammerle, H., 304, 308 Hammerstad, J., 553, 573 Hamosh, M., 642, 648 Hamrick, W. D., 172, 175, 177, 191, 196, 410, 430 Hamuy, T. P., 410, 435 Han, L., 472, 477, 495, 500, 619, 626 Han, L. Y., 511, 516, 517, 519, 523 Hanamori, T., 740, 741, 747, 748, 755, 757 Hanamura, Y., 24, 46, 120, 136 Hancock, D., 972, 976 Hancox, J., 365, 375 Haneberg, B., 554, 568 Hankins, W. G., xxxi, xxxvi, 396, 398, 906, 913, 925, 927, 928, 931 Hann, C. S., 858, 859 Hannibal, J., 786, 802 Hannigan, J. H. 843 Hannon, K., xxvii, xxxv, 101, 102, 108, 119, 132 Hannover, A., 654, 672 Hanoune, J., 80, 87 Hanrahan, L. A., 471, 475 Hansel, D. E., 76, 87 Hansen, A., 34, 40 Hansen, B., 554, 563, 568 Hansen, L. F., 991, 997, 999 Hanski, E., xxvi, xl, 76, 80, 89 Hansma, P. J., 281, 293 Hanson, I. M., 116, 133 Hanson, L. 839, 842 Hanson, N. P., 542, 543 Hantsch, J 515, 519 Hanukoglu, I., 57, 71, 559, 570 Hao, S. P., 940, 957 Hao, Y. P., 149, 159 Haouari, N., 828, 829, 842 Happer, C., 602, 610 Hara, I., 941, 957 Hara, J., 285, 293 Hara, T. J., 34, 49, 659, 660, 675, 677 Harada, H., 232, 245 Harada, N., 584, 589, 712, 729

Harada, S., 670, 672, 720, 726, 741, 746, 755, 764, 767, 768, 779 Harada, Y., 59, 72 Hard, C., 713, 725 Hardcastle, P. F., 451, 455 Hardebo, J. E., 553, 566 Hardelin, J. P., 127, 134, 1003, 1012, 1016, 1017, 1022, 1023, 1025 Harden, R., 582, 589 Harder, D. B., 709, 726, 730, 873, 877 Harder, U., 284, 294 Hardesty, I., 1002, 1022 Hardin, E. E., 526, 538, 543 Harding, G. F. A., 686, 700 Harding, J. W., 96, 108, 109, 616, 623, 624 Hardy, H., 147, 148, 156, 165, 169, 171–173, 177 Hardy, S. L., 950, 955 Hari, R., 241, 245, 246, 259, 266, 271 Harkavay-Friedman, J., 493, 499 Harkema, J. R., 52, 54, 55, 60, 67, 69, 70 Harker, L.A., 942, 955 Harkins, S. W., 212, 228 Harkness, L., 831, 843 Harkness, R. S., 922, 931 Harlnsen, A. G., 54, 69 Harmatz, P., 361, 377 Harner, S. G., 938, 346, 954, 955 Harnsberger, H. R., 595, 609 Harper, A. E., 884, 899 Harper, H. W., 825, 842 Harper, R., xv, xxxvii Harriman, A. E., xxix, xxxvii, 867, 869, 870, 877 Harrington, A., xv, xxxvii, 254, 270 Harrington, C., 28, 43 Harrington, J. M., 526, 546 Harris, G., 830, 842, 886, 897 Harris, G. J., 601, 610 Harris, G. W., 349, 375 Harris, H., 784, 801, 848, 849, 859 Harris, H. E., 468–469, 476 Harris, L. J., 254, 270 Harris, S. R., 554, 563, 569 Harris, W., 667, 672 Harrision, L. M., 494, 499 Harrison, A. K., 551, 557, 561, 570 Harrison, P. J., 491, 498, 510, 515, 520, 600, 610 Harrison, R., 445, 454 Harrison, T., 509, 521 Harrison, T. A., 139, 163, 184, 186, 196, 200 Harrison, W. T., xxvi, xxxiii, 556, 565 Harron, A., 471, 474, 867, 872, 876, 892, 895 Hart, B. L., 909, 928 Hart, J.R., xxxii, xliii, 480, 482, 501 Hartfiel, L. M., 884, 902 Hartley, C. A., xxix, xliii, 256, 259, 263, 265–267, 272, 424, 436, 452, 458 Hartman, D., xxxi, xxxvii, 38, 43, 463, 476

Author Index Haruma, K., 887, 900 Harvey, C., 797, 802 Harvey, J., 482, 499 Harvey, S., 351, 362, 380, 971, 977 Hasama, B., xxiv, xxxvii Hasan, A., 493, 499 Hasan, G., 56–58, 73, 335, 341 Haschek, W. M., 60, 62, 69, 552, 559, 569 Hasegawa, M., 444, 450, 451, 456, 458, 510, 513, 514, 523 Hasegawa, S., xxxi, xxxvii, 37, 38, 43, 49, 463, 477 Hasegawa, T., 553, 562, 571, 573 Hashibia, M., 467, 476, 629, 637 Hashida, M., 553, 561, 562, 571 Hashimoto, M., 78, 91, 145, 164, 287, 294, 396, 400 Hashkes, P. J., 888, 897 Hasler, A. D., 426, 430 Hassab, M. H., 9, 15 Hasselmo, M. E., 148, 159, 170, 173, 177, 178, 190–193, 194–197, 200, 410, 413, 424–426, 428, 430, 432, 680, 701 Hastie, N. D., 116, 133 Hastings, L., 33, 45, 63, 70, 101, 103, 111, 388, 392, 394, 396, 398, 400, 551, 555, 556, 567, 568, 576, 578, 581–584, 588, 589, 591, 619, 626 Hastings, N. B., xxviii, xliii, 622, 624 Hataguchi, Y., 151, 158, 183, 197 Hatanaka, T., 351, 376 Hatfield, J. V., 299, 307 Hatfield, T., 420, 430 Hathcock, S. L., Jr., 305, 307 Hatini, V., 116, 133 Hato, N., 942, 956, 957 Hatt, H., xxv, xliv, 26, 43, 82, 87, 243, 249, 287, 293, 304, 308 Hatton, G. I., 169, 170, 180 Hatton, M., 647, 648, 649 Hattori, T., 952, 955, 957 Hau, K. M., 991, 997, 998 Haugen, I. L., 554, 568 Haughey, N. J., 511, 515, 520 Haun, F., 416, 417, 432 Hausdorff, W. P., 84, 87 Hauser, G., xxix, xxxvii Hauser, G. J., 311, 317, 323, 833, 842 Hausfeld, J. N., 54, 71 Hausser-Hauw, C., 950, 955 Hautvast, J. G., 888, 903 Hautzinger, M., 236, 244, 491, 496 Havens, B. R., 285, 293 Hawkes, C. H., 238, 245, 248, 479, 482, 485, 486, 488, 490, 498, 501, 509, 510, 514, 519, 520, 522 Haxby, J. V., 481, 495, 499 Haxhiu, M. A., 450, 455 Hayama, T., 736, 755, 756, 916, 930 Hayashi, H., 121, 123, 125, 138 Hayashi, S., 356, 377 Hayashi, T., 887, 897

1053 Hayashida, T., 485, 496 Haycock, J. W., 151, 152, 156, 183, 196 Hayden, M. R., 596, 610 Hayden, R. E., 602, 611 Hayek, R., 551, 555, 568 Hayes, C. L., 61, 69 Hayes, R. B., 559, 568 Hayes, U., 921, 925 Hayes, U. L., 915, 921, 926 Haykin, S., 191, 195 Haynes, N. B., 368, 374 Hayney, M. S., 330, 341 Hayward, M., 788, 801 Hazlett, B. A., 356, 382 Hbe, K., 709, 728 He, J. W., 82, 87 He, L., 581, 589 He, W., 64, 72, 717 Head, R., 103, 111, 618, 625 Heading, R. C., 923, 928 Heald, A. E., xxxi, xlii, 605, 610, 788, 803, 951, 956 Healey, B. G., 299, 308 Healy, B., 593, 610 Heaney, R. K., 857, 859 Heaslip, R. J., 82, 85 Heather, J. D., 253, 270 Hebhardt, P., 789, 801 Hecht, B. R., 330, 341 Heck, G. L., xxv, xxxvii, 643, 648, 715, 718, 720–722, 725, 726, 728–730, 776, 779, 782 Heck, H. D., 56, 67 Heck, H. F., 713, 725 Hedrick, P. W., 340, 341 Heemskerk, J., 21, 44 Heffron, S., 788, 803, 951, 956 Heidel, A., xvi, xxxvii Heiderich, F., 825, 842 Heilmann, S., 236, 246 Heimer, L., 147, 148, 153, 156, 164, 165, 167, 169–173, 177, 178, 180, 413, 438, 618, 624 Heinonen, T., 442, 454 Heinrichs, S. C., 884, 896 Heinsbroek, R. P. W., 918, 933 Heinsen, H., 510, 521 Heintz, C., 209, 211, 227 Heinz, B., 447, 458 Heinz, E. R., 602, 609 Heinzow, B., 525, 528, 543 Heise, S., 358, 376 Heist, H. E., 35, 43, 46, 120, 136 Heitzenrohder, C., 386, 398 Heizmann, C. W., 140, 142, 144, 151, 152, 158, 163 Hejmancik, J. K., 1012, 1026 Heldin, C.-H., 101, 113 Heldman, H., 559, 570 Heldman, R., 536, 545 Heldmann, J., 57, 71 Helekar, B. S., 83, 89 Helenius, H., 511, 515, 517, 522

Helfand, S. L., 335, 341 Helfrich, T., 32, 36, 48, 100, 112, 126, 137 Helgren, M., 762, 779 Hellekant, G., 658, 659, 662, 663, 670, 671, 672, 674, 713, 714, 725, 726, 741, 755 Heller, A. D., 471, 476, 892, 899 Heller, J., 77, 87 Heller, M. B., 553, 562, 570 Hellhammer, D. H., 359, 377 Hellman, T. J., 665, 672, 824, 842 Helm, J. F., 644, 648 Helmholtz, H., 654, 672 Helmreich, R. L., 365, 377 Helms, J. A., 788, 790, 792, 801, 953, 955 Helsmoortel, J., 960, 965 Helson, H., 210, 225 Heltberg, J., 910, 929 Hemdal, P., 483, 498 Hempel-Jørgensen, A., 987, 998 Hempstead, J., 618, 625 Hempstead, J. L., 103, 106, 109, 111, 122, 133, 560, 568 Hen, R., 103, 108 Henderson, P. W., 316, 323, 355, 376, 908, 927 Henderson, R. F., 54, 69, 582, 590 Henderson, S. A., 798, 800, 848, 849, 851–853, 856–858, 858, 859 Henderson, V. W., 480, 498 Hendricks, L. A., 827–829, 840, 842 Hendricks, S. J., 769, 776, 779 Hendrickx, A. G., 982, 997 Hendrix, D. E., 230, 244, 245, 246 Hendrix, D. V., 244, 246 Hendrix, G. M., 142, 162 Hendry, S. H. C., 151, 152, 156, 183, 196 Hendry, C., xxiv, xxxiv Henegar, J. R., 97, 98, 110, 564, 570, 616, 617, 620, 622, 623–625 Hengehold, A. K., 416, 435 Henggeler, S., 913, 924 Henk, W. G., 60, 63, 72, 553, 572, 583, 591 Henkin, R. I., 257, 266, 268, 269–271, 291, 293, 424, 432, 470, 471, 475, 579, 589, 593, 604, 612, 620, 625, 645, 646, 648–650, 661, 664, 675, 676, 710, 716, 727, 784, 793, 794, 797, 801, 890–894, 897, 899–902, 950, 952, 955, 956, 963, 965 Henneberger, P. K., 529, 532, 537, 542, 545 Hennessey, J. W., 918, 929 Henning, H., 386, 398 Henning, H. E., 617, 623 Henquell, D. 269 Henriksson, A., 553, 566 Henriksson, J., 60, 69, 72, 551, 554, 555, 556, 568, 572 Hensley, M. J., 441, 458 Henson, R. N., 258, 270 Henton, W. W., xxxi, xxxvii, 987, 998

1054 Henzler, D. M., 661, 664, 673 Hepper, P. G., 309, 312, 315–318, 323, 326, 336, 338, 340, 341, 355, 377, 833, 834, 842 Her, C., 59, 73, 875, 877 Herberhold, C., 230, 232, 245, 960, 965 Herbert, J., 356, 381 Herbison, A. E., 921, 932 Heredia, M., 104, 112, 129, 130, 137 Herkenham, M., 152, 160 Herlyn, D., 599, 608 Herman, C. P., 882, 887, 896, 898 Herman, M. M., 515, 517, 518, 520, 521 Hermann, E. A., 583, 590 Hermann, F., 654, 672 Hernadi, A., 192, 193, 199, 688, 697, 704 Hernadi, I., 695, 704, 829, 844, 948, 956, 959, 966 Hernández, S. M., 987, 993, 997 Herness, M. S., 645, 649, 662, 664, 672, 673, 697, 698, 701, 711, 715, 719, 721, 726, 737, 755, 791, 801, 864, 877 Herold, S., 516, 520 Heron, I., 554, 564 Heron, S., 305, 307 Herrada, G., xxv, xxxvii, xli, 972, 977 Herrick, C. J., 148, 157, 652, 672 Herrick, C. L., 1001, 1022 Herrmann, A. D., 147, 153, 157 Herrnstein, R. J., xxxiii, 359, 377, 873, 877 Herrup, K., 95, 113, 266, 273 Hersch, M., 665, 672, 824, 825, 842 Herschel, M., 828, 842 Herscovitch, P., 253, 272 Hersh, L. B., 54, 71, 153, 161, 190, 198 Hertling, E., 318, 321, 326 Hertz, J., 784, 801 Hertzberg, E. L., 105, 111 Hervada, A. R., 837, 842 Hervé, D., xxv, xxxvii Herz, R. S., 424, 430 Herzer, M., 515, 519 Herzog, C., 414, 425, 430, 431, 434, 619, 624 Herzog, C. D., 917, 921, 925 Herzog, S., 551, 557, 570 Hess, A., 983, 998 Hess, K., 462, 477 Hess, M. F., 390, 392, 397, 398 Hess, U. S., 412, 425, 431 Hess, W. A., 343, 485, 501 Hesse, J., 299, 307 Hesselbrock, V. M., 885, 886, 898 Hesseltine, G. R., 62, 63, 67 Hester, L. D., 76, 80–81, 90, 125, 136 Heth, G., 318, 320, 323, 326, 336, 341, 394, 395, 398, 404, 407 Hetherington, C. M., 116, 134 Hetherington, M., 696, 703, 882, 901 Hetrick, W. P., 516, 518

Author Index Hettema, J. M., 330, 341 Hettinger, T. P., 644, 649, 714, 721, 729, 740, 746, 747, 755, 756, 783–785, 787, 788, 790, 792–796, 800, 801, 803 Heurteaux, C., 721, 730 Heuser, G., 536, 544 Hevner, R., xxvii, xxxiv, 130, 131 Hewitt, J., 342 Hext, P. M., 583, 589 Heye, B., 1017, 1021 Heymann, H., 784, 794, 801 Heymans, G., 819, 821 Heyser, C. J., 361, 377 Heyser, D., 660, 673 Heywood, P. G., 215, 226, 580, 584, 590, 629, 632, 635, 637, 638 Hibert, O., 482, 499 Hichwa, R. D. 269, 491, 496 Hickey, D. C., 536, 539, 546 Hidaka, H., 26, 41, 83, 85, 88, 462, 476, 629, 633, 637 Higashi, T., 875, 877 Higgins, C. S., xxviii, xxxvii Higgins, J., 210, 225, 788, 801, 853, 854, 859 Higgins, S., 305, 306 High, W. M., 604, 611, 631, 632, 637 Highkin, M. K., 551, 561, 569 Highsmith, E., 532, 546 Hightower, S. I., 596, 609 Hiji, T., 713, 726 Hijman, R., 514, 519 Hilberg, O., 443, 444, 450, 455, 456, 467, 475 Hildebrand, J. G., 185, 186, 193, 195, 200, 351, 377 Hileman, B., 537, 542, 544 Hiley, E., 554, 563, 566 Hill, A. J., 882, 895, 897 Hill, B. J., 149, 164 Hill, C. R., 414, 436 Hill, C. S., 84, 87 Hill, D. L., 720, 722, 726, 729, 734, 751, 754, 755, 759, 764, 766, 768–771, 773–778, 779–782, 830, 842, 886, 897 Hill, M. W. 800 Hill, R. E., 116, 133 Hill, T. W., 394, 400, 418, 438 Hill, W. D., 505–507, 509, 513, 514, 519, 521, 523 Hillmann, D.E., 661, 673 Hills, K., 911, 931 Hills, T. T., 738, 755 Hillyard, S. A., 230, 233, 248 Hilsinger R.L. Jr., 942, 954 Hilsted, J., 554, 563, 568 Hinde, R. A. 844, 907, 931 Hinderer, K. H., 11, 12, 15, 439, 441, 458, 982, 996 Hinds, J. W., xxvii, xxxvii, 36, 43, 94, 95, 97, 98, 109, 119, 120, 123, 125,

128, 129, 134, 339, 341, 564, 565, 568, 617, 623 Hinds, P. L., xxvii, xxxvii, 36, 43, 94, 95, 97, 98, 109, 123, 125, 134, 341, 564, 568 Hines, E. L., 305, 307 Hines, R. N., 56, 72 Hing, S. C., 37, 44 Hingco, E. E., 145, 146, 158, 182, 197 Hino, A., 714, 727 Hinrichsen, K. V., 665, 672 Hinriksdottir, I., 467, 475 Hinton, G. E., 301, 308, 681, 701 Hinton, M. R., 423, 431, 432 Hipkin, L. J., 332, 341 Hirai, K., 551, 570 Hirai, S., 512, 522 Hirai, T., 36, 43 Hiraishi, T., 995, 997 Hirano, M., 514, 522 Hirano, T., 961, 965 Hirata, K., 82, 87, 662, 674, 789, 800, 960, 962, 965 Hirata, Y., 660, 662, 672 Hirayama, K., 952, 955 Hirning, M., xxvii, xxxvi, 103, 107, 109 Hirning, M. H., 516, 520 Hirono, J., 76, 78, 88, 182, 185, 198, 199, 287, 288, 293, 294 Hirono, S., 276, 292, 294 Hirose, M., 553, 569 Hirotsu, T., 85, 87 Hirsch, D. J., 58, 73, 75, 81–82, 87, 90 Hirsch, J. T., 422, 427 Hirsch, J. D., 124, 134 Hirschberg, A., 452, 455 Hirth, F., 116, 134 Hirzy, J., 525, 533, 544 His, W., 1014, 1023 Hitsumoto, Y., 942, 957 Hjortkjaer, R. K., 553, 565 Hladik, C. M., 698, 701, 847, 859 Hlavati, I., 153, 158 Hnik, P., 764, 781 Ho, K. Y., 451, 458 Ho, S., xxvii, xxxix, 107, 110 Ho, T. I., 600, 611 Hobbs, C. H., 62, 72, 583, 588 Hobbs, E. J., 583, 589 Hobbs, P. J., 300, 305, 307 Hobson, A., 686, 700 Hochban, W., 444, 455, 456 Hock, C., 508, 509, 521 Hodge, M. C., 583, 589 Hodgson, E., 61, 70 Hodgson, M., 525, 546, 982, 994, 996–998 Hodgson, E. S., xxiv, xxxvi Hodos, W., 167, 172, 176, 209, 225, 354, 377, 866, 877 Hoedt-Rasmussen, K. Hoek, H. W., 516, 523 Hoeppner, T. J., 552, 558, 568

Author Index Hoey, N. E., 867, 872, 876 Hof, P. R., 483, 498, 505, 520 Hofer, H., 658, 672 Hofer, M. A., 310, 323, 326 Höfer, D., 664, 672 Hoffer, B. J., 101, 111 Hoffert, M., 553, 573 Hoffman, A.A., 334, 341, 342 Hoffman, C. K., xxii, xxxvii Hoffman, E., xxviii, xli Hoffman, E. J., xxviii, xliv, 253, 273, 594, 609 Hoffman, H. J., 461, 475, 634, 637, 935, 955 Hoffman, J. M., 789, 803 Hoffman, K., 789, 801 Hoffman, M. H., 1002, 1023 Hoffman, W. H., 170, 177 Hoffmann, A., 655, 656, 673, 824, 842 Hoffmann, H., 908, 932 Hoffmann, M.-C. 111, 112 Hoffmeyer, L. B., 828, 829, 841 Hofmann, F., 80, 90, 715, 729 Hogan, B. L., 116, 133, 134 Hogan, J. A., 908, 928 Hogan, W. J., 644, 648 Hogg, I. D., 982, 998 Hohman, R. J., 54, 70, 71 Hohmann, C. F., 394, 396, 397 Hoiby, E. A., 554, 568 Hokanson, J. A., 450, 457 Hokfelt, T., 103, 109, 261, 272, 514, 520 Holbrook, E. H., 33, 43, 100–102, 106, 109, 118, 119, 121, 122, 134 Holcomb, J., 96, 98, 103, 109 Holcomb, J. D., 121, 133 Hold, B., 321, 323 Holder, M. D., 396, 398, 420, 431, 839, 844 Holder, N., 116, 135 Holekamp, T. F., 621, 625 Holinka, C. F., 359, 377 Holl, A., 551, 557, 568 Holland, A. J., 483, 500 Holland, G., 709, 728 Holland, P., 554, 563, 566 Holland, P. C., 911, 928 Holland, Y., 552, 558, 561, 573 Holland, V. F., xxv, xxxvii Hollander, F., 887, 897 Hollander, G. R., 910, 918, 932 Holldobler, B., 343 Hollemann, T., 116, 133 Holler, C., 341 Holley, A., 102, 109, 130, 131, 145, 158, 174, 179, 182, 184, 187, 191, 195, 197–200, 213, 226, 258, 264, 265, 266, 272, 388, 397, 410, 413, 421, 424, 427, 433–435, 551, 569, 982, 996 Hollister, J. M., 509, 516, 519, 521 Hollopeter, G., 76, 85 Holloway, W. R., 415, 416, 437

1055 Holm, A. K., 831, 844 Holman, B. L., 600, 608, 610 Holman, E. W., 914, 931 Holman, L., 536, 545 Holmes, G., 255, 270 Holmes, P. V., 619, 624 Holmes, W. G., 309, 323, 336, 343 Holmstrom, M., 467, 475 Holsboer, F., 416, 432, 619, 626 Holst, J., 554, 568 Holstrom, M., 585, 589 Holt, J. A., 365, 374 Holt, J. P., 441, 455 Holt, S., 923, 928 Holtgreve-Grez, H., xxiv, xlii, 644, 649, 658, 664, 676 Holtmaat, A. J., 616, 625 Holtz, J., 586, 589 Holtz, N., 553, 573 Holy, T. E., 971, 979 Holzinger, K. J., 342 Homady, M. H., 362, 373 Homma, H., 32, 36, 45, 56, 59, 65, 71 Homma S., 888, 894 Honda, N., 942, 957 Honda, Y. 48, 440, 458 Honer, W. G. 271, 342, 516, 517, 520, 521 Hong, J.-Y., 57, 72 Hong, S. C., 14, 15, 229, 230, 233, 243, 247 Honig, W. K., 869, 876 Hönigschmied, J., xxi, xliv, 655, 666, 673, 676, 773, 781 Honkanen, T., 1004, 1022 Hoogland, P. V., 514, 521 Hooks, L. D., 831, 840, 858, 858 Hoon, M. A., xxv, xxxvii, xl, 709–711, 714, 716, 717, 723, 724–726, 728, 798, 798, 848, 858 Hooper, J. E., 765, 779 Hoover, D. R., 536, 537, 545 Hope, B. T., 79, 87 Hoppe, P. C., 367, 377 Hopwood, J. H., 367, 378 Horak, F., 444, 455 Hörandner, H., 23, 43 Horiguchi, S., 585, 590 Horikawa, I., 101, 102, 111 Horikawa, Y., 791, 804 Horimoto, S., 662, 675 Horisberger, J. D., 719, 725 Horn, M., 93, 112 Horn, S., 58, 61, 70, 76, 88, 559, 569 Horn, W., xxii, xxxvii, 784, 801 Hornack, K., 794, 803 Horne, J., 788, 802 Horner, G., 299, 302, 306–307 Horner, W. G., 491, 492, 493, 498, 499 Hornigold, N., 709, 710, 717, 728 Horning, M. S., 149, 151, 163, 164 Hornstein, I., 276, 293 Hornum, I., 910, 929

Hornung, D.E., 127, 134, 185, 201, 213, 226, 266, 267, 270, 271, 273, 392, 394, 395, 400, 418, 438, 442, 445, 447, 455 Horowitz, J. M., 167, 180 Horowitz, L. F., 100, 112, 117, 120, 121, 137, 551, 560, 568 Horrall, R. M., 426, 430 Horsburgh, G., 116, 134 Horton, J., 255, 270 Horton, L. J., 829, 842 Hoseman, W., 468, 473, 475 Hoshi, H., 601, 611 Hoshiyama, M., 242, 248, 259, 266, 272 Hosley, M. A., 670, 673, 767, 768, 780 Hosoya, Y., xxiv, xxxvii, 230, 245 Hotchkiss, J. A., 54, 57, 60, 69, 70, 72 Hotelling, H., 301, 307 Hotz, P., 586, 589 Hou, V., 76 Houart, C., 116, 137 Hou-Jensen, H., 655, 656, 673, 784, 801 Houlihan, D. J., 491, 492, 498 Hounsfield, G. N., xxviii, xxxvii, 253, 270 Houpt, T. A., 918, 928 Houser, C. R., 153, 161 Houslay, M. D., 27, 43, 83, 88 Hovemann, B. T., xxv, xliv, 57, 69 Howard, M. J., 552, 569, 621, 625 Howard, R. O., xxix, xlv, 256, 258, 259, 266, 267, 270, 273 Howard, W. E., 404, 407 Howe, H. A., xxvi, xxxiv, 556, 565 Hoye, R., 963, 965 Hoyt, W. F., 255, 270 Hsia, A. Y., 149, 157, 184, 196, 514, 521 Hsia, Y., 750, 751, 755 Hsu, P., 101, 109, 481, 498 Hsu, Y.-T., 83, 87 Hu, D., 412, 430 Hu, G., 116, 138 Hu, H., 536, 545, 581, 588 Hu, H. Y., 128, 134 Hu, S., 698, 702 Hu, Z. L., 659, 673 Hua, Z., 55, 57, 61, 69, 72 Huang, A., 557, 568 Huang, C. M., 266, 270 Huang, E. J., 762, 780 Huang, J., 83, 89 Huang, L., 710, 713, 714, 717, 726, 728 Huang, Q., 509, 521 Huang, S., 103, 108 Huang, W., 184, 195, 392, 398, 484, 485, 494, 497, 515, 520 Huard, J. M., 33, 35, 43, 47, 52, 69, 76, 87, 106, 109, 617, 624 Hubbard, A. E., 359, 376 Hubbard, M. J., 510, 521 Hubener, F., 221, 226, 385, 398 Huber, G. C., 1001–1004, 1023 Huber, S. I., 484, 486, 495, 496 Hubert, H. B., 331, 342

1056 Hubert, W., 359, 377 Huby, F. J. 306, 307 Huch, V., 284, 286, 293, 294 Huck, U. W., 320, 323, 404, 407, 422, 431 Hudnell, H. K., 239, 248, 987, 992, 993, 997 Hudnell, K., 539, 543, 564, 565, 587, 588 Hudry, J., 174, 179, 264, 272 Hudson, A. L., 183, 194 Hudson, R., 218, 219, 226, 310, 315, 317, 322–324, 355, 369, 370, 373–377, 381, 410, 432, 830, 833, 834, 838, 840, 842 Huel, G., 581, 589 Huet, J.-C., xxiv, xxxiv Huether, G., 619, 627 Huettner, J. E., 621, 624 Huffmann, E., 244, 247 Huganir, R. L., 84, 88 Hugdahl, K., 254, 270 Huggins, G. R., 209, 216, 217, 224, 228, 469, 474 Hughes, J. R., 230, 245, 246 Hughes, S. E., 670, 673, 767, 773, 780, 781 Hugoson, A., 645, 649 Hui, C. C., 117, 134 Huisman, E., 514, 521 Huk, W. J., 241, 248 Hukkanen, J., 57, 70 Hulbert, J. N., 305, 307 Hulihan, T. J., 422, 437 Hulme, E. C., 154, 162 Hulse, S. H., 865, 877 Hum, D. W., 58, 70 Hummel, C., 235, 246, 259, 271, 424, 431 Hummel, T., xxxii, xxxv, 14, 15, 37, 44, 190, 197, 204, 212, 215, 226, 229–240, 243, 244, 246–248, 256, 259, 264–266, 268, 270, 273, 344, 388, 399, 444, 445, 452, 455, 456, 462–463, 472, 475–477, 485, 486, 491, 494, 495, 496–500, 538, 545, 587, 589, 593, 594, 613, 791, 797, 798, 800, 801, 987, 990, 997, 998 Humpel, C., 101, 111 Humphrey, T., 127, 128, 134, 311, 324, 983, 998, 1006, 1007, 1023 Humphreys-Beher, M. G., 643, 649 Humphries, R. E., 970, 977 Huneycutt, B. S., 557, 568 Hunink, M. G., 597, 598, 610 Hunt, J. R., 667, 673 Hunt, N. L., 392, 397, 398 Hunt, P. S., 390, 392, 398 Hunt, T., 914, 928 Hunt, W. A., 915, 918, 931 Hunter, A. J., 170, 178, 191, 196, 417, 418, 431 Hunter, D. D., 75–76, 86, 106, 108, 119, 131 Hunter, D. G., 33, 41 Hunter, G., 445, 455

Author Index Hunter, R. P., 554, 563, 571 Hunter, J., xxii, xxxvii Hunziker, W., 140, 151, 158 Huque, T., 81, 88, 709, 710, 717, 723, 725, 727, 729 Hurst, E. W., xxvi, xxxvii, 556, 566 Hurst, J. L., 358, 376, 970, 971, 977, 978 Hurt, K. J., 101–103, 105, 112, 619, 626 Hurtig, H. I., 215, 225, 484, 485, 497, 514, 520, 521 Hurtt, M. E., 38, 43, 99, 109 Hurwitz, T.A., 491, 492, 493, 496, 498, 499 Husmann, M., 105, 111, 125, 135 Hussain, A. A., 553, 562, 568 Hussain, M. A., 553, 562, 568 Hussein, M., 388, 398 Hutchings, M. R., 909, 929 Hutchings, R. T., 7, 15 Hutchins, G. D., 480, 482, 498 Hutchinson, I., 825, 842, 843, 845 Hutchinson, L. E., 549, 550, 561, 570 Hutt, A. J., 286, 293 Huttenbrink, K. B., 472, 477 Hutter, A., 257, 266, 269, 271, 424, 432 Huttner, A., 621, 623 Hutton, A., 394, 398 Hutton, H. E., 923, 928 Huttunen, M. O., 516, 522 Huws, R., 492, 498 Huxley, H. E., 643, 648 Huylebroeck, D., 101, 113 Hvidberg, A., 554, 563, 568 Hwang, P. M., xxiv, xli, 77, 82, 89, 709, 727 Hwang, T.-K., 105, 111 Hyatt, R. E., 439, 441, 458 Hyde, J. F., 119, 133 Hyde, J. M., 839, 844 Hyde, R., 549, 550, 561, 570 Hyde, S 258, 269 Hyde, T. M., 510, 517, 518, 521 Hyder, F., 258, 273 Hylander, B., 893, 896 Hyman, B. T., 510, 512, 513, 516, 517, 519, 521, 522, 600, 610 Ibanez, C. F., 772, 781 Ibanez, V., 601, 608 Ichikawa, M., 27, 44, 351, 382, 972, 975, 976, 978, 979, 1003, 1005, 1024 Ichimura, K., 33, 46 Ichise, M., 602, 611 Ida, S., 359, 383 Ide, S. E., 514, 522 Ido, S., 767, 782 Ido, T., xxviii, xli Idvall, J., 553, 562, 565 Ieraci, A., 101, 111 Igarashi, S., 26, 34, 49 Igarashi, Y., 54, 70 Iida, M., 658, 673 Iida, T., 244, 249

Iino, M., xxiv, xliii, 173, 174, 180, 255, 263, 264, 273, 412, 421, 436, 437, 692, 704 Iino, S., 83 Iino, Y., 85, 87, 88 Iiri, T., 291, 294 Ikan, R., 394, 395, 398, 404, 407 Ikeda, J., 83, 89 Ikeda, K., 462, 471, 476, 629, 633, 637, 689, 701 Ikeda, M., 785, 789–791, 804, 938, 948, 949, 956 Ikeda, S., 806, 822 Ikehara, A., 644, 649 Ikeoka, H., 585, 590 Ikuta, F., 48 Ilicali, O. C., 443, 456 Iliffe, C. P., 358, 376 Illig, K. R., 187, 188, 190, 192, 196, 197 Illum, P., 443, 456 Ilmoniemi, R. J. 245 Imai, Y., 410, 437 Imaizumi, K., 102, 105, 110 Imaizumi, T., 29, 43, 105, 109 Imamura, K., 121, 135, 184, 186, 192, 196, 198, 221, 226, 560, 570 Imfeld, T. N., 784, 801 Imler, M., 886, 902 Imoto, T., 713, 723, 730 Impagnatiello, F., 621, 625 Impey, S., 27, 49, 80, 90 Imran, M. B., 266, 267, 272 Inaki, K., xxv, xliv, 287, 294 Inamitsu, M., 37, 38, 46, 631, 637 Inamura, K., 972, 978 Incerti, B., 127, 133, 1017, 1021, 1022 Ingber, D., 103, 108 Ingelman-Sundberg, M., 64, 65, 70 Ingi, T., 76, 82, 88 Ingram, D. K., 919, 928 Ingram, R. H., 441, 458 Ingrand, P., 482, 496 Ingvar, D. H., xxviii, xxxviii, 253, 270, 271 Ingvar, G. H., xxviii, xxxvii Ingvar, M., 413, 436 Inoh, N., 59, 72 Inokuchi, A., 172, 178, 982, 998 Inoue, K., 266, 267, 272 Inoue, M., 717 Insausti, R., 680, 701 Insel, T. R., 422, 430, 432, 437, 438 Institute of Medicine (IOM) 534, 545 Ionescu, E., 887, 897 Iougi, Y., 717, 727 Ip, N. Y., 762, 779 Iqbal, K., 553, 563, 566 Iravani, J., 55, 69 Iregren, A., 581, 589 Irvine, R. F., 81, 85 Irwin, C. L., 642, 649 Irwin, I., 487, 499 Irwin, M., 886, 896 Irwin, R. D., 583, 590

Author Index Isaac, W. L., 421, 433 Isaacks, N., 346, 382 Isaacs, A. J., 923, 928 Isaacson, J. S., 151, 157, 185, 196 Isaacson, R. L., 411, 435 Isackson, P., 102, 105, 109 Isacoff, E. Y., 969, 979 Isaka, H., 553, 568 Iseki, K., 553, 562, 568 Iseki, M., 507, 512, 521 Ishii, E. K., 461, 475, 634, 637, 935, 955 Ishii, R., 884, 897, 901 Ishii, T., 182, 200, 972, 978 Ishikawa, K., 444, 458 Ishiko, N., 740, 755 Ishimaru, T., 101, 102, 111, 232, 246 Ishizuka, Y., 26, 34, 49, 507, 509, 523, 715, 728 Islam, A. T., 82, 87 Ismail, H., 634, 637 Isolauri, E., 942, 955 Isolauri, J., 942, 955 Itakura, C., 36, 43 Itani, J., 908, 928 Itano, A., 320, 326 Itaya, S. K., 551, 556, 568 Itil, T. M., 367, 381 Ito, M., 910, 932 Ito, S., 736, 755, 916, 930 Ito, Y., 242, 248, 259, 266, 272 Itoh, H., 266, 271 Itoh, M., 266, 267, 271–273, 716, 727, 789, 800, 960, 962, 965 Iuga, L., xxxi, xlii, 788, 803 Ivankovic, S., 552, 568 Ivanova, S. F., 916, 928 Iverius, P. H., 892, 896 Ivic, L., xxv, xlv, 78, 91, 145, 164, 287, 294, 396, 400 Ivry, R., 266, 270 Ivy, G., 411, 436 Iwabuchi, K., 709, 711, 715, 716, 727, 728 Iwai, A., 509, 512, 523 Iwamoto, E. T., 915, 928 Iwanaga, I., 124, 137 Iwanega, T., 28, 48, 124, 138, 662, 663, 677 Iwasaki, K., 713, 727, 894, 897, 898 Iwasaki, N., 27, 44 Iwasaki, S., 661, 673 Iwashita, K., 23, 24, 30, 36, 46 Iwata, H., 102, 105, 110 Iwata, S., 451, 452, 456 Iwatsubo, T., 509, 522 Iwema, C. L., 616, 626 Iyanagi, T., 58, 70 Iyengar, B., 713, 727 Izard, M. K., 348, 377 Izrael, Z., 413, 436 Jabbour, W., 38, 39, 41, 505, 519 Jaber, M., xxv, xxxvii

1057 Jaberi, P., 76, 81–84, 89 Jackman, A., 764, 781 Jackowski, A., 144, 157 Jacks, S. C., 789, 801 Jackson, C., 602, 611 Jackson, C. G., 946, 955 Jackson, G. P., 890, 903 Jackson, J. A., 488, 498 Jackson, J. C., 263, 269 Jackson, J. E., 21, 22, 33, 34, 36, 45 Jackson, J. H., 629, 637 Jackson, R. T., xxii, xxxvii, 256, 270, 550, 554, 569 Jackson, T., 1001, 1006, 1021 Jackson, W., 647, 648 Jacobberger, J., 105, 109 Jacobowitz, D. M., 140, 151, 158 Jacobs, G., 442, 457 Jacobs, H. L., 882, 900 Jacobs, J., 908, 914, 931 Jacobs, J. B., 466, 468, 475 Jacobs, K. M., 684, 701, 736, 747, 755 Jacobs, R. D., 472, 474, 889, 896 Jacobsen, G., 557, 570 Jacobsen, P. B., 838, 845 Jacobson, A.G., 116, 134 Jacobson, I., 185, 197 Jacobson, J., 554, 563, 569 Jacobson, L., 349, 377 Jacobson, M., 125, 133 Jacobson, R. R., 602, 610 Jacques, P. F., 893, 897 Jacquin, M., 764, 782 Jacquin, M. F., 983, 998 Jaeger, C. B., 661, 673 Jaenisch, R., 761, 762, 767, 779, 780 Jafek, B.W., xxxi, xxxvii, xl, 3, 5, 15, 16, 21–23, 27, 30, 33, 34, 37, 38, 43–47, 121–123, 134, 135, 140, 160, 463, 466, 472, 476, 600, 610, 631, 638, 662, 673, 825, 843 Jafek, T. B., 466, 476 Jägel, B., 667, 673 Jager, S., 444, 455 Jagnow, C. J., 834, 838, 844 Jagust, W. J., 593, 594, 601, 610 Jahan-Pawar, B., 555, 567 Jahr, C. E., 153, 157 Jaillardon, E., 55, 69 Jajek, V. W., 600, 610 Jakes, R., 510, 513, 514, 523 Jakinovich, W. Jr., 714–716, 727, 730, 875, 877 Jaklevic, R. C., 281, 293 Jakob, H., 517, 521 Jakoby, W. B., 57, 63, 66 Jakubowski, M., 660, 661, 673, 677 Jalowayski, A., 889, 896 Jalowayski, A. A., 463, 472–473, 474, 982, 997 James, C. E., 832, 842, 843 James, F. M., 945, 955 James, I. E. A., 982, 998

James, R. A., 392, 399, 578, 582, 583, 588, 591 James, W. P. T., 887, 901 James 276, 293 Jan, L. Y., 1005, 1023 Jan, Y. N., 1005, 1023 Jancso, N., 983, 998 Jancso-Gabor, A., 983, 998 Janday, B. S., 241, 246 Jandova, A., 855, 860 Jani, M. K., 58, 72 Jankovic, J., 488, 498 Jankovic, J. T., 542, 545 Jankovski, A., 143, 157, 166, 178, 517, 519 Jankowski, C. K., 77, 86 Janota, M., 656, 676 Janowitz, H. D., 882, 887, 897 Jansen, A. S., 557, 571 Jansen, E., 923, 926 Janson, A. M., 621, 624 Janus, C., 356, 377 Jany, G., 299, 307 Jaramillo, C. J., 483, 500 Jarolim, K. L., 552, 569 Jarrard, L. E., 174, 175, 177, 421, 429 Jarus, G. D., 605, 610 Jarvik, M. E., 554, 571 Järyholm, B., 542, 543 Jason, L. A., 537, 541, 545 Jasper, H., 948, 950, 956 Jastreboff, P. J., 145, 157 Jaumann, M. P., 538, 545, 587, 589 Jawad, M. S., 452, 454 Jaworsky, D. E., 27, 42, 79, 84, 87, 88 Jay, J. R., 825, 842 Jaye, M., 57, 71, 559, 570 Jaynes, J., 355, 372 Jayyosi, Z., 583, 590 Jean, C., 554, 569 Jeannet, P. Y., 553, 562, 569 Jeanraud, B., 887, 897 Jech, R., 484, 500 Jedlitschky, G., 58, 69 Jeffery, E. H., 60, 62, 69, 552, 559, 569 Jeffery, P. K., 33, 43 Jeffery, R. W., 886, 897 Jeffery, W., 660, 673 Jehl, C., 213, 226 Jehl, J. W., xxviii, xxxviii Jelks, G. W., 9, 16 Jellinek, J. S., 219, 220, 226 Jellison, W., 463, 473, 476 Jemiolo, B., 351, 361, 362, 377, 380, 971, 977 Jendoubi, M., 621, 624 Jenike, M. A., 686, 702 Jenkins, G. N., 887, 897 Jenkins, J. J., 348, 375 Jenkins, N. A., 116, 133, 136 Jenks, B. G., 76, 86 Jennes, L., 152, 164, 1003, 1007, 1023 Jennings, H. S., xv, xxxvii, 403, 408

1058 Jennings, J. W., 354, 377, 410, 431, 434 Jennings, R. A., 389, 391, 400, 987, 999 Jennings-White, C., 969, 978 Jensen, E. W., 888, 894 Jensen, K., 911, 931 Jensen, K. F., 392, 399, 583, 590 Jensen, R. K., 553, 569 Jernigan, P., 417, 431 Jernigan, T. L., 495, 497, 602, 610, 886, 896 Jerome, N., 883, 897 Jersey, G. C., 582, 590 Jesmanowicz, A., 258, 269 Jessell, M., 123, 136 Jessell, T. M. 998 Jetton, M. M., 893, 895 Jewett, D. C., 826, 829, 844 Jezzard, P., 258, 270 Jia, C. P., 972, 977 Jiang, D., 230, 248 Jiang, M., 153, 157, 187, 197 Jiang, M. R., 413, 424, 431 Jiang, T., 314, 326 Jiang, X. Z., 582, 583, 588 Jiang, Z., 510, 513, 522 Jiang, T., 834, 845 Jimenez, J., 238, 247 Jimma, L. A., 472, 477, 495, 500, 619, 626 Jin, J., 149, 164 Jingami, S., 717, 727 Jinich, S., 483, 500 Jinks, A., 220, 221, 222, 226, 825, 842, 845 Jino, M., 604, 612 Jin-Xiang, H., 580, 591 Joachim, C., 482, 499, 513, 522 Joerges, J., 182, 183, 193, 197, 220, 226 Joh, T. H., 103, 113, 131, 564, 565, 572, 617, 618, 623, 626, 918, 928 Johanson, C. E., 886, 896 Johanson, I. B., 837, 845 Johansson, B., 209, 225 Johansson, C., 887, 903 Johansson, C. B., 620, 621, 623, 624 Johansson, I., 204, 223 Johansson, O., 103, 109, 514, 520, 663, 670 John, D. T., 552, 569 Johns, M. E., 596, 611 Johns, T., 698, 701 Johnson, A., 311, 317, 324, 355, 356, 379, 479, 485, 496, 833, 843, 844 Johnson, A. R., 529, 536, 537, 539, 541, 542, 545, 546 Johnson, B. A., 145, 146, 157, 158, 182, 187, 197 Johnson, C. C., 556, 564, 568 Johnson, D. M. G., 187, 188, 192, 197 Johnson, D. R., 117, 127, 134 Johnson, E. W., xxxi, xxxvii, 27, 33, 38, 43, 123, 134, 463, 476, 600, 610, 662, 673, 825, 843, 969, 977 Johnson, F. Jr., 217, 224

Author Index Johnson, G. R., 392, 397 Johnson, J. E., 121, 133, 773, 780 Johnson, K., 536, 545 Johnson, K. A., 582, 589, 602, 611 Johnson, R. C., 331, 343 Johnson, R. S., 83, 89 Johnson, S., 299, 308 Johnson, S. L., 886, 897 Johnson, T. M., 961, 965 Johnson, T. N., 680, 701 Johnson, W. G., 514, 522, 888, 897 Johnston, C. C., 829, 842 Johnston, E., 660, 675 Johnston, J. B., 1002, 1003, 1005, 1006, 1023 Johnston, J. W. Jr., xvi, xxix, xxxvii, xliii, 204, 206, 213, 226, 228, 230, 245, 801, 841 Johnston, R. A., 969, 970, 977 Johnston, R. E., xxix, xxxvii, 318, 320, 323, 326, 336, 341, 342, 350, 357, 364, 377, 379, 380, 388, 398, 417, 422, 425, 431, 434, 550, 551, 557, 566 Jokinen, M. P., 583, 588 Jolayemi, E. T., 832, 844 Jolles, P. R., 593, 594, 610 Jones, A. M., 57, 58, 66 Jones, A. S., 12, 15, 441, 443, 450, 452, 453, 455, 456, 458, 982, 996 Jones, B., 492, 493, 496, 498 Jones, B. E., 170, 178, 179 Jones, B. P., 214, 226, 424, 431, 602, 610, 886, 897, 898 Jones, D. J., 923, 928 Jones, D. T., xxv, xxxvii, 24, 43, 79, 88, 333, 342, 559, 569, 716, 727 Jones, E. G., 151, 152, 156, 183, 196, 516, 518 Jones, F., 887, 900, 901 Jones, F. N., xvi, xxxvii, 215, 218, 226 Jones, J. G., 440, 455 Jones, K., 536, 545 Jones, K. R., 761, 762, 767, 780 Jones, N. S., 563, 569 Jones, R. B., 361, 377 Jones, R. L., 554, 563, 569 Jones, S. W., 1005, 1023 Jones, T., 253, 270 Jones, M. H., xvi, xxxvii Jones-Gotman, M., xxix, xliii, xlv, 190, 192, 201, 214, 226, 258, 259, 262–268, 270–273, 424, 431, 698, 699, 704, 705, 797, 803, 948, 957 Jonson, B., 451, 453 Jonsson, A., 305, 306 Jonsson, L., 392, 399 Joo, F., 153, 158 Joppa, M., 359, 379 Jordan, H. A., 886, 898 Jordan, T., 116, 133 Jorgensen, F. N., 370, 376 Jorgensen, K. L., 1013, 1025

Jorissen, M., 55, 69, 562, 572 Josefsen, D., 62, 69 Joseph, S. K., 81, 88 Josephs, O., 258, 270 Jourdan, F., 33, 43, 96, 98, 101, 103, 108, 109, 111, 145, 151, 153, 154, 156, 158, 159, 162, 164, 172, 179, 182, 184, 186, 188, 194, 195, 197, 199, 622, 626, 1003, 1025 Jousmaki, V., 241, 246, 259, 266, 271 Joutsiniemi, S. L., 241, 245, 259, 266, 271 Jowett, A., 656, 673 Joyce, E. M., 602, 610 Joyner, A. L., 117, 121, 133, 134, 621, 624 Joyner, D. R., 55, 70, 578, 589 Juangadhwalla, M., 394, 396, 397 Juel-Nielsen, N., 332, 342 Jugo, S. B., 3, 15 Juhling, M. H., 642, 649 Juilfs, D. M., 27, 41, 43, 82–83, 86, 88 Julien, R., 554, 563, 571 Julius, D., 983, 996 Julliard, A. K., 1017, 1022 Jung, C., 57, 67 Jung, D. H., 451, 456 Jung, G., 304, 308 Jung, H-S., 765, 780 Jung, I. H., 55, 71 Jung, M. W., 170, 178, 190, 197 Jung, T. M., 469, 476 Juni, K., 553, 562, 571, 573 Jurisch, A., xxxiii, 652, 655, 670, 673 Jurs, P., 299, 308 Jurs, P. C., 234, 245, 984–987, 991, 996, 997 Jutzi, A., 553, 562, 569 Kaada, B. R., xxiv, xxxvii, Kaakkola, S., 505, 506, 521 Kaasalainen, V., 553, 562, 572 Kaba, H., 320, 322, 365, 368, 373, 377, 970, 973, 975, 976, 977, 978 Kaban, L. B., 946, 957 Kachele, D. L., 773, 774, 775, 780 Kadmon, G., 105, 111, 123, 125, 135 Kadoya, M., 594, 612 Kaegler, M., 983, 999 Kaetsu, I., 242, 249 Kafitz, K., 104, 105, 110 Kafitz, K. W., 141, 158 Kagamiyama, H., 121, 123, 125, 138 Kagawa, H., 662, 664, 672 Kageyama, R., 119, 121, 132 Kagimoto, K., 57, 71 Kagoshima, M., 553, 562, 568 Kahn, A., 551, 567 Kaie, H., 314, 324 Kaigawa, Y., 767, 782 Kailscher, O., 386, 398 Kainz, J., 6, 15 Kaiser, F., 554, 563, 571 Kaissling, K.-E., xxiv, xxix, xxxviii

Author Index Kaitz, M., 321, 324 Kajiura, H., 720, 728, 828–830, 843 Kakigi, R., 242, 248, 259, 266, 272 Kakihana, R., 365, 377 Kalaria, R. N., 513, 521 Kalat, J. W., 905, 908, 910, 928, 931 Kale, S., 468, 475 Kalenian, M., 443, 451, 459 Kalil, R., 618, 624 Kaliner, M. A., 54, 70, 71 Kalinoski, D. L., 26, 33, 42, 43, 81, 88, 660, 672, 723, 725 Kalinowski, A. G., 492, 500 Kalkstein, D., 600, 612 Kalkstein, D. S., 481, 491, 492, 495, 496, 501 Kalkstein, S., 891, 895 Kallas, H. E., 554, 563, 569 Kallert, S., 791, 801 Kallmann, F., 1016, 1023 Kallmann, F. J., 127, 134, 603, 610 Kalmus, H., 318, 324, 784, 791, 798, 801, 826, 843, 848, 849, 852, 853, 856, 859, 891, 898 Kalra, P. S., 922, 926 Kalra, S. P., 922, 926 Kalso, E., 553, 562, 572 Kaluza, J. F., 182, 197, 287, 293 Kamami, Y. V., 443, 455 Kamataki, T., 57, 71 Kamateh, D. K., 832, 845 Kamath, S. K., 893, 902 Kamen, J. M., 807, 821 Kameyama, K., 709, 711, 727 Kamide, M., 215, 225 Kaminski, L. C., 849, 859 Kaminsky, L. S., 57, 61–64, 69, 73 Kaminsky, Z., 514, 520 Kamzalov, S. S., 54, 71 Kanabayashi, T., 553, 568 Kanaki, K., 150, 158 Kanamura, S., 244, 249 Kanazawa, H., 661, 673 Kanazawa, K. K., 300, 306 Kandel, E. R. 998 Kane, W. J., 942, 955 Kaneda, H., 783, 787, 793, 797, 801 Kaneko, A., 712, 730 Kaneko, N., 394, 400, 411, 436 Kaneko, S., 62, 68 Kanemaru, N., 764, 767, 768, 779 Kanemoto, H., 233, 249 Kaneoke, Y., 242, 248, 259, 266, 272 Kaner, H. C., 356, 357, 376 Kanmori, K., 359, 383 Kanno, Y., 661, 674 Kanter, E. D., 190, 197, 425, 431 Kantola, A., 291, 293 Kaplan, A. R., 848, 855, 857, 858, 859 Kaplan, C., 289, 294 Kaplan, J., 894, 901 Kaplan, J. M., xxv, xxxiii, 862, 864–866, 876, 877, 879

1059 Kaplan, M., 130, 133 Kaplan, M. R., 620, 625 Kapur, A., 190, 197 Karachunski, P. I., 553, 569 Karadi, Z., 174, 178, 182, 197, 680, 688, 689, 701, 702, 704 Karcsu, S., 154, 158 Kare, M. R., xvi, xli, 24, 47, 341, 343, 344, 682, 689, 690, 701, 702, 705, 845, 848, 855, 859, 936, 953, 955, 956, 982, 997, 999 Kareken, D. A., 480, 482, 498 Karl, K. J., 550, 453 Karlan, M. S., 36, 45, 93, 109, 616, 625 Karlson, P., xxx, xxxviii, 345, 346, 348, 350, 351, 361, 377 Karlstrom, H., 621, 623 Karniol, I. G., 910, 914, 929 Karpen, J. W., 80, 86 Karram, K., 621, 623 Karrer, T., 854, 856, 860 Karrer, T. A., xxxi, xlv, 938, 957 Kasa, P., 153, 154, 158, 512, 521 Kasahara, T., 894, 898 Kasahara, Y., 713, 725, 746, 755, 764, 767, 768, 779 Kasai, H., 182, 200 Kasambir, T., 916, 930 Kasbekar N., 951, 954 Kase, N., 472, 476 Kashiwadani, H., 145, 158, 185, 186, 197 Kashiwayanagi, M., 61, 70, 150, 158, 936, 955 Kasick, J., 442, 457 Kasoff, S. S., 469, 474 Kasowski, H. J., 130, 134, 140, 158 Kasper, M., 663–666, 677 Kasperbauer, J. L., 441, 455 Kasprzak, M., 339, 342 Kasschau, R. A., 785, 802 Kassel, E. E., 600, 610 Kassel, R. R., 452, 455 Kastl, P. E., 583, 590 Kaszniak, A. W., 486, 500 Kata, A. C., 762, 779 Katagiri, Y., 620, 624 Katahira, K., 835, 840 Kato, H., 884, 898 Kato, R., 452, 456 Kato, S., 62, 68 Kato, T., 101, 102, 111, 232, 245, 746, 758 Katoh, K., 78, 83, 88, 89, 186, 197 Katon, W. J., 532, 535, 546 Katsarou, Z., 485, 496 Katschinski, M., 887, 898 Katsui, Y., 690, 691, 696, 703 Katsukawa, H., 720, 728, 875, 877 Katsuki, T., 659, 673 Katz, A., 710, 727 Katz, D. B., 670, 673 Katz, D. M., 762, 779 Katz, H., xxx, xliv Katz, H. M., 394, 400, 409, 410, 436

Katz, L., 512, 513, 522, 893, 895 Katz, L. C., 145, 146, 155, 162, 182, 199, 259, 272 Katz, R. J., 914, 929 Katz, S. H., 994, 998 Katz, Y., xxx, xliv Katzman, R., 479, 499 Kauer, J., 299, 306–308 Kauer, J. S., xxiv, xxix, xxviii, xxxvii, xlii, xliii, 22, 33, 41, 42, 75, 76, 86, 106, 108, 118, 119, 124, 131, 137, 141, 145, 155, 157–159, 163, 182, 184–186, 190, 194–197, 200, 220, 226, 228, 299, 306–308, 505, 506, 509, 523, 560, 563, 572, 573, 616, 626 Kaufman, L., 241, 244 Kaufman, M. H., 621, 623, 624, 761, 765, 780 Kaufman, M. R., 887, 897 Kaufman, R. H., 923, 928 Kaulbach, H., 54, 70 Kaupp, U. B., 27, 39, 45, 46, 76, 80, 83, 86–89, 722, 729 Kavaliers, M., 921, 926 Kawabata, T. T., 57, 66 Kawabuchi, M., 82, 87 Kawad, T., 884, 903 Kawaguchi, T., 553, 562, 571, 573, 968, 978 Kawamura, S., 83, 89 Kawamura, Y., xxiv, xxxvi, 689, 690, 701, 745, 746, 758, 845, 918, 933 Kawana, E., 561, 573 Kawanabe, M., 551, 570 Kawano, T., 103, 107, 123, 131, 134, 135, 150, 160, 564, 565, 569, 618, 623, 624 Kawasaki, M., 121, 123, 125, 138, 233, 249 Kawashima, R., 266, 267, 271, 272 Kawashima, S., 150, 158 Kay, A. R., xxviii, xl Kay, L. M., 174, 175, 178, 187, 191, 197, 412, 421, 426, 431, 538, 545 Kaya, N., 664, 673 Kaye, W. H., 922, 929 Kayser, R., 439, 455 Kazanskaya, O. V., 116, 138 Kaziro, Y., 716, 727 Kazprzak, M., 321, 324 Keck, T., 439, 456 Keding, H. J. 306, 307 Keefer, L. H., 410, 431 Keele, C. A. 998 Keenan, C. M., xxvii, xxxviii, 582, 589 Keesey, J. C., xxx, xxxiv, Keeton, D. A., 26, 29, 30, 31, 33, 45 Keffer, L. H., 354, 377 Kehlet, H., 888, 899 Kehoe, P., 906–907, 929 Kehrl, J. H., 24, 26, 27, 43, 48 Keifer, M. C., 564, 565

1060 Keiger, C. J., 983, 999 Keil, W., 310, 315, 324 Keinanen, R., 82, 88 Keino, H., 517, 522 Keith, D. A., 946, 947, 956 Keith, D. M., 340, 341 Keith, L. G., 340, 341 Keles, N., 443, 456 Keller, A., 149, 158 Kelley, K. W., 909, 915, 921, 929, 931 Kelling, S. T., 789, 801 Kellogg, M. S., 714, 726 Kelly, A. B., 602, 604, 610 Kelly, D. P., xxvii, xxxviii, 582, 589, 591 Kelly, J. P., 149, 158 Kelly, K. M., 424, 433 Kelly, K. S., 420, 421, 433 Kelly-McNeil, K., 529, 538, 544 Kemali, M., 165, 171, 178 Kemink, J. L., 962, 965 Kemm, R. E., 153, 159 Kemmer, T., 887, 898 Kemnitz, J. W., 919, 929 Kemp, J. M., 680, 701 Kemp, S. E., 884, 898 Kempermann, G., 619, 620, 624, 625 Kempler, D., 602, 611 Kemppainen, R. J., 471, 475 Kendal-Reed, M., 982, 983, 990, 997–999 Kendler, K. S., 330, 341 Kendler, K., 918, 929 Kendrick, K., 360, 377 Kendrick, K. M., 320, 324, 413, 419, 423, 424, 427, 428, 431, 432, 975, 976, 977 Kenigfest, N. B., 149, 153, 159 Kennedy, B., 450, 456 Kennedy, C., xxviii, xxxviii, xliii Kennedy, D. A., 392, 397, 400 Kennedy, D. W., 2, 5, 6, 9, 15, 37, 38, 39, 44, 45, 451, 456, 593, 594, 596, 609, 611 Kenneth, J. H., xx, xxviii Kennedy, L. M., 713, 729, 795, 800 Kennedy, R. C., xxxiii, 142, 155 Kennett, D. J., 419, 430 Kent, P. F., 98, 110, 119, 134, 355, 383, 394, 398, 400, 463, 476 Kent, S., 909, 929 Kent, W. D., 921, 926 Kerchner, M., 423, 431 Kerjaschki, D., 23, 43 Kern, E. B., 4, 15, 441, 451, 454, 455 Kern, R. C., 26, 31, 33, 38, 43, 54, 62, 68, 72, 445, 456, 466, 476, 595, 610 Kerr, T. P., 26, 33, 43 Kerzner, B., 642, 649 Kesavanathan, J., 442, 456 Keshavan, M., 491, 492, 498 Kesler, M., 420, 436 Kessen, W., 828, 844 Kesslak, J. P., 480, 481, 485, 489, 491–493, 496, 498, 501, 601, 610

Author Index Kessler, J A., 101, 103, 113 Kessler, J. B., 555, 569 Kessler, M., 411, 436 Kessler, S., 361, 365, 377 Kettenmann, B., 235, 236, 238, 241, 246, 248, 259, 266, 271, 424, 431, 494, 498, 990, 998 Kety, S. S., xxviii, xxxviii, xliii, 253, 271 Keverne, E. B., 26, 44, 120, 123, 136, 187, 194, 320, 322, 324, 346, 350, 351, 363, 365, 367, 368, 373, 375, 377–381, 413, 421, 423, 427, 431, 432, 434, 969–972, 975, 976, 976–978, 982, 998, 1003, 1023 Kevetter, G. A., 173, 178 Key, B., 105, 112, 120, 125, 126, 136, 137, 560, 569, 620, 624 Key, S., 1013, 1022 Keyhani, K., 448, 449, 453, 456 Keyl, P. M., 527, 544 Khaffaf, S. M., 300, 305, 307 Khan, R. M., 190, 200, 256, 267, 272, 273, 452, 458 Khen, M., 58, 61, 66, 70, 76, 88, 559, 569 Khetarpal, K., 472, 474 Khew-Goodall, Y.-S., 125, 138 Khopkar, S. M. 306–307 Khoshnood, B., 828, 842 Kiang, N. Y.-S., 740, 755 Kida, A., 949, 955 Kida, H., 242, 249 Kida, S., 417, 420, 436 Kido, D. K., 601, 610 Kiefer, H., 287, 293 Kiefer, S. W., 420, 427, 838, 843, 885, 886, 898, 917–918, 925, 929 Kier, E. L., 167, 178 Kier, L. B., 714, 727 Kiernan, J. A., 38, 42 Kiesow, F., xxi, xxii, xxxviii, 805, 816, 821 Kikuyama, S., 1003, 1008, 1018, 1023 Kilburn, K. H., 581, 585, 587, 588, 589 Killburn, K., 539, 543 Killenberg, P. G., 893, 895, 950, 954 Kilpatrick, T. J., 619, 626 Kim, C. S., 451, 456 Kim, D. J., 664, 673 Kim, H., 130, 134, 140, 158 Kim, I., 698, 702 Kim, I. J., 450, 458 Kim, J. J., 516, 518, 916, 929 Kim, K. H., 1003, 1006, 1023, 1025 Kim, K. N., 664, 671 Kim, M. M., 971, 977 Kim, N., 170, 177, 389, 392, 398, 413, 428 Kim, P. J., 210, 225 Kim, S. G., 64, 71, 254, 269, 271 Kim, S. U., 621, 624 Kim, S. W., 263, 273 Kim, Y. H., 768, 778

Kimbell, J. S., 55, 56, 67, 70, 447, 456, 578, 589 Kimble, D. P., 123, 137, 315, 326, 355, 382, 410, 431, 838, 845, 907, 932 Kimeldorf, D. J., xxix, xxxi, xxxvi, 873, 877, 906, 927 Kimelman, B. R., 361, 377, 422, 431 Kimizuka, A., 689, 705 Kimmel, S. A., 832, 843 Kimmelman, C. P., xxxi, xxxv, 2, 16, 98, 111, 172, 178, 216, 224, 311, 325, 439, 440, 454, 456, 468, 476, 593, 602, 609, 610, 622, 625, 629, 635, 637, 787, 788, 790, 791, 795, 799, 890, 894, 896, 935, 938, 945, 950, 951, 954, 959, 961, 963, 965, 982, 998 Kimmerle, G., 63, 72 Kimura, A., 439, 457 Kimura, H., 153, 161 Kimura, K., xxiv, xxxviii Kimura, Y., 63, 70, 232, 246, 392, 397, 399, 551, 557, 570 Kindermann, U., 370, 377, 410, 423, 426, 432, 433 King, B. S., 1003, 1006, 1023–1025 King, E., 482, 499, 512, 513, 522 King, J. A., 362, 367, 378 King, K. R., 882, 901 King, M. S., 736, 754, 755 King, P., 709, 710, 717, 728 King, S. R., 152, 160 Kinget, R., 562, 572 Kini, A. D., 221, 223 Kinnamon, I. C., 983, 997 Kinnamon, J. C., 661–664, 672, 673, 675, 769, 779 Kinnamon, S. C., xxiv, xxv, xxxvii, 661, 664, 671, 673, 709, 712, 714, 715, 717–719, 721–723, 725–728, 730, 737, 738, 754, 755, 776, 780 Kinomura, S., 266, 271 Kinsella, B. A., 854, 860 Kinzie, J. M., 149, 151, 158, 162, 185, 199 Kipen, H. M., 526, 527, 529, 530, 533, 535, 538, 539, 541, 544, 546 Kippin, T. E., 357, 378, 423, 431 Kirk, J. M., 472, 477 Kirk, Smith, M., x, 249 Kirkby, H. M., 204, 224 Kirkes. W. S., xxii, xxxviii Kirkpatrick, B. W., 422, 432, 437 Kirk-Smith, M. D., 321, 324, 350, 378 Kisaki, H., 942, 957 Kishi, K., 142, 160, 168, 179, 188, 198 Kishi, M., 663, 673 Kishikawa, H., 554, 570 Kishikawa, M., 507, 512, 521 Kishimoto, J., 26, 44, 120, 123, 136 Kita, K., 952, 955 Kitagawa, M., 714, 727 Kitagoh, H., 946, 956

Author Index Kitamura, M., 554, 570 Kitamura, R., 745, 758 Kitamura, T., 204, 228 Kittel, P., 94, 95, 98, 100, 106, 110, 119, 125, 135 Kittel, P. W., xxvii, xxxix, 36, 44 Kitterle, F. L., 254, 270 Kittle, G., 445, 456 Kittok, R., 359, 373 Kiyomitsu, Y., 745, 758 Kjærgaard, S. K., 987, 998 Kjeilen, J., 647, 649 Klajner, F., 887, 898 Klauke, N., 24, 26, 47 Kleene, S. J., 24, 43, 84, 88, 511, 521 Kleiderlein, J. J., 514, 520 Kleifield, E. I., 883, 898 Klein, D., 406, 408 Klein, S. B., 920, 930 Klein, V. R., 603, 608, 610 Kleitman, N., 450, 456 Klemm, T., 76, 81, 86 Klemm, W. R., 244, 246 Klenoff, J. R., 130, 134, 140, 158 Klevitsky, R., 664, 676 Kleykers, R. W. G., 815, 816, 820, 822 Kleyman, T. R., 718, 727 Kligman, A., 216, 224 Klimas, N. G., 538, 543 Klimek, L., 190, 197, 444, 458, 467, 468, 476 Kline, J. P., 329, 342, 526, 538, 543 Kling, A., 410, 411, 435 Kling, J. W., 747, 750, 751, 753, 754 Klinger, K. W., 343 Klingmuller, D., 472, 476, 593, 594, 603, 609, 610 Klioze, S., 554, 572 Klitzner, M. D., 807, 821 Klopping, H. L., 282, 293 Klose, U., 261, 269 Klucy, R. S., 921, 926 Kluger, M. J., 909, 929 Klumpp, P. A., 864, 879 Knapp, S. A., 57, 66 Knecht, M., 14, 15, 37, 44, 229, 230, 233, 243, 246, 247 Knight, J., xxvi, xl, 551, 555, 566 Knobil, E. 976 Knöpfli, R. U., 788, 793, 802 Knöppel, H. 999 Knoth, R. L., 602, 611 Knowles, J. A., 909, 929 Knudsen, A., 829, 844 Knupfer, L., 480, 481, 495, 498 Knutila, J. 245 Ko, A. I., 513, 522 Ko, K. N., 581, 591 Kobal, G., xxiv, xxxvii, 37, 44, 190, 193, 197, 198, 204, 212, 215, 226, 229–244, 244–249, 259, 264, 266, 271, 388, 399, 424, 431, 444, 445, 452, 455, 456, 462–463, 466,

1061 475–477 485, 491, 494, 496, 498, 538, 539, 543, 545, 587, 588, 589, 633, 637, 699, 702, 791, 797, 798, 801, 983, 987, 990, 998, 999 Kobashi, M., 915, 924 Kobayakawa, T., 235, 244, 783, 787, 793, 797, 801 Kobayashi, H., 553, 569 Kobayashi, M., 83, 89 Kobayashi, S., 83, 88, 659, 675 Koch, A., 618, 625, 645, 649 Koch, C., 139, 163 Kock, K., 644, 645, 648, 649, 658, 676, 698, 701 Kocsis, J., 105, 109 Kocsis, J. D., 28, 29, 42, 43, 140, 156 Koczapski, A. B., 492, 493, 499 Koda, L. Y., 916, 933 Kodama, H., 887, 889, 900 Kodama, J., 766, 781 Kodama, N., 232, 248 Kodis, M., 329, 342 Koefoed-Johnsen, V., 718, 727 Koehl, M. A. R., 185, 198 Koelega, H. S., 215, 216, 226 Koelliker, R. A., 652, 654, 673 Koelling, R. A., xxix, xxxi, xxxvi, 396, 398, 873, 877, 906, 910–911, 917, 927 Koenig, O., 193, 199, 258, 261–266, 272, 424, 435 Koentges, G., 350, 351, 373, 971, 972, 973, 976, 979 Kogan, J. H., 417, 420, 432, 436 Koger, S. M., 414, 415, 432 Kogure, S., 182, 197, 263, 264, 273 Koh, S. D., 867, 869, 870, 871, 877 Kohbara, J., 723, 725 Kohl, D., 298, 300, 307 Kohl, J. V., 329, 342 Kohler, C. G., 484, 493, 494, 498 Kohno, Y., 359, 380 Kohonen, T., 302, 307 Koike, M., 553, 568 Koistinaho, J., 82, 88 Koivistoinen, P., 807, 820 Koizuka, I., 242, 247, 259, 269, 271 Kojima, H., 153, 161 Kojima, S., 36, 43, 952, 955 Kojima, Y., 961, 965 Kokmen, E., 469, 475 Kolb, B., 193, 197, 411, 418, 432, 437 Kolesar, G. B., 583, 589 Kolesnikov, S. S., 715, 727 Koliatsos, V. E., 618, 623 Kolkow, N. D., 600, 608, 610 Koller, W. C., 486, 495, 496, 500, 601, 611 Kollmann, P. A., 291, 293 Kolodiy, N., 471, 474, 867, 872, 876, 892, 895 Kolstad, K., 29, 48, 152, 163, 514, 523 Koltai, P. J., 2, 15

Komaroff, A. L., 536, 545 Komatsu, M., 182, 200 Komatsu, T., 551, 557, 570 Komisaruk, B. R., xxiv, xxxvii, Komitowski, D., 552, 568 Komori, M., 451, 456 Kompella, U. B., 549, 550, 561, 563, 570 Kompter, C., 551, 557, 570 Kondo, H., 467, 476, 629, 637 Kondo, Y., 364, 378, 444, 451, 452, 456 Kondoh, N., 554, 570 Kong, M., 713, 714, 717, 728 Konturek, J. W., 887, 898 Konturek, S. J., 887, 898 Konzelmann, S., 100, 110, 112 Koob, G. F., 416, 428, 432 Kooistra, A., 211, 225 Koolhaas, J. M., 416, 422, 427, 429, 432, 433 Koop, D. R., 53, 61, 64, 68, 70 Koopmans, H. S., 892, 902 Koopmans, S. J., 422, 433 Koops, H. S., 891, 900 Kopala, L. C. 271, 331, 342, 491–493, 495, 496, 498, 499, 961, 965 Kopelman, M. D., 602, 610 Kopka, S. L., 872, 877 Kopp, B., 887, 898 Koppel, J., 919, 929 Korchmar, D. L., 798, 800, 856, 859 Koren, D., 491, 492, 501 Koritnik, D. R., 359, 380 Korley, J. N., 713, 714, 717, 729 Kornack, D. R., 515, 521 Korner, A. F., 837, 843 Korol, D. L., 618, 624 Korsching, S. I., xxiv, xxxvii, 126, 133 Korsmit, M., 423, 429 Korte, G. E., 661, 673 Korten, J. J., 485, 499 Kortsha, G. X., 556, 564, 568 Korzekwa, K., 61, 73 Kosaka, K., 140, 142, 144, 151, 152, 158, 163, 184, 197, 618, 624 Kosaka, T., 140, 142, 144, 151, 152, 158, 163, 183, 184, 197, 618, 624 Kosel, S., 509, 514, 521 Koshimo, H., 585, 590 Koshimoto, H., 78, 83, 88, 89, 186, 197 Koshland, D. E., Jr., xv, xlii Kosik, K., 563, 572 Kosik, K. S., 124, 137, 505, 506, 509, 523 Koskela, S., 57, 70 Koski, M. A., 336, 342 Koss, E., 481, 499 Kossel, A. H., 776, 780 Kosten, T., 470, 476, 917, 918, 929 Koster, N., 101–103, 110 Koster, N. L., 149, 158, 184, 197, 634, 637 Köster, E. P., 204, 213, 216, 217, 219, 220, 226, 234, 247, 256, 271, 795, 801

1062 Kostiainen, R., 994, 998 Kostyu, D., 342 Kotlus, B., 717, 725 Kotrschal, K., 659, 673 Kott, J. N., 105, 113 Kotz, C. M., 826, 829, 844 Koujitani, T., 553, 569 Koura, M., 553, 568 Koutsilieris, M., 554, 572 Kovacs, I., 154, 158 Kovacs, L., 923, 930 Kovacs, T., 512, 521 Kovner, R., 732, 755 Kowall, N. W., 555, 569 Kowianski, P., 172–174, 178 Koyama, H., 31, 33, 44 Koyama, N., xxvi, xxviii, 716, 727 Koyama, S., 242, 248, 259, 266, 272 Koymans, L., 57, 71 Kozasa, T., 716, 727 Koziell, D. A., 170, 177 Krabe, T., 593, 594, 603, 610 Kracko, D. A., 64, 71, 73 Kraegen, E. W., 888, 895, 898 Kraft, P., 276, 293 Krahe, T., 472, 476 Kral, P., 913, 933 Krames, L., xxix, xxxviii, 403, 404, 407, 422, 427, 905, 930 Kramies, L., 906, 927 Kranen, B., 812, 821 Krantic, S., 103, 108 Kranzler, H. R., 885, 886, 898 Krarup, B., 784, 786, 801 Krarup, T., 554, 563, 568 Krasnegor, N. A. 845 Kratschmer, F., 982, 984, 998 Kratskin, I. L., 63, 70, 139, 146–149, 153, 158, 159, 162 Krauel, K., 233, 234, 236, 239, 240, 247, 248 Kraus, D. H., 597, 611 Kraus, W., 629, 637 Krause, C. F. Th., 18, 44 Krause C. J., 942, 955 Krause, R., 654, 673 Krause, W., xxii, xxxvi, 18, 44 Kraut, M. A., 259, 264–266, 273 Krautwurst, D., xxv, xxxviii, 78, 88, 287, 293 Kravetz, M. A., 363, 376 Kream, R., 27, 41, 123, 131, 150, 155, 396, 397 Kream, R. M. 131, 134, 564, 569 Krebs, D., 343 Kreider, M., 392, 398, 494, 495, 496, 499 Kreiss, D. S., xxvi, xxxiv Kremen, W. S., 492, 501 Kremer, M., 97, 110 Kremmer, E., 27, 45, 83, 89, 722, 729 Krengel, M., 495, 498 Kreshak, A., 790, 795, 799, 953, 954

Author Index Kreshak, A. A., 495, 497, 616, 623, 629, 630, 631, 634, 637 Krestel, D., 392, 399 Krettek, J. E., 167, 170, 172, 173, 178, 192, 197 Kretz, O., 711, 719, 727 Kreumer, W. M. I., 891, 900 Kreutzberg, G. W., 558, 568, 573 Kreutzer, R., 527, 530, 532, 538, 545, 546, 586, 590 Krieckhaus, E. E., 908, 929 Krieger, J., xxv, xli, 77, 88, 287, 293, 294, 553, 573, 972, 977 Krienke, J. D., 392, 399 Kril, J., 602, 610 Krimer, L. S., 517, 518, 521 Krimm, R. F., 741, 756, 768, 770–773, 780 Krinke, G., 894, 898 Krishna, N. S., 54, 58, 70, 507, 510, 520 Krishnan, K. R., 601, 611 Kristal, A., 856, 860 Kristal, M. B., 356, 378 Kristensen, S., 451, 456 Kristensson, K., 552, 557, 558, 560, 569 Kroeze, J. H. A., 785, 800, 807, 817–819, 821, 845 Kroger, H., 267, 273, 450, 457, 472, 477, 619, 626, 633, 638, 789, 797, 800, 801 Krohn, P. L., 919, 929 Kroll, B. J., 807, 821 Kromer-Vogt, L. J., 505, 509, 513, 521 Krondl, M., 857, 860 Kroner, C., 76, 90, 709, 710, 729, 971, 977 Kroner, T., 313, 324 Krozowski, Z. S., 54, 72 Kruckenberg, S., 21, 46 Krug, R., 240, 247 Kruger, R., 509, 514, 521 Krupinski, J., 24, 44 Kruysse, A., 582, 588 Krygier-Brevart, V., 558, 573 Kryspin-Exner, I., 485, 499, 891, 898 Kubick, S., xxv, xli, 126, 134, 287, 294 Kubie, J. L., xxiv, xxxvii Kubo, T., 259, 269, 271 Kubovy, M., 816, 817, 821 Kucera, J., 764, 781 Kuchar, S., 919, 929 Kucharski, D., 171, 178, 190, 197, 908, 932 Kuczkowiak, M., 287, 293 Kuening, J., 450, 456 Kuenzel, W. J., 1003, 1023 Kufera, A., 354, 381, 394, 400, 410, 436 Kuffler, S. W., 1005, 1023 Kuhl, D., xxviii, xli Kuhlenbeck, H., xxviii, xxxvi Kuhn, F., 7, 15 Kuhn, H. G., 619, 625 Kuhn, T. S., 530, 545

Kuhn, W., 509, 514, 521 Kühnel, W., 36, 45 Kukekov, V., 93, 112 Kukekov, V. G., 621, 625 Kulaga, H., 119, 121, 124, 137 Kulich, R., 946, 947, 956 Kulkarni-Narla, A., 507, 510, 521 Külpe, O., 805, 821 Kumanyika, S. K., 891, 899 Kumar, S., 553, 563, 566 Kumarsingh, R., 983, 992, 995, 997 Kumasaka, T., 712, 717, 723, 725, 727, 729 Kumraiah, V., 882, 900 Kunder, J., 231, 247 Kung, D., 647, 648 Kunishima, N., 717, 727 Kunko, P.M., 406, 407 Kuno, M., 81, 88 Kuntz, I. D., 291, 293 Kunz, R. D., 915, 921, 926 Kunze, W. A. A., 153, 159 Kunzle, H., 165, 169, 173, 178, 179 Kuo, C, K., 285, 293 Kuo, J. F., xxxiii Kuo-LeBlank, A. 1025 Kuperstein, M., 412, 428 Kupfer, D., 515, 521 Kupniewski, D., 320, 327, 356, 383 Kurahashi, T., 27, 44, 83–84, 88, 220, 226, 712, 730 Kurahashi, T., xxiv, xxxviii Kuraoka, A., 82, 87 Kurihara, K., xxvi, xxviii, 61, 70, 101, 102, 108, 150, 158, 661, 663, 672, 673, 713, 727, 916, 918, 933, 936, 955 Kurihara, Y., 710, 713, 716, 723, 727, 728, 730 Kuriloff, D. B., 600, 611 Kurioka, Y., 233, 249 Kurisu, K., 664, 677 Kurita, N., 450, 456 Kuriyama, K., 359, 383 Kurland, L. T., 480, 484, 487, 488, 497, 499, 501, 563, 567, 923, 930 Kuroda, M., 192, 199 Kurt, T. L., 539, 545 Kurth, C., 892, 896 Kurth, C. L., 886, 897 Kurtz, D. B., 204, 213, 226, 228 Kurz, M. E., 392, 399 Kusakabe, M., 621, 625 Kusakabe, T., 968, 978 Kusakabe, Y., 709, 711, 714–716, 723, 727, 728 Kusano, K., 1013, 1025 Kushner, M., 513, 516, 520 Kussmaul, A., 826, 843 Kutcher, S., 539, 544 Kuttner, A., 182, 183, 193, 197, 220, 226 Kutty, R. K., 82, 88 Kuwabara, Y., 366, 372

Author Index Kuwano, R., 26, 34, 49 Kuznicki, J. T., 221, 226, 788, 801, 815, 817, 821 Kveton, J. F., xxxi, xxxviii, xlv, 784, 787, 802, 938, 945, 955, 957 Kwan, A., 768, 769, 782 Kwiecien, N., 887, 898 Kwong, K. K., 260, 271 Kyin, M., 420, 435 Kyle, A. L., 389, 399, 1005, 1023 Kyriazakis, I., 909, 929 La Du, B. N., 538, 539, 544 Laasonen, E., 442, 454 Labarca, P., 75, 88, 715, 729 Labarthe, D., 923, 930 Labov, J., 858, 858 Labov, J. B., 364, 378 Labovitz, D., 516, 523 Labroue, M., 893, 898 LaBuda, M. C., 330, 342 Labyak, S., 774, 781 Lacadie, C. M., 259, 264–266, 270 Laccourreye, L., 38, 39, 41, 505, 519 Lacroix, J. S., 54, 72 Lader, M. H., 910, 914, 929 Ladowsky, R. L., 910, 915, 917, 929, 930 LaDu, T. J., 923, 928 Laeng, B., 882, 898 Laethem, C. L., 61, 64, 70 Laethem, R. M., 61, 64, 70 Lafay, F., 551, 557, 565, 569 Laffort, P., 218, 226, 227, 815, 822 LaForge, J., 440, 458 Lafreniere, D., 467, 472, 476 Lagercrantz, H., 320, 324 Lago, P., 236, 247 Lai, E., 116, 133, 137 Lai, J. S., 581, 591 Lai, M. T., 509, 521 Lai, S., 257, 270 Laing, D. G., 38, 46, 145, 163, 183, 194, 217–222, 223, 226–228, 256, 266, 267, 271, 391–394, 399, 400, 471, 476, 494, 499, 590, 807, 817, 821, 822, 825, 832, 842, 843, 845, 884, 892, 899, 901, 999 Laing, P., 516, 521 Laippala, P., 451, 458 Laitinen, J. T., 82, 88 Lakoski, J. M., 920–923, 928 Lallemand, S., 314, 323 Lallemand, Y., 116, 131 Lalonde, E., 656, 658, 673 Lalonde, E. R., 784, 802, 825, 843 LaMacchio, M., 990, 998 LaMantia, A., 105, 113 LaMantia, A.-S., 21, 44, 62, 73, 125, 138 LaMantia, T. J., 486, 499, 601, 611 Lambe, J., 281, 293 Lamberty, Y., 416, 436 Lamblin, C., 440, 456 Lambujon, J., 893, 899

1063 Lamey, P. J., 786, 791, 801 Lamielle, M., 528, 530, 536, 545 Lammertsma, A. A., 267, 269 Lamp, C., 723, 725 Lanahan, A. A., 514, 520 Lancashire, B., 450, 452, 454 Lancer, J. M., 450, 452, 455, 456 Lancet, D., xxvi, xl, xliii, 17, 31, 43, 44, 46, 57, 58, 61, 66, 70, 71, 73, 76, 79–80, 88, 89, 100, 110, 124, 136, 145, 157, 159, 280, 289, 293, 341, 445, 457, 467, 473, 475, 477, 491, 492, 498, 559, 569, 570, 573, 712, 715, 716, 730 Lancisi, J. M., xix, xxxviii Land, L. J., 552, 558, 561, 569 Land, R. B., 367, 374 Land, D. G., xv, xxxvii Landahl, H. D., 449, 456 Landau, W. M., 253, 271 Landgraf, R., xxviii, xxxviii, 367, 382, 416, 428, 429, 432, 563, 565 Landin, A., 664, 673 Landin, A. M., 690, 700, 723, 725 Landmesser, L., 124, 136 Landsberg, R., 468, 475 Lane, A., 443, 451, 456, 459 Lane, A. P., 80, 86, 467, 468, 476, 973, 977 Lane, R. J., 553, 569 Lang, A. E., 484, 499 Lang, B., 886, 895 Lang, C. J., 232, 238, 244, 485, 496 Lang, J., 3, 5, 8–12, 14, 15 Lang, M. A., 64, 67 Lang, N. P., 788, 793, 802 Lange, J., 392, 399 Langford, H. G., 891, 898 Langford, P. E., 365, 374 Langlais, P. J., 424, 433 Langlois, D., 310, 315, 323, 369, 370, 374, 379, 381 Langlois, J. C., 392, 399 Langston, J. W., 486, 487, 497, 499, 564, 569, 571 Lankford, K., 105, 109 Lankford, K. L., 29, 43 Lantos, P. L., 505, 512, 521, 605, 609 Lantz, T. D., 542, 543 Lanuza, E., 148, 159, 168, 178 Lanza, D., 443, 451, 456, 459 Lanza, D. C., 2, 3, 5, 7, 10, 15, 37, 38, 39, 44, 45, 467, 476 Lanzieri, C. F., 597, 598, 611 Lapenson, D. P., 61, 73 Lapointe, F., 99, 110, 127, 134 Larabee, T. M., 57, 71 Larsell, O., 1002–1004, 1023 Larsen, E. H., 718, 726 Larsen, K., 451, 456 Larsen, K. W., 484, 499 Larsen, M., 392, 399 Larsen, W. J., 142, 162

Larsen-Su, S., 56, 72 Larson, A. L., 646, 648 Larson, E. B., xxxii, xxxvii, 341, 483, 498 Larson, J., 170, 178, 188, 190, 191, 197, 198, 412, 413, 426, 432, 433 Larson, J. L., 582, 583, 589, 590 Larson, P., 600, 609 Larson, P. M., 481, 491, 492, 501 Larson, S. M., 594, 609 Larsson, B. S., 551, 554, 555, 556, 572 Larsson, M., 174, 179, 258, 262–264, 266, 267, 268, 272, 341, 424, 435, 482, 499 Larsson, O., 784, 800 Larsson, P., 56, 60, 65, 70, 552, 569 Lasek, R. J., 558, 566 Lash, I., 644, 648 Lashley, K. S., 387, 399, 410, 432 Lashuay, N., 527, 530, 532, 545 Lasiter, P. S., 773–776, 780, 782, 839, 843, 917, 925 Laska, M., 218, 219, 221, 226, 385, 398, 410, 432, 839, 843 Laskaris, G., 941, 955 Lasker, B. R., 480, 481, 500 Laskin, C. R., 833, 843., 845, 886, 902 Lassen, N. A., xxviii, xxxviii, 253, 271 Lau, D., 670, 674, 771, 781, 782 Laugwitz, H. J., 30, 31, 41, 100, 107 Laugwitz, K. L., 76, 90 Laurent, G., 185, 186, 187, 197, 200, 261, 273, 329, 342, 412, 421, 426, 431 Laurie, D. J., 150, 159 Lauriello, M., 443, 457 Laurinen, P., 784, 804 Lauritsen, K. B., 910, 929 Lauterbur, P. C., xxviii, xxxviii, 253, 271 Lavedan, C., 514, 522 Lavenex, P., 418, 432 Lavenne, F., 174, 179, 193, 199, 258, 261, 264, 265, 266, 272, 424, 435 Laveran, C. L. A., xix, xxxviii Lavi, E., 551, 561, 569 Lavie, P., 440, 456 Lavin, J. H., 882, 898 Lavin, M. J., 909, 929 Lavond, D. G., 915–916, 921, 929, 933 Law, J. S., 710, 716, 727 Lawler, C. P., 394, 396, 400 Lawless, H. T., 211, 226, 783–785, 788, 794, 795, 799, 801, 802, 807, 813, 821, 848, 850, 859, 962, 963, 965, 982, 997 Lawrence, C., 1017, 1022 Lawrence, C. D., 420, 433 Laws, E. R. Jr., 938, 946, 954, 955 Lawson, N., 346, 382 Lawson, W., 9, 16 Lawton, A., 664, 670, 674, 677, 771, 781, 782 Lawton, M. P., 280, 293 Lax, M. B., 529, 532, 537, 542, 545 Laxhuber, L., 304, 308

1064 Layer, P., 887, 898 Laywell, E., 93, 112 Laywell, E. D., 621, 625 Lazard, D., 57, 58, 61, 70, 71, 76, 88, 559, 569, 570 Lazarovits, J., 58, 61, 70, 76, 88, 559, 569 Lazdunski, M., 721, 730 Lazzarini, A. M., 514, 522 Le, B. D., 948, 955 Le Bars, D., 258, 264–266, 272, 424, 435 Le Bihan, D., 797, 800 Le Camus, J. 845 Le Douarin, N. M., 115, 132 Le Jeune, H., 153, 154, 159 Le Lievre, G., 893, 898 Le Magnen, J., xxx, xxxviii, 216, 226, 421, 434 Le Moal, M., 416, 428, 432, 515, 521 Le Moine, C., xxv, xxxvii Le Neindre, P., 423, 434 Le Paslier, D., 127, 134, 1001, 1017, 1023 Le Roux, L., 472, 477 Leach, C. L., 583, 590 Leaderer, B. P., 634, 637 Lean, M. E. J., 887, 901 Leard, B., 920, 930 Leathem, J. H., 923, 924 Lebas, F., 317, 323, 370, 374 Lebenthal, E., 642, 649, 887, 898 Lebihan, D., 948, 954, 960, 962, 965 LeBlanc, J., 888, 896, 898, 902 Lebovics, R. S., 119, 121, 124, 137 Lebowitz, R. A., 466, 468, 475 Lecanuet, J. P., 312, 326, 845 Ledent, C., xxv, xxxvii, xli Lederer, F. L., xx, xxxviii Ledger, W. D., 363, 376 LeDoux, J. E., 414, 432 LeDuc, B., 413, 432 Leduc, D., 831, 840 Lee, A., 260, 271, 423, 432 Lee, C. H., 54, 70 Lee, C. T., 352, 361, 378 Lee, C. Y., 714, 729 Lee, D. H., 443, 453 Lee, E., 116, 132 Lee, H. M., 466, 476 Lee, H.-C., 417, 431 Lee, J., 390, 392, 397, 400, 413, 435, 915, 918, 931 Lee, J. H., 5, 15, 37, 44, 505, 506, 509, 521, 698, 705, 797, 804 Lee, K., xxiv, xxxviii Lee, K. H., 101, 110 Lee, K. K. H., 126, 134 Lee, K. S., 55, 71, 468, 476 Lee, K-F., 761, 762, 779 Lee, M. H., 450, 458 Lee, M. L., 581, 588 Lee, N. S., 443, 453 Lee, P. C., 642, 649 Lee, S., 970, 977, 979

Author Index Lee, S. H., 466, 476 Lee, S. van der, xxix, xxxviii, 346, 378 Lee, V. M., 505–507, 508–510, 512–517, 519–523 Lee, V. M. Y., 124, 137, 563, 572 Lee, V.-Y., 98, 109 Lee, W. W., 206, 215, 225, 462, 474, 495, 497, 616, 623, 629, 630, 631, 634, 635, 637 Lee, W.-H., 101, 113 Lee, Y., 102, 105, 110 Lee, T. M., xxviii, xl Lees, A., 238, 249, 488, 501 LeFavour, G., 893, 895 Lefkowitz, R. J., 27, 33, 42, 46, 47, 84, 85, 87–90 LeFloch, J. P., 893, 898 Lefort, A., xxv, xli Legg, J. W., 629, 637 LeGoff, D. B., 887, 898 Legouis, R., 127, 134, 1017, 1023 Legrand, C., 620, 623 Lehman, C. 998 Lehman, C. D., xxxi, xxxviii, 784, 787, 802 Lehman, M. N., 14, 16, 37, 46, 472, 477, 1002, 1003, 1007, 1024 Lehrach, C.M., 486, 497 Lehrman, D. S., 352, 378 Lehrner, J., 462, 476, 482, 485, 499 Lehrner, J. P., 839, 843, 891, 898 Leiacker, R., 439, 456 Leibel, R., 885, 895 Leibovici, M., 99, 110, 127, 134 Leichner, P., 887, 898 Leidahl, L. C., 359, 377, 378 Leigh, A. D., 604, 611, 629, 637, 959, 965 Leinders-Zufall, T., xxv, xxvi, xxxvii, xxxix, xxxv, 26, 49, 80–81, 86, 88, 91, 511, 523, 971, 973, 977 Leiser, R., 26, 49 Leiter, L. A., 882, 895 Leitsch, J. M., 807, 822 Leitz, E., 653 Lejour, M., 1014, 1015, 1023 Lellis, C., 509, 521 LeMagnen, J., xxx, xxxviii, 187, 199, 266, 271, 349, 357, 378, 403, 404, 408, 682, 702, 734, 751, 754, 883, 888, 898, 899 Lemaire, M., 416, 417, 432 Lemaistre, M., 117, 136 Lemann, W., 605, 611 LeMaster, A. M., 773, 780 Lemay, A., 554, 569, 572 Lemay, B., 515, 520 LeMay, M., 601, 610 Lembeck, F., 907, 932 LeMeur, M., 760, 779 Lemon, C., 153, 163 Lenard, L., 192, 193, 199, 680, 688, 689, 697, 701, 702, 704, 829, 844

Lendahl, U., 620, 621, 623, 624 Lengersdorf, A., 444, 458 Lenington, S., 318, 324, 336, 341, 342 Lenz, H., 36, 44 Lenzi, G. L., 253, 270 Leon, M., xxix, xxxix, 145, 146, 153, 157, 158, 164, 182, 184, 186, 187, 196, 197, 200, 201, 310, 320, 324, 326, 355, 359, 360, 374, 378, 404, 408, 420, 431, 558, 560, 568, 617, 618, 622, 624, 627 Leonard, B. E., 149, 158, 919, 926 Leonard, C. M., 679, 702, 704, 1005, 1025 Leonard, G., 204, 224, 467, 468, 472, 476, 593, 610, 633, 637, 889, 896 Leopold, D. A., 14, 15, 37, 38, 44, 47, 127, 134, 137, 229, 230, 233, 243, 247, 266, 271, 440, 442, 445, 447, 452, 457, 455, 456, 457, 463, 472, 476, 477, 606, 611, 632, 638, 795, 804 Leopold, H., 21, 46 LePendu, J., 123, 131 Leplow, B., 238, 247 Lepole, D. A., 1017, 1025 Leppik, I. E., 686, 700 Lepri, J. J., 389, 400, 1005, 1025 Leprohon, C. E., 884, 898 Lera, M. F., 789, 799 Lerner, E. A., 472, 477 Lerner, M. R., xxv, xliv, 78, 90 Leroy, E., 514, 522 LeRoy, J., 537, 541, 545 Lery, L. S., 526, 546 Lesch, K. P., 119, 121, 124, 137 Lesch, P., xxvii, xliv Leshem, M., 833, 843, 886, 898 Leshner, A. I., 310, 322, 359, 363, 373, 376 Leslie, R. D. G., 923, 928 Lesser, R. P., 602, 609 Lester, D. S., 119, 121, 124, 137 Lester, H. S., 80, 86 Lestienne, R., 192, 194 Letourneau, A. M., 4, 15 Lett, B. T., 914, 929 Letterio, F. C., xxxi, xliii, 863, 878 Lettvin, J. Y., xxiv, xxxvi Leung, M., 886, 903 Leung, P. M. B., 884, 899 Leung, W.-Y., 994, 996 Levene, M., 828, 829, 842 Levene, M. I., 829, 844 Levenson, M., 715, 728 Lévesque, M. F., 686, 704 Leveteau, J., xxiv, xxxixi, 145, 159, 171, 172, 174, 176, 178 Leveugle, B., 488, 496 Levi, P. E., 56, 64, 68, 553, 559, 567 Levilliers, J., 127, 134, 1017, 1023 Levi-Montalcini, R., 100, 110 Levin, H., 983, 994, 998

Author Index Levin, H. S., 604, 611, 631, 632, 637 Levine, A. S., 826, 844 Levine, H., 442, 457 Levine, H. L., 600, 611 Levine, J. D. 996 Levine, L., 362, 367, 378 Levine, M., 642, 647, 648, 649 Levine, M. A., 471, 474, 477 Levine, M. W., 862, 876 Levine, P. A., 594, 597, 611 Levine, P. H., 534, 544 Levine, R. R., 129, 133 Levine, S., 367, 378, 918, 929 Levine, S., 918, 929 Levine, S. C., 442, 457 Levitsky, D. A., 419, 420, 436 Levitt, P., 518, 522 Levy, A. V., 491, 496 Levy, F., 172, 178, 320, 324, 343, ?, 423, 426, 429, 431, 432 Levy, I., 204, 225, 469, 475, 580, 588, 985, 997 Levy, L. M., 257, 266, 269, 271, 424, 432, 797, 801 Levy, M. H., 887, 897 Levy, N., 24, 25, 26, 27, 28, 33, 43, 45, 78, 89 Levy, R. L., 553, 562, 569 Levy, S. M., 909–910, 926 Levy-Lahad, E., 513, 522 Lewindon, N., 831, 843 Lewindon, P. J., 831, 843 Lewine, J. D. 269, 270 Lewis, B. D., 148, 155 Lewis, C., 211, 223 Lewis, D., 667, 673 Lewis, D. A., 517, 519, 520, 522 Lewis, J. L., 54–56, 59, 60, 64, 67, 70, 71, 583, 590 Lewis, N. S., 300, 307 Lewis, R., 621, 625 Lewis, R. F., 601, 610 Lewis J. L., 550, 569 Lewitt, M. S., 471, 476, 892, 899 Lewy, F. H., 513, 521 Leyden, J., 216, 224 Leydig, F., xx, xxxviii, 652, 673 Leypold, B. G., 971, 977 Leyton, L., 440, 457 Leznoff, A., 539, 545 Li, C., xxxii, xxxv, 127, 138, 184, 195, 392, 398, 472, 476, 478, 484, 485, 489, 490, 494, 497, 503, 515, 520, 593, 594, 599, 606, 607, 609, 611, 613 Li, C.-S., 736, 756, 757, 970, 973, 977 Li, D., 491, 493, 496 Li, H., 419, 432 Li, J., 80, 86 Li, M., 423, 432 Li, P., 511, 521 Li, S., 256, 257, 273, 717, 724 Li, S. H., 81, 86

1065 Li, T., 885, 902 Li, W. F., 539, 544 Li, X., 717, 723, 724, 727 Li, X.-J., 645, 649, 658, 673 Li, X. M., 538, 546 Li, Y., xxvii, xxxix, 105, 110, 119, 121, 124, 137 Li, Y. M., 509, 521 Li, Y. W., 557, 565 Liang, H. C., 57, 62, 65, 68, 552, 559, 567 Liang, J.-J., 101, 108, 151, 156, 184, 195 Libbey, M., 394, 396, 397 Liberini, P., 513, 514, 518, 521 Liberles, S. D., 713, 714, 717, 728 Libert, F., xxv, xli Lichtarge, O., 291, 294 Lichtenstein, A., 824, 826, 830, 843 Liddell, K., 319, 324 Liddle, P. F., 516, 520 Liden, E., 462, 477 Lidow, M. S., 24, 44, 123, 132 Lieb, W. R., 988, 997 Liebenthal, R., 448, 454 Lieberman, A. R., 144, 157 Liebl, D. J., 762, 771, 780 Lieblich, I., 829, 843 Lieblich, J. M., 603, 611 Liebsch, G., 416, 432 Liedo, P-M., xxviii, xxxvi, xlii Liesegang, T. J., 469, 475 Liesi, P., 93, 110, 125, 134 Lieu, T., 413, 432 Lievre, C. A., 127, 134 Liggett, J. R., 387, 396, 399 Lignelli, A., 262, 265, 266, 271, 491, 492, 499 Liley, A. W., 883, 885, 899 Lilliston, L. G., 352, 356, 379 Lilly, M., 334, 342 Lim, D. A., 620, 624 Lim, D. W., 450, 458 Lim, H. H., 466, 476 Lim, M. L., 596, 609 Liman, E. R., xxv, xxxix, 27, 45, 80, 88, 351, 378, 971, 977 Lin, C. S., 257, 266, 269, 271, 424, 432, 797, 801 Lin, D. M., xxvii, xxxix, 35, 36, 44, 125, 126, 134, 969, 979 Lin, P. J., 97, 98, 110, 564, 570 Lin, S., 516, 523 Lin, S. C., 469, 475 Lin, W., 719, 722, 723, 727, 776, 780 Lindauer, M ., 343 Lindberg, L., 794, 799 Lindemann, B., xxv, xxvi, xxxiii, xxxix, xliii, 26, 44, 658, 664, 673, 710, 711, 712, 714–722, 725–727, 729, 730, 737, 753, 756, 777, 778 Lindemeier, J., xxv, xxxvii, 717, 726 Linden, D. J., 80, 89 Linden, R. W., 655, 673

Lindley, M. G., 714, 729, 793, 803, 813, 822 Lindorfer, H. W., 1002, 1003, 1023 Lindquist, N. G., 552, 554–556, 558, 569 Lindqvist, E., 101, 111 Lindsay, D. R., 423, 431 Lindsay, K. A., 919, 929 Lindsay, R. M., 28, 41, 773, 782 Lindskog, S., 101, 111, 670, 674 Lindvall, T., 4, 15, 204, 218, 223, 525, 542, 543, 544, 585, 587, 807, 814, 820 Lindzey, G., xxxiii, 404, 408 Ling, B. N., 718, 727 Linker, E., 882, 900 Linnaeus, C., xix, xxxix Linnarsson, S., 620, 625 Lino, M., 182, 188, 192, 198, 200 Linster, C., 148, 149, 156, 159, 170, 173, 178, 187, 190–192, 196, 197, 394, 395, 396, 399, 413, 424, 426, 430, 432 Lio, P., 260, 271 Liou, S. H., 581, 591 Lipinskas, T. W., 53, 57, 61, 62, 64, 65, 69, 72, 552, 568, 582, 589 Lipkin, V. M., 54, 71 Lipowska, M., 172, 173, 174, 178 Lippa, C. F., 480, 500, 509, 522 Lippoldt, R., 645, 648 Lipsett, L., 682, 704 Lipsey, J. R., 481, 489, 499 Lipsitt, L. P., 314, 324, 828, 840, 841, 842, 845, 906, 917, 932 Lipsky, C., 892, 896 Lipsky, J. J., 341 Lipton, P. A., 174, 175, 178, 192, 193, 198, 418, 419, 426, 433 Lipton, R. B., 553, 562 569 Lishman, W. A., 602, 610 Litaudon, P., 188, 190, 191, 198, 255, 259, 271 Litingtung, Y., 116, 132 Litterio, M., 343 Little, A. C., 891, 899 Little, J. R., 597, 611 Little, S., 119, 133 Little, S. S., 101, 102, 111 Littlejohn, M. C., 450, 457 Liu, A. K. 269, 270 Liu, C., 61, 70 Liu, C.-j., 151, 159 Liu, D., 511, 515, 520 Liu, G. L., 266, 270 Liu, H., 560, 571, 736, 756, 757 Liu, H-X., 765, 766, 781 Liu, J., 553, 567 Liu, K. L., 126, 134 Liu, L., 698, 702, 721, 727 Liu, M. A., 554, 563, 569 Liu, N., 103, 110, 511, 515, 519, 620, 625 Liu, Q., 292, 294 Liu, S., 621, 625

1066 Liu, W., 100, 105, 109 Liu, W. L., 142, 151, 159 Liu, W. M., 970, 979 Liu, X., xxxii, xxxv, 483, 497, 762, 767, 780 Liu, Y., 422, 430 Liu, Z., 713, 714, 717, 728 Livermore, A., 217, 219, 220, 226, 227, 232, 233, 235, 239, 246, 247, 388, 399, 486, 499, 500 Livesey, J., xxvii, xl, 36, 46, 93, 98, 103, 111 Ljungdahl, A., 103, 109, 511, 514, 520 Ljunggren, G., 210, 227 Lledo, P.-M., 143, 149, 151, 154, 155–157, 162, 184, 196, 509, 514, 521 Llinás, R., 661, 673 Llobet, E., 305, 306 Llorens, J., 60, 68, 553, 567, 583, 589 Lloyd, G. A., 598, 611 Lloyd, J. A., 366, 374 Lloyd-Thomas, A., 350, 365, 378, 970, 977 Lo, L. C., 121, 133 Lobb, B., 786, 802 Lobel, D., 77, 88, 972, 977 Lobel, E., 797, 800 Lobocki, C. A., 619, 626 LoBrutto, R., 599, 608 Locatelli, A., 312, 326, 423, 432 Lock, E. A., 57, 58, 64, 65, 66, 67, 72 Lockhead, E., 715, 718, 729 Lockhead, G. R., 817, 821 Locy, W. A., 1001, 1003, 1023 Lodi, L., 63, 70 Loeb, C., 505, 506, 523, 563, 572 Loeb, L. S., xxx, xxxiv, 352, 357, 374, 403, 404, 407 Loerzel, A. J., 488, 496 Loescher, A. R., 946, 953, 956 Loeve, K., 713, 730 Loew, G. H., 291, 293 Loewy, A. D., 557, 571, 572 Lofaso, F., 444, 457 Loffredo, F., 658, 675 Lofgren, L., 586, 590 Logothetis, J., 485, 496 Logothetis, N. K., 193, 198 Logue, A. W., 905, 910, 929 Logvinenko A. V., 311, 324 Lohse, M. J., 84, 88 Lois, C., xxviii, xxxix, 128, 134, 143, 159 LoLordo, V. M., 420, 428 Lomax, L. G., 582, 589 Lombard, J., 244, 249 Lombardi, J. R., 970, 979 Lomo, J., 62, 69 Lonai, P., 101, 111 London, R. M., 735, 756 Long, C. J., 405, 408, 413, 433 Long, D., 209, 211, 227 Long, L. Jr., 713, 725

Author Index Long, P. J., 369, 382 Longcope, C., 919, 930 Longo, V., 55–58, 61, 63–65, 66, 68, 70, 71, 561, 567 Longson, D., 511, 517, 522 Longstreth, W. T., Jr., 564, 565 Lonnoy, O., 101, 113 Loo, A. T., 98, 110, 119, 134, 463, 476 Loop, T., 116, 134 Loopuijt, L. D., 512, 522 Looy, H., 857, 859, 882, 899 Lopes da Silva, F. H., 241, 247 Lopez, A., 923, 931 Lopez, M. F., 355, 375, 885, 896 Lopez-Mascaraque, L., 104, 112, 166, 169, 171, 177, 180 López-Sela, P., 831, 840 Loraine, J. A., 922, 931 Lord, T., 321, 324, 339, 342 Lorentzen, M., 888, 899 Lorenz, K., 347, 352, 378, 828, 839, 843 Lorig, T. S., 204, 227, 232, 233, 240, 244, 247, 255, 271 Lorino, A. M., 444, 457 Lorino, H., 444, 457 Lorry, D., xix, xxxix Lötsch, J., 231, 238, 245, 246, 247, 466, 477, 990, 998 Lott, D. F., 367, 378 Lott, I., 320, 326 Lotze, M., 261, 269 Loudon, C., 185, 198 Loughney, K., 27, 49, 82, 90 Louie, S., 54, 67 Louisot, P., 58, 70 Louis-Sylvestre, J., 682, 702, 882, 888, 894, 896, 899 Loumaye, E., 1004, 1023 Lounasmaa, O. V., 245 Louon, A., 553, 569 Loury, M. C., 595, 611 Love, L., 5, 15, 441, 453 Lovelace, C. T., 394, 399, 410, 433 Lovell, M. A., 5, 15, 21, 23, 30, 33, 34, 37, 44, 45 Lovén, C., xx, xxxiv, 652, 655, 673 Lowe, G., xxv–xxvii, xxxix, xliv, 24, 26, 36, 37, 44, 49, 75, 76, 80, 83–84, 88, 90, 126, 134, 220, 226, 397, 400 Lowe, M. R., 883, 898 Lowell, M. A., 140, 160 Lowenthal, D. T., 554, 563, 569 Lower, R., xxii, xxxix Lowery, L. D., 599, 609 Lowery, P., 305, 307 Lowlicht, R. A., xxxi, xxxviii, 784, 787, 802 Lowry, L. D., xxxiii, 209, 217, 221, 224, 227, 228, 234, 243, 245, 248, 466–467, 469, 474, 783, 788, 790, 795, 799, 984–987, 991, 997 Lowry, O. H., 147, 153, 157, 170, 177

Lozano, D. I., 882, 899 Lozano, G. A., 909, 929 Lu, J., xxvii, xxxix, 107, 110 Lu, S., 664, 673 Lu, X. C., 145, 159, 389, 392, 394, 399, 411, 414, 433 Lubke, J., 142, 162 Lubow, R. E., 361, 377, 422, 431 Lucas, F., 906, 929 Lucas, G. A., 914, 929 Lucas, J. A., 469, 475 Lucchina, L. A., 788, 802, 853–855, 857, 859, 860, 961, 965 Lucchini, R., 581, 589 Luce, R. D., xv, xxxiii Lucero, D. P., 354, 379, 392, 399 Lucero, M., 101, 102, 103, 110, 112 Luchansky, L. L., 1013, 1025 Lucki, I., 392, 398, 494, 496 Luders H., 950, 954 Ludvisgon, H. W., 406, 408 Ludwig, M., 416, 429 Luiten, P. G., 170, 177, 422, 433 Lukach, B. M., 542, 543 Luke, B., 340, 341 Lukyanov, S. A., 116, 138 Lumeng, L., 885, 902 Lumia, A. R., 310, 326 Lumsden, A. 779 Lund, V. J., 7, 15, 444, 450, 454, 457, 467–468, 475, 476 Lundberg, C., 515, 520 Lundberg, J. M., 784, 800, 983, 998 Lundblad, L., 983, 998 Lundgren, J. D., 54, 71 Lundgren, K. H., 152, 164 Lundh, B., 560, 569 Lundstrom, K., 723, 728 Lung, M. A., 440, 457 Luo, G., 61, 70 Luostarinen, M., 942, 955 Lupi, R., 710, 716 Lupo, D., 63, 70 Luppi, P. H., 170, 176 Lüscher, M., xxx, xxxviii, 345, 346, 348, 350, 351, 361, 377 Lush, I. E., 709, 710, 717, 728 Lush, D., xxvi, xxxiv Lusis, A., 539, 544 Luskin, M. B., xxviii, xxxix, 33, 35, 43, 47, 53, 69, 76, 87, 93, 109, 110, 112, 128, 135, 143, 146, 147, 159, 167–174, 178, 515, 522, 617, 620, 624, 627 Lussana, F., 666, 673, 674 Luszyk, D., 319, 323, 341 Lutes, S. D., 244, 246 Lutfalla, G., 1017, 1023 Lutherat, P. J., 605, 609 Lütjen, K., 525, 528, 543 Lutter, C. D., 359, 373 Lutz, R. W., 256, 273, 990, 998 Ly, A., 850, 851, 856, 857, 859

Author Index Lyall, V., 722, 728, 729 Lydell, K., 357, 372, 378, 404, 407 Lyden, A., 552, 558, 569 Lygonis, C. S., 470, 476 Lykken, D. T., 330, 342 Lyman, R. F., 342 Lynch, G., 170, 178, 187, 188, 190, 191, 193, 197–199, 410–414, 425, 426, 426, 431–436, 481, 485, 489, 491, 496, 498 Lyon, M. F., 116, 134 Lyons, B. M., 598, 611 Lyons, K., 486, 500 Lysakowski, A., 190, 198 Ma, D., 190, 196, 413, 430 Ma, J. X., 61, 71 Ma, M., 396, 399 Ma, Q., 760, 780 Ma, W., 351, 381, 971, 973, 977 Maassab, H. F.554, 563, 570 Maaswinkel, H., 417, 433 Mac, L. P., 948, 954, 960, 962, 965 Mac Leod, P., 171, 178 Macagnano, A., 299, 306 MacAirt, J. G., 885, 903 Macallum, D. K., 763, 764, 765, 780, 781 Macaluso, D. A., 949, 956 Macauley, S. P., 643, 649 MacCallum, D. K., 762, 765, 766, 772, 777–779, 781 Maccia, C., 535, 544 MacDonald, A. C., 894, 897 Macdonald, D. W., xxx, xxxiv, 341, 357, 373, 422, 427 MacDonald, G. J., 760, 782 MacDonald, J. F., 712, 729 MacDonald, K. P. A., xxvii, xxxix, xl, 36, 46, 93, 98, 103, 110, 111 Macdonald, R., 116, 135, 137 Mace, J. W., 833, 843 Macfarlane, A., 338, 342, 312, 314, 317, 324 MacFarlane, A. J., 834, 843 MacFarlane, T. W., 893, 901, 950, 956 MacFie, H. J. H., 817, 821 MacGregor, R. R., 891, 899 Machida, Y., 554, 561, 571 Machon, R. A., 505, 516, 522 Maciewicz, R. J., 946, 947, 956 Mackay, S., 466, 475 Mackay, T. F., 334, 342 Mackay-Sim, A., xxiv, xxvii, xxxvi, xxxix, xl, 13, 14, 15, 35, 36, 44, 46, 93–98, 100–103, 105–107, 107, 109–111, 119, 125, 126, 135, 140, 159, 220, 227, 392, 397, 471, 474, 516, 520, 620, 623, 625, 661, 671, 712, 728, 892, 899, 1015, 1021 MacKenzie, G., 564, 565 Mackenzie, I. C. K., 784, 802 Mackenzie, P. I., 58, 70 Mackintosh, J. H., 361, 378

1067 Mackintosh, N. J., 873, 877 Maclean, C. M., 207, 224 MacLean, D. B., 919, 930 Maclennan, A. J., 917, 933 MacLeod, K. B., 915, 931 MacLeod, N. K., 124, 135, 973, 977 MacLeod, P., xxiv, xxxi, xxxvii, xxxix, 145, 159, 188, 196, 734, 751, 754, 797, 800, 813, 821, 875, 877 MacLeod, R. B., xxix, xxxvii, 867, 869, 870, 877 Macmillan, N., 209, 227 MacNichol, E. F., Jr., 750, 756 MacNiven, E., 366, 375, 378 Maconi, P., 422, 427 Macrides, F., xxx, xxxv, 103, 108, 131, 134, 139–142, 146–149, 151, 152, 155, 156, 160, 162, 165, 167–171, 173, 176, 177, 179, 185, 198, 369, 381, 412, 433, 552, 558, 564, 565, 566, 569, 618, 624, 970, 978, 1003, 1025 Macturk, R. H., 461, 475, 634, 637, 935, 955 Macynski, A., 647, 648 Maczka, M., 887, 898 Maddalena, J., 1017, 1021 Maddison, S. P., 680, 687, 688, 691, 692, 694, 695, 701, 704 Maddrey, W. C., 889, 892, 893, 896 Maderdrut, J. S., 124, 136 Madgio, J. C., 709, 726 Madowitz, M. D., 235, 236, 247, 342, 462, 476 Madsbad, S., 554, 563, 568, 888, 894, 899 Maeda, G., 167, 180 Maeda, T., 664, 677 Maeder-Ingvar, M., 553, 562, 569 Maekawa, K., 826, 828, 830, 843 Maes, F. W., 715, 718, 729 Maes, H., 342 Maesako, K., 444, 458 Maeshima, K., 787, 793, 801 Maestrini, E., 127, 133, 1017, 1022 Maffei, G., 656, 671 Maga, J. A., 723, 728, 828, 843 Magdalou, J., 58, 70 Magendie, F., xxii, xxxix, 653, 658, 666, 674, 981, 998 Magherini, P. C., 149, 155 Magli, M. C., 621, 623 Magnuson, T., 128, 134 Magoon, M. W., 330, 341 Magoul, R., 152, 154 Magrassi, L., 98, 107, 110, 130, 135 Magson, L. D., 882, 897 Mahajan, H., 472, 474 Mahajan, S. K., 893, 899 Mahalik, T., 95, 98, 110 Mahanthappa, N. K., xxvii, xxxix, 102, 103, 106, 110, 119, 121, 135 Maheux, R., 554, 569

Mahr, R. N., 479, 480, 491–493, 498, 499 Mai, J. K., 259, 271 Maier, S., 736, 756 Maigret, C., 416, 437 Mailhos, A., 116, 136 Mailman, R. B., 394, 396, 400 Mainardi, D., xxix, xxxix, 320, 324, 349, 352, 356, 378, 404, 408 Mainardi, M., 316, 324, 377 Maines, M. D., 82, 87, 88 Mair, E. A., 3, 16 Mair, R. G., 174, 180, 184, 186, 194, 198, 201, 394, 400, 411, 414, 415, 424, 426, 432, 433, 438, 469, 475, 494, 495, 499, 602, 611 Maitani, Y., 554, 561, 571 Majer, M., 232, 238, 244, 485, 496 Makary, C., 495, 498 Makena, A. M., 921, 926 Makin, J. W., 216, 227, 313, 317, 324, 325, 338, 342, 357, 380 Makino, C., 793, 803 Makita, K., 61, 71 Makris, N., 260, 271 Makura, Z. G. G., 947, 957 Malamud, D., 644, 648 Malaspina, D., 262, 265, 266, 271, 491–493, 499 Malczewski, R. M., 583, 589 Maldjian, J. A., 259, 264–266, 273, 344, 594, 599, 613 Maleeh, M., 867, 870, 879 Malemud, C. J., 130, 133 Malenfant, S. A., 909, 927 Males, J. L., 471, 476, 603, 611 Malfertheiner, P., 887, 898 Malherbe, P., 150, 161, 723, 728 Malhoutra, S. L., 887, 899 Malick, A. W., 553, 563, 566 Mallamaci, A., 116, 132 Maller, O., xvi, xli, 828–830, 832, 841, 843, 848, 855, 859, 883, 886, 896 Mallet, P., 321, 324 Malm, L., 450, 451, 453, 457 Malmberg, H., 445, 458, 467, 477, 505, 506, 521 Malnic, B., xxv, xxxix, 76, 78, 88, 182, 185, 198, 287, 288, 293, 351, 381 Malone, G. T., 211, 226 Malonek, D., 259, 271 Malpighi, M., xx, xxi, xxxiv, 651, 652, 674 Maltais, F., 440, 457 Malun, D., 129, 135 Malz, J., 57, 69 Mamikoglu, B., 442, 444, 457 Man, S. F., 55, 71, 554, 563, 569 Mancardi, G. L., 505, 506, 523, 563, 572 Mancuso, M., 38, 44 Mandel, I. D., 939, 954 Mandelkow, E., 508, 509, 521 Mandelkow, T., 891, 903 Mandell, M., 537, 545

1068 Mandenius, C. F., 305, 306 Mandl, A. M., 369, 379 Mania-Farnell, B., 24, 44, 103, 110, 121, 135 Manicom, R., 551, 567 Mann, D. G., 4, 16 Mann, D. M., 509, 522 Mann, S. B., 597, 611 Mann, W. J., 190, 197, 444, 445, 454, 467, 468, 476 Mannara, C., 553, 563, 566 Manning, R., 647, 649 Mannon, L. J., xxxii, xxxv, 489, 490, 497, 503, 520, 606, 609 Manolidis, S., 946, 955 Manoochehri, S., 618, 625 Mansfeld, G., 267, 271 Mansour, A., 560, 570 Mansueto, C. S., 472, 477 Manzo, P. A., 686, 702 Maone, T. R., 826, 843, 883, 899 Mar, A., 367, 379 Maracek, J. F., 81, 88 Maran, A. G. D., 7, 15 Marasan, M., 320, 324 Marasco, E., 357, 362, 374 Marchand, J. E., 141, 142, 160, 162, 185, 198 Marchese, S., 77, 88 Marchionni, M., 104, 111 Marchlewska-Koj, A., xxix, xxxix, 366, 379, 970, 977 Marcos, H. J. A., 30, 42 Marcotte, E. R., 619, 626 Marcus, A., 462, 474 Marcus, J., xxxi, xxxiv, 387, 394, 397 Marder, K., xxxii, xxxv, 483, 497 Marek, G. J., 170, 178 Maremmani, C., 54, 68, 101–103, 109 Marentette, L. J., 469, 476 Margalit, T., 57, 58, 71, 100, 110, 559, 570 Margarone, J., 647, 648 Margolick, J. B., 536, 537, 545 Margolin, S. G., 887, 897 Margolis, F. L., 27, 41, 44, 54, 70, 75, 76, 81, 85, 87, 88, 96, 103, 106, 107, 109–111, 123–125, 131, 131–135, 140, 147–150, 152, 155, 158, 160, 162, 216, 221, 224, 225, 233, 245, 341, 392, 393, 396, 397, 400, 505, 514, 520, 550, 551, 552, 554, 557–560, 564, 565–567, 570, 573, 616, 618, 623, 624–626, 660, 672, 729 Margolis-Nunno, H., 1001, 1022 Margolskee, R. F., xxvi, xxxix, xlii, 664, 675, 677, 708–711, 713–719, 725–730, 936, 956 Marini, S., 56–58, 61, 71 Marino, L., 828, 844 Marino, S., 798, 803, 849, 850, 860 Marin-Padilla, M., 104, 110

Author Index Mariotti, F., 365, 382 Mark, G. P., 684, 701, 734, 735, 736, 747, 755, 756, 757, 783, 803 Mark, H., 600, 610 Mark, M., 907, 931 Markert, L. M., 118, 138, 394, 395, 400, 418, 438 Markham, J., 253, 272 Markison, S., 462, 476, 734, 738, 741, 756, 758, 766, 782, 865, 868, 874, 875, 877, 879 Markley, E. J., 890, 891, 899 Markopoulou, K., 329, 344, 484, 499 Marks, L. E., 204, 211, 227, 371, 379, 788, 790, 793, 795, 796, 798, 799–801, 803, 818, 822, 848, 853, 854, 856, 858, 887, 900 Marks, W. B., 750, 756 Marler, M., 355, 381 Marlier, L., 314–316, 324, 326, 355, 381, 833, 834, 843, 845 Marlin, D. W., 908, 924 Maronpot, R. R., 52, 60, 62, 63, 66, 72, 553, 565 Marotta, A., 494, 500 Marouli, C., 525, 528, 543 Maroun, M., 833, 843, 886, 898 Marowitz, L. A., 643, 649 Marowitz, Z., 420, 432 Marquardt, D. W., 241, 247 Marquez, A., 509, 518, 522 Marr, J. N., 320, 324, 349, 352, 356, 374, 379, 404, 408 Marrama, P., 359, 374 Marsan, M., xxix, xxxix, 349, 356, 378, 404, 408 Marsden, C. D., 484, 485, 500 Marsden, H. M., 366, 379 Marsh, J., 645, 649 Marsh, R. E., 404, 407 Marsh, R. R., 829, 830, 840 Marshak, G., 445, 457, 467, 473, 475, 477 Marshall, D. A., 354, 379, 388, 392, 399, 400, 747, 748, 754, 983, 999 Marshall, J. F., 558, 560, 568, 617, 624 Marshall, R. S., 262, 265, 266, 271, 491, 492, 499 Marshall, R.W., 305, 307 Martel, J. C., 76, 86 Martez, S. J., 444, 456 Marti, E., 124, 131 Martin, A. J., 267, 269 Martin, B. M., 645, 650 Martin, F., 468, 475 Martin, G. R., 621, 625 Martin, H. N., xxii, xxxix Martin, I. G., 348, 352, 379, 422, 433 Martin, J. A., 507, 509, 514, 519 Martin, J. E., 238, 245, 490, 498 Martin, J. H. 998 Martin, J. R., 915, 929 Martin, L., 581, 589

Martin, L. T., 420, 433 Martin, M. V., 64, 69 Martin, S., 885, 899 Martin, W. R., 253, 272 Martin du Pan, R., 826, 843 Martinez, B. A., 494, 499 Martinez, M., 359, 382 Martinez, R., 658, 676 Martinez, S., 117, 136 Martinez-Guijarro, F. J., 154, 156 Martínez-Marcos, A., 973, 977, 978 Martinez-Sanchis, S., 359, 382 Martini, F., 58, 66 Martini, J. J., 38, 48 Martini, R., 664, 674 Martins, D., 257, 266, 271, 424, 432 Martzke, J. S., 491, 492, 498, 499, 961, 965, 966 Maruenda, J., 554, 563, 569 Marui, T., 746, 755 Maruniak, J. A., 64, 65, 73, 97, 98, 103, 107, 110, 113, 184, 194, 204, 227, 358, 368, 373, 379., 388, 400, 559, 560, 564, 565, 570, 616–618, 620, 622, 623–625, 970, 978, 983, 999 Marwaha, R., 472, 474 Mascarella, S. W., 276, 293 Masieri, S., 444, 456 Masini, C. V., 619, 624 Masliah, E., 509, 523 Masmela, J. R., 310, 326 Mason, J. R., 388, 400, 419, 430, 563, 569, 982, 983, 997, 999 Mason, R., 193, 199, 421, 435, 688, 692, 694, 695, 704 Mass, E., 952, 955 Massa, N., 17, 44 Massien, C., 882, 896 Masson, M., 488, 496 Mast, T. E., 470, 474 Masterton, R. B., 751, 754 Masuko, S., 659, 673 Mata, J., 238, 247 Mataga, N., 184, 186, 196, 198, 221, 226 Matava, M. J., 444, 457 Mateer, C. A., 962, 966 Mateo, J. M., 336, 342, 357, 379 Matern, G., 238, 247 Matfin, G., 471, 477 Math, M. F., 983, 998 Matha, K. V., 212, 223, 494, 495 Mathe, F., 234, 244 Mather, P., 914, 925 Matheson, W. R., 394, 400 Mathews, D. F., 184, 186, 198 Mathews, J. R., 472, 477 Mathews, P., 411, 428, 429 Mathews, W. C., 463, 473 Mathieson, B. J., 320, 327, 356, 383 Mathison S., 549, 550, 561, 563, 570 Mathog, R. H., 600, 611 Matia, D. C., 233, 247 Matochik, J. A., 357, 380

Author Index Matoltsy, A. G., 660, 675 Matschinsky, F. M., 147, 153, 157, 170, 177 Matsuda, H., 968, 978 Matsuda, T., xxxi, xxxix, 467, 476, 629, 637, 788, 789, 802, 938, 952, 955 Matsuda, Y., 182, 200 Matsui, H., 266, 271 Matsui, M., 32, 36, 45, 56, 59, 65, 71, 72 Matsumiya, K., 554, 570 Matsumoto, I., 709, 711, 716, 727 Matsumoto, K., 419, 432, 664, 677, 766, 781 Matsumoto, S., 659, 675 Matsunami, H., 351, 379, 709, 710, 713, 714, 717, 728, 798, 802, 972, 978 Matsuo, R., 639, 644, 649, 723, 730, 745, 758, 887, 899, 918, 933 Matsuoka, I., 723, 728 Matsuoka, M., 27, 44, 351, 382, 975, 976, 978 Matsushita, F., 971, 978 Matsuyama, A., 102, 105, 110 Matsuzaki, O., 24, 26, 44, 79, 88, 123, 135, 344 Matsuzaki, T., 31, 33, 44 Mattern, C. F., 661, 675 Mattern, C. F. T., 646, 648, 963, 965 Mattern, R., 82, 85 Mattes, R. D., 471, 476, 732, 755, 792, 802, 826, 843, 858, 858, 881–883, 885–892, 899, 902, 906, 910, 929, 936, 950, 952, 953, 955, 956 Mattes-Kulig, D. A., 890, 891, 900 Matthaei, K., 102, 109 Matthews, J. R., 619, 626 Matthias, C., 238, 247 Matthiesen, A. S., 313, 327 Mattila, P. M., 505, 515, 522 Mattson, M. P., 511, 515, 520 Mattson, R., 268, 270, 494, 496 Matulionis, D. H., 96, 110, 616, 625 Matute, C., 151, 159 Matyas, E. P., 394, 400, 418, 438 Mauderli, F., 946, 954 Mauguiere, F., 258, 264, 265, 266, 272, 424, 435 Maury, M., 116, 131 Max, M., 710, 713, 714, 717, 726, 728 Maxson, S. C., 318, 326 Maxwell, L., 462, 473 Maxwell, R., 686, 700 May, B., 314, 322, 355, 372 May, M., 789, 802, 962, 965 Mayberg, H. S., 536, 545 Maydew, N., 828, 842 Mayer, D., 785, 789, 791, 799 Mayeux, R., xxxii, xxxv Mazaika, T. J., 553, 566 Mazan, S., 116, 131 Mazur, A., 359, 373 Mazzaccaro, A., 56–58, 61, 71 Mazzarello, V., 824, 844

1069 Mbiene, J.-P., 761–766, 768, 769, 771, 779, 780 McAllister, T. W., 961, 965 McArdle, J. J., 331, 343 McArthur, L. H., 884, 897, 900, 906, 927 McBride, R. L., 168, 179, 188, 200, 218, 219, 226, 227, 807, 817, 821, 822 McBride, S. A., 192, 198, 394, 399, 411, 425, 433 McBurney, D. H., xvi, xxxix, 644, 649, 734, 751, 756, 783, 785, 788, 794, 797, 799, 800, 802, 803, 816, 822, 847, 848, 859 McCaffrey, R. J., xxxii, xxxix, 480, 482, 499 McCaffrey, T. V., 440, 451, 454, 457, 942, 955 McCallum, W. C., 233, 249 McCann, M. J., 910, 929, 932 McCarley, R. W., 492, 501 McCarthy, J. F. 996 McCartney, W., xvi, xxxix, 276, 293, 386, 399 McCarty, R., 103, 111, 151, 161, 184, 194, 320, 324, 336, 342, 356, 379, 618, 626 McCaughey, S. A., 683, 684, 701, 702, 736, 744, 755, 756 McCaul, K. D., 359, 379 McClain, J., 762, 779 McClanahan, R. H., 550, 567 McClearn, G. E., 343 McCleary R. A., 862, 877 McClellan, R. O., 56, 67 McCleskey, E. W 983, 998 McClintock, M. K., 350, 382 McClintock, T. S., 24, 44 McClure, D., 366, 379 McCollum, J., 188, 191, 198, 412, 426, 433 McConnell, R. J., 471, 476, 620, 625, 892, 900, 950, 956 McConville, P., 795, 803 McCormack, L. J., 468–469, 476 McCormick, J. P., 318, 324, 339, 342 McCormick, M. S., 947, 957 McCormick, W. C., xxxii, xxxvii, 341, 483, 498 McCosh, J. K., 556, 569 McCotter, R. E., 1002, 1005, 1023 McCoy, D. F., 420, 433 McCraig, L. F., 883, 900 McCulloch, M. A., 968, 979 McCurry, S. M., xxxii, xxxvii, 341, 483, 498 McCutcheon, N. B., 790, 804, 882, 900 McDaniel, M. R., 835, 843 McDonald, C. J., 644, 648 McDonald, F. D., 893, 899 McDonald, J. W., 621, 625 McDonald, P., 27, 33, 46, 84, 89 McDonald, R. J., 173, 180, 414, 436, 916, 929

McDonald, W. I., 489, 501, 936, 954 McDonald, W. M., 601, 611 McEntee, W. J., 424, 433, 495, 499 McEntegart, C., 300, 308 McFadden, K. A., 116, 133 McFadden, S. A., 539, 545 McFarlane, I., 1004, 1022 McGadey, J., 36, 41 McGaugh, J. L., 414, 425, 427, 916, 924 McGeady, S. J., 466, 467, 474, 599, 609 McGeer, P. L., 153, 161, 555, 571 McGehee, D., 154, 156 McGlone, F., 193, 198, 259, 264, 266, 271, 698, 699, 700, 702, 797, 802 McGlone, F. P., 236, 248 McGlone, J. J., 315, 325, 915, 921, 931 McGlone, R. E., 470, 475 McGorry, P. D., 492, 493, 496 McGrath, J., xxvii, xxxvi, xl, 36, 46, 93, 98, 103, 107, 109, 111, 516, 520 McGrath, J. J., 13, 14, 15, 466, 475 McGregor, I. S., 394, 400 McGue, M., 331, 332, 342 McIlhany, M., 170, 178 McIndoo, N. E., 403, 408 McKay, R. D., 621, 623 McKeel, D.W., 513, 522 McKenna, M. J., 582, 590 McKenzie, J. S., 148, 153, 159, 161, 170, 179, 418, 434 McKeown, D., 37, 49, 206, 212, 215, 225, 341, 462, 470–471, 474, 478, 483–486, 495, 497, 499–501, 599, 613, 631, 633, 635, 637, 638 McKhann, G. M., 479, 489, 499, 501, 936, 954 McKibben, P. S., 1001, 1002, 1005, 1023 McKinnon, P. J., xxvi, xxxix, 664, 675, 709–711, 716, 717, 728, 729 McKinnon, R. A., 57, 62, 65, 68, 552, 559, 567 McLachlan, J. A., 923, 929 McLaren, J. W., 469, 475 McLaughlin, F. J., 318, 321, 325, 343 McLaughlin, S., 936, 956 McLaughlin, S. K., xxvi, xxxix, 664, 675, 709–711, 716, 717, 728, 729, 764, 780 McLaughlin, T., xvii, xix, xxxix McLaurin, J. W., 439, 441, 458 McLean, H., xxviii, xxxvi McLean, J. H., 60, 71, 143, 148, 152, 153, 156, 160, 551, 557, 570 McLean, S., 152, 160 McLellan, R. K., 541, 545 McLelland, J. L., 681, 701 McLennan, H., 153, 160 McMackin, D., 268, 270, 424, 431 McMahon, A. P., 765, 778, 779 McMahon, D. B. T., 874, 878 McMartin C., 549, 550, 561, 570 McMinn, R. M. H., 7, 15

1070 McNamara, M. E., 888, 898 McNaughton, P., 983, 996 McNelly, N. A., xxvii, xxxvii, 36, 43, 94, 95, 97, 98, 109, 125, 134, 341, 564, 568 McPhee, L., 882, 895 McPheeters, M. M., 661, 673, 746, 756, 776, 780 McRae, T., 601, 609 McShane, R. H., 482, 499, 513, 522 McTamney, J., 587, 591 Meachum, C. L., 910–911, 917, 929 Mead, P. S., 883, 900 Meador-Woodruff, J. H., 560, 570 Mediavilla, C., 917, 929 Medinsky, M., 539, 543, 587, 588 Mednick, S. A., 516, 521, 522 Medrano, C. J., 581, 589 Meehan, J., 788, 803 Meehan, K., 721, 729 Meertz, E., 518, 519 Meggs, W. J., 530, 532, 538, 539, 543, 545, 587, 588, 589 Meghan, M., 946, 957 Mehansho, H., 644, 649 Mehdorn, H. M., 469, 474 Mehler, M. F., 101, 103, 113 Mehra, Y. N., 597, 611 Mehta, N., 483, 498, 946, 947, 956 Mehta, R. H., 444, 457 Meier, G. W., 310, 325 Meier, R., 105, 112 Meijs, J. W., 241, 247 Meinel, W., 33, 42, 658, 672 Meinken, C., 287, 293 Meiri, K. F., 96, 112, 119, 137 Meisami, E., xxxiii, 98, 111, 118, 135, 142, 143, 160, 161, 166, 178, 511, 512, 522, 564, 570, 615–618, 620, 625, 626, 969, 976, 978 Meiselman, H. L., xxxix, 216, 224, 751, 756, 785, 788, 799, 802, 806, 822, 840, 853, 859 Meister, M., 145, 160, 971, 979 Meitinger, T., 1017, 1021 Mela, D. J., 884, 885, 900 Melanson, S. W., 553, 562, 570 Melia, K. R., 413, 433 Mellert, T. K., 35, 42, 54, 71, 560, 570, 577, 589 Mellick, P. W., 583, 590 Mellier, D., 312, 325, 355, 380 Melnick, S. L., 941, 956 Melnick, S. M., 818, 820 Meloan, C. E., 285, 293 Melrose, D. R., 346, 379 Melrose, R., 419, 426 Melton, III., L. J., 923, 928 Meltzer, H. Y., 923, 928 Melville, G. N., 55, 69 Men, D., 103, 111, 151, 161, 618, 626 Mena, L., 536, 544

Author Index Menco, B. P. M., xxv, xxxiv, 17, 19–38, 41–47, 75, 78, 80, 88, 89, 96, 97, 108, 111, 119, 120, 123, 124, 125, 132, 135, 662, 664, 674 Mendelsohn, M., 36, 49, 78, 89, 126, 130, 135, 138, 146, 157, 160, 164, 289, 294, 296, 307, 354, 379 Mendelson, J., 600, 610, 864, 877, 879 Mendelson, T., 321, 325, 343 Mendoza, A. A., 122, 135 Mendoza, A. S., 36, 45 Mendoza, R., 320, 326 Menendez, C. E., 471, 476, 620, 625, 892, 900, 950, 956 Meneses, J. J., xxvii, xxxiv, 117, 130, 131, 136 Meng, E. C., 291, 294 Mengod, G., 152, 161 Menini, A., 27, 44, 287, 293 Mennella, J. A., 311, 314, 317, 322, 324, 355, 356, 379, 829, 830, 833–839, 840, 843, 844, 885, 900 Mennin, S. P., 923, 929 Menon, R., 254, 269, 271 Mentenopoulos, G., 485, 496 Menzel, R., 182, 183, 193, 197, 220, 226, 298, 307 Mercer, M. E., 839, 844 Meredith, M., xxix, xxx, xxxix, 184, 186, 198, 348, 350, 351, 379, 383, 404, 408, 558, 559, 570, 967, 968, 970, 973–975, 977, 978, 982, 998, 1005, 1025 Mergler, D., 581, 586, 589 Merida, M., 54, 71 Merig, J., 646, 648 Merkel, F., xxi, xxxix, 660, 674 Merkle, H., 254, 269, 271 Merkulova, M. I., 54, 71 Merkus, F. W., 553, 554, 562, 563, 570, 572 Merlio, J-P., 762, 779 Merwin, A. A., 920–921, 929 Mery, S., 582, 589 Mesholam, R. I., 479, 480, 491, 499 Messerklinger, W., 1, 7, 12, 16 Mester, A. F., xxxi, xxxv, 216, 224, 440, 454, 463, 471, 474, 480, 483, 484, 495, 496, 497, 499, 500, 629, 635, 637, 787, 788, 790, 791, 795, 799, 890, 894, 896, 935, 938, 945, 950, 951, 954, 959, 961, 963, 965 Mestre, N., 169, 178 Mesulam, M. M., 242, 247, 603, 611, 680, 686, 697, 702 Metcalf, J. F., xxvii, xxxvii, 75, 87, 93, 94, 109 Mettenleiter, T. C., 557, 571 Metter, E. J., 602, 611 Metzher, M. A., 828, 841 Meulstee, J., 485, 499 Meurisse, M., 172, 178, 423, 432

Mevio, E., 450, 454 Meyer, D. L., 1001, 1002, 1003, 1023 Meyer, E., xxix, xlv, 190, 201, 259, 264, 266, 273, 699, 705 Meyer, J., 342 Meyer, M., 763, 782 Meyer, M. R., 27, 45, 83, 89 Meyer, R. P., 165, 178 Meyer, U. A., 57, 71 Meyer Naess, L., 554, 568 Meyer-Franke, A., 620, 625 Meyerhof, W., 722, 729 Meyerhoff, W. L. Meyermann, R., 82, 85 Meyerowitz, B. E., 909, 910, 926 Meza, C. V., 313, 324 Mezza, R. C., 38, 47, 65, 72, 106, 112, 119, 122, 137, 145, 162, 616, 626 Mezzanotte, W. S., 441, 456 Miakiewicz, S. L., 921, 930 Miao, Z., 971, 977 Miarzynska, M., 580, 584, 585, 591 Michael, K. L., 299, 307 Michael, R. P., 346, 363, 373, 375, 377, 379, 423, 437 Michaelsen, T. E., 554, 568 Michaels-Marston, K. S., xxxii, xxxv, 483, 497 Michas, J., 97, 108, 118, 133 Michaud, B., 416, 432 Michel, D., 96, 111 Michel, W. C., 81, 86 Michener, C. D., 343 Middleton, C. B., 238, 245 Middleton, F. A., 518, 522 Middleton, R. A., 893, 900, 950, 956 Miele, J., 920, 930 Mieleszko, K. E., 1017, 1025 Mieleszko Szumowski, K. E., 33, 38, 43, 47, 118, 119, 121, 122, 127, 134, 137 Mierson, S., xxv, xxxvii, 715, 726, 728, 776, 781 Miescher, E. A., 82, 85 Miettinen, S., 82, 88 Mifka, P., 629, 638 Migdal, S. D., 893, 899 Miguel, E. C., 472, 477, 686, 702 Mihalick, S. M., 392, 399 Mike, V., 320, 327, 356, 383 Mikhail, L., 143, 160, 512, 522 Mikkola, I., 116, 135 Mikos, E., 887, 898 Mikoshiba, K., xxv, xlv, 32, 36, 45, 56, 59, 65, 71, 72, 78, 81, 87, 91, 145, 164, 287, 294, 396, 400, 971, 978 Mikulka, P. J., 920, 930 Milgram, N. W., 905–906, 927, 930 Millar, A. R., 970, 978 Millar, L. C., 312, 323 Millar, R. P., 1012, 1024 Millasseau, P., 127, 134, 1017, 1023

Author Index Miller, A., 833, 844 Miller, C. S., 525–530, 532–542, 543, 545, 546, 587, 588 Miller, D., 790, 802 Miller, F. J., 584, 590 Miller, G. K., 583, 590 Miller, I. J., Jr, xvi, xxxi, xxxix, xlv, 655–658, 660, 664, 667, 674, 677, 709, 728, 740–742, 747, 748, 755, 756, 784, 789, 802, 804, 825, 828, 844, 849–854, 858, 860, 937, 943, 953, 956, 957, 963, 965 Miller, J. S., 420, 421, 433 Miller, K. K., 772, 773, 780 Miller, M. L., 33, 45, 388, 392, 394, 396, 398, 400, 556, 567, 578, 581–584, 588, 589, 591 Miller, R., 945, 957 Miller, R. A., 583, 590 Miller, R. R., 582, 583, 590 Miller, S., 786, 791, 801 Miller, S. D., 864, 877 Miller, S. L., xxxi, xl, 786, 802, 937, 956 Miller, S. M., 951, 956 Millhouse, O. E., 167, 172, 178 Milligan, G. J., 329, 340 Milliken, H. I., 491, 498, 499 Millman, B., 404, 408 Millman, L., 907, 931 Millot, J. L. 269 Mills, S. E., 38, 48 Mills, T. M., 553, 562, 570 Milne-Edwards, H., 19, 45 Milunicova, A., 855, 860 Mimmack, M., 120, 123, 136, 423, 431 Min, N., 151, 155, 564, 566 Min, Y. G., 55, 71, 451, 456, 468, 476 Minami, Y., 721, 730 Ming, D., 664, 677, 710, 711, 716, 717, 728 Minnema, D. J., 388, 394, 396, 398, 578, 583, 589 Minnich, D. E., xxix, xl Minor, A. V., xxv, xxxiv Minsky, M., 296, 307 Mintun, M. A., 253, 270, 272, 593, 595, 612 Miragall, F., 24, 45, 105, 111, 122, 123, 125, 135 Miranda, M. I., 420, 429 Mireles, R., 686, 700 Mirnics, K., 518, 522 Miro, J. L., 618, 625, 960, 965 Mirza, N., xxxi, xxxv, xl, 450, 457, 785, 786, 789, 800, 802, 937, 955, 956 Misaka, T., 715, 728 Mischinger, A., 909, 927 Miselis, R. R., 916, 932 Miserendino, M. J. D., 413, 433 Mishkin, M., 165, 168, 171, 172, 180, 410, 414, 427, 433, 680, 686, 701, 704

1071 Mishra, A., xxxii, xxxv, 466, 474, 595, 609 Misiewicz, J. J., 887, 901 Misselbrook, T. H., 300, 305, 307 Mistretta, C. M., 641, 649, 658, 670, 671, 674, 742, 758, 759, 761–769, 771–775, 777, 778, 778–782, 825, 830, 842, 843, 844, 886, 897 Mitchell, C. S., 536, 537, 545 Mitchell, D., 643, 649 Mitchell, J., 81, 89 Mitchell, J. E., 923, 930 Mito, Y., 938, 949, 957 Mitra, A. K., 31, 48, 562, 572 Mitsuhashi, F., 659, 675 Mitsui, S., 145, 160 Mitsumori, K., 553, 569, 578, 583, 591 Mittelbronn, M., 82, 85 Mittendorf, R., 828, 842 Mitzel, H. C., 526, 532–534, 537, 538, 540, 545 Miura, H., 714, 727 Miwa, N., 83, 89 Miwa, T., 101, 102, 111, 152, 160, 215, 225, 232, 246, 392, 397, 399, 634, 638 Miyakawa, M., 1002, 1003, 1023 Miyamoto, K., 660, 676 Miyamoto, T., xxv, xli, 76, 81, 89, 90, 712, 719–722, 728, 729 Miyanhi, R., 233, 249 Miyaoka, Y., 888, 894 Miyasaka, A., 723, 730 Miyawaki, A., 32, 36, 45, 56, 59, 65, 71, 72, 81, 87, 971, 978 Miyazano, K., 101, 113 Miyazawa, H., 266, 271 Miyoshi, M. A., 709, 728 Miyoshi, N., 915, 924 Miyoshi, Y., 37, 49 Mize, S. J. S., 795, 800, 812, 813, 821 Mizobuchi, M., 942, 956, 957 Mizukoshi, T., 875, 877 Mizuno, M., 485, 496 Mobbs, C.V., 505, 520 Moberg, P. J., xxxii, xliv, 238, 245, 269, 270, 271, 273, 479, 480–482, 489, 491–495, 496–499, 512, 515–517, 519, 522, 523, 602, 603, 611, 612 Mochizuki, K., 720, 728 Mochizuki, R., 259, 269, 271 Mochizuki, Y., 656, 674 Mody, F. V., 554, 571 Mody, I., 712, 729 Moe, K., 447, 458 Moe, K. E., 906, 930 Moel, D. I., 832, 845, 893, 902 Mogi, G., 54, 67 Mohammadian, P., 231, 247 Mohan, C., 483, 501 Mohler, H., 150, 156 Mohns, T., 392, 399

Mohr, U., 63, 72 Mohs, R. C., 483, 501 Moinuddin, R., 450, 452, 455 Moir, A. A., 450, 452, 455 Mok, M. Y., 350, 380 Mokrusch, T., 238, 246 Molday, R. S., 83, 87 Molgaard, C. A., 64, 69 Mølhave, L., 525, 528, 543, 987, 998 Molin, C., 539, 544 Molina, F., 917, 929 Molina, J. C., 315, 322 355, 375, 885, 896 Moll, B., 190, 197, 467, 468, 476 Moll, R., 659, 674 Molle, G., 26, 41 Molle, M., 554, 563, 571 Moller, R., 469, 476 Mollereau, C., xxv, xli, Mollicone, R., 123, 131 Moltz, H., 310, 324, 359, 378, 404, 408 Mombaerts, P., xxv, xxxv, 24, 28, 45, 78, 89, 126, 130, 135, 137, 138, 145, 146, 160, 182, 198, 275, 291, 294, 296, 307, 334, 342, 350, 354, 379, 380, 972, 973, 978 Momma, S., 620, 624 Monaco, H. L., 78, 86, 280, 293 Monaghan, D. T., 151, 156 Monahan, E., 318, 326 Monard, D., 105, 112 Monastryrskaia, K., 723, 728 Monath, T. P., 551, 557, 561, 570 Mönch, B., 525, 528, 543 Moncomble, A.S., 369, 379 Moncrieff, R. W., 218, 227, 243, 247, 279, 291, 294 Mondragon, M., 3, 15 Moniot, B., 1017, 1022 Monk, J., 526, 545 Monnier, P., 443, 457 Monroe, C., 217, 228, 461, 474, 580, 584, 585, 590 Monroe, S., 736, 754 Mons, N., 80, 86, 89 Monsch, A. U., 462, 477, 480, 481, 500 Montagner, H., 318, 321, 326 Montague, A. A., 149, 160 Montararo, A., 941, 954 Montavon, P., 663, 674 Monte, P., 334, 342 Monte-Alegre, S., 888, 901 Montella, P. G., 397, 399 Montgomery, E. B., Jr., 486, 499, 500, 601, 611 Montgomery, G. G., 698, 701 Montgomery, K., 424, 431, 886, 897, 898 Monti Graziadei, G. A., 36, 43, 93, 94, 97, 98, 100, 106, 109, 118, 119, 121–123, 125, 129, 130, 133, 135, 141, 157, 616, 625 Monti-Bloch, L., 311, 324, 969, 978, 979

1072 Monticello, T. M., 38, 43, 59, 71, 99, 109, 447, 456 Monti-Graziadei, A. G., 53, 62, 69 Monti-Graziadei, G. A., 75–76, 87, 1015, 1022 Montkowski, A., 416, 432 Montmayeur, J. P., 551, 560, 568, 709, 710, 713, 714, 717, 728, 798, 802 Montoro, J. B., 359, 382 Monzingo, F. L., 784, 798 Moody, W. J., Jr., 426, 433 Mook, D. G., 906, 930 Moon, B.K., 451, 456 Moon, C., 76, 81–84, 89, 833, 842 Moon, C. N., 945, 956 Moon, Y., 99, 111 Moon, Y. W., 564, 570 Mooney, K. E., 172, 178 Mooney, M. P., 3, 16, 969, 979 Moor, J. G., 887, 900 Moore, B. O., 887, 896 Moore, F. L., 1001, 1003, 1005, 1007, 1023, 1024 Moore, J. D., 321, 325, 338, 343 Moore, K. L., 116, 117, 118, 135 Moore, M., 885, 900 Moore, M. E., 882, 900 Moore, M. J., 908, 925 Moore, P. A., 185, 199 Moore, P. J., 885, 886, 898 Moore, R. E., 404, 408 Moore, R. Y., 170, 177, 178, 179 Moore, S., 557, 570 Moore-Gillon, V., xxxi, xxxv, 216, 224, 440, 454, 463, 471, 474, 480, 495, 496, 497, 629, 635, 637, 787, 788, 790, 791, 795, 799, 890, 894, 896, 935, 938, 945, 950, 951, 954, 959, 961, 963, 965 Moorhead, J. F., 536, 545 Moorman, J. R., 603, 608, 611 Moosler, A., 1004, 1022 Mor, G., 173, 179 Mora, F., 680, 688, 692, 700, 703 Morales, J. A., 551, 557, 570 Morales, M. T., 305, 306 Moran, D. T., xxxi, xxxvii, xl, 3, 5, 15, 16, 21–23, 30, 33, 34, 37–39, 43–47, 121, 122, 135, 140, 160, 311, 324, 463, 466, 476, 600, 610, 631, 638 Moran, M., 828, 830, 831, 840, 886, 894 Moran, M. J., 363, 376 Mordes, J. P., 919, 930 Morecraft, R. J., 680, 702 Morel, K., 103, 113, 184, 194, 564, 565, 618, 623 Moren, A., 101, 113 Moretti, C., 686, 702 Moretti, E., 243, 244 Morfit, C., xvi, xl Morgan, C. D., 233, 235–237, 240, 245, 247, 342, 462, 472, 476, 480, 481, 500

Author Index Morgan, J., 106, 109 Morgan, J. I., 33, 34, 41, 118, 121–123, 132, 133, 560, 568 Morgan, K. T., 38, 43, 52, 54–56, 59, 60, 67, 68, 70, 71, 99, 109, 392, 399, 447, 456, 553, 567, 578, 582, 583, 588, 589, 590, 591 Morgan, T. O., 886, 896 Morgan, W. T., 990, 998 Morgenlander, J. C., 483, 496 Morgenson, M., xxxi, xxxv, 785, 789, 800, 937, 955 Mori, H., 946, 957 Mori, I., 551, 557, 570 Mori, J., 102, 113 Mori, K., xxiv, xl, 28, 32, 36, 46, 54, 67, 78, 83, 88, 89, 121, 123, 125, 130, 135, 138, 139, 142, 144, 145, 148, 158, 160, 163, 164, 168, 179, 182–188, 190, 193, 196–201, 221, 226, 280, 294, 560, 570, 972, 973, 975, 977–979 Mori, T., 723, 728 Mori, Y., 351, 382, 975, 976, 978 Moriguchi, I., 276, 292, 294 Moriguchi, T., 394, 398 Moriizumi, T., 101, 102, 111, 152, 160 Morikawa, K., 717, 727 Morikawa, T., 440, 458 Morilak, D. A., 152, 160 Morimoto, K., 889, 990 Morimoto, R. I., 26, 28, 30–33, 38, 41, 145, 155 Morimoto, T., 887, 899 Morin, N., 950, 954 Morin, D., 583, 590 Morin, P.-P., 426, 433 Morin, X., 760, 779 Morishita, H., 343 Moriyama, H., 581, 589 Morley, S. D., 645, 649 Mormede, P., 885, 901 Morrell, J. I., 1002, 1003, 1004, 1007, 1009, 1024 Morris, E. T., xviii, xl Morris, J. B., 54, 55, 64, 65, 67, 71 Morris, J. B., 551, 567 Morris, J. C., 513, 522 Morris, J. J. 992, 995 Morris, N., 223, 228 Morris, N. M., 363, 379 Morris, P., 884, 900 Morris, R., 916, 930 Morris, R. G., 354, 380, 410, 413, 420, 421, 433, 435 Morris, R. W., 712, 713, 725 Morris, S., 444, 452, 454, 456 Morrison, D. A., 440, 456 Morrison, E. E., 18, 21, 23, 24, 26, 27, 30–37, 42, 46, 48, 130, 136 Morrison, G. R., xxxi, xl, 732, 748, 756, 867–871, 873, 875, 877 Morrison, J. H., 483, 498

Morrison, K., 491, 499 Morrison, R., 525, 533, 544 Morrison, R. R., 406, 408 Morris-Wiman, J., 643, 649, 670, 674, 767, 781, 939, 954 Morrow, G. R., 551, 567 Morrow, L. A., 525, 538, 543, 546 Morrow-Tesch, J., 315, 325 Morse, C. C., 583, 590 Morse, J. R., 679, 680, 685, 686, 700, 702, 948, 954, 956 Morse, J. W., 553, 562, 570 Morshead, C. M., 616, 620, 623, 625, 128, 136 Morton, C. C., 848, 860 Morton, J. R. C., 369, 379 Morton, R., xix, xl Morton, R. P., 786, 791, 803 Morton, T. H., 208, 209, 225, 421, 424, 428 Morton, W. E., 539, 545 Morys, J., 172, 173, 174, 178 Moses, A., 341 Moses, A. M., 471, 474, 477 Mosges, R., 444, 458 Mosheva, T., 492, 493, 501 Moskat, L. J., 794, 802 Moskowitz, H. R., 211, 227, 214, 218–220, 226, 227, 424, 431, 602, 610, 690, 704, 740, 756, 807, 822, 882, 883, 886, 892, 897, 898, 900, 901 Mosnik, D., 480, 482, 498 Moss, O. R., 392, 399, 582, 591 Moss, R. L., 57, 61, 69, 971, 978 Moss, R. W., 527, 546 Mossman, C. A., 357, 379 Mossman, K. L., 130, 136, 891, 900 Mosso, A., 252, 271 Mosso, A., xxviii, xl Moss-Salentijn, L., 9, 16 Mostafa, B. E., 443, 456 Motoki, D., 887, 900 Motokizawa, F., xxiv, xxxvi, xl, 170, 179, 243, 247 Mott, A. E., 232, 244, 440, 445, 456, 463, 467, 472, 476, 606, 611, 632, 638, 783, 784, 786–793, 800–803, 952, 953, 955 Mottin, Y., 495, 500, 960, 961, 962, 966 Moudrian, L., 142, 163 Moulton, D. G., xvi, xxiv, xxix, xxvii, xxxi, xxxvi, xxxvii, xxxiv, xxxix, xl, xliii, 61, 66, 75–76, 89, 93–95, 98, 103, 111, 112, 145, 158, 186, 197, 204, 206, 213, 220, 227, 228, 353, 373, 386, 391, 392, 397, 398, 399, 615, 623, 625, 801, 841, 985, 999 Mouly, A. M., 174, 175, 176, 179, 187, 188, 190, 191, 195, 198, 410, 413, 433 Mountcastle, V. B., 913, 930 Moura, L. A., 81, 86

Author Index Mouradian, L. E., 171, 179 Mouton, P. R., 618, 623 Moy, K., 491, 492, 493, 501 Moyse, A., 318, 321, 326 Moyse, E., 96, 98, 101, 103, 108, 109, 111, 151, 156, 183, 184, 195 Mozell, M. M., xxiv, xxxi, xl, 55, 69, 185, 201, 216, 227, 266, 267, 270, 271, 273, 392, 394, 395, 399, 400, 418, 438, 442, 445, 448–450, 452, 453, 455–457 Mozes, S., 919, 929 Mrksich, M., 103, 108 Mroueh, A., 472, 476 Mrowinski, D., 230, 234, 238, 244, 245, 247 Mucignat-Caretta, C., 351, 359, 369, 374, 379, 382, 976, 978 Mueller, C. A., 585, 588 Mueller, K. L., 709, 710, 711, 724, 798, 798, 848, 858 Mufson, E. J., 242, 247, 488, 496, 680, 686, 697, 702 Mufti, W. A., 330, 340 Mugford, R. A., 61, 66, 352, 353, 361, 373, 379, 380 Mugnaini, E., 151, 160 Mugnaini, M. T., 30, 42 Muhlhauser, B., 311, 317, 323 Muir, C., 367, 375, 383 Mulbauer, B., 833, 842 Mulder, I., 340 Mulder, N. H., 891, 900 Mullani, N. A., xxviii, xxxviii, xliv, 253, 273, 600, 608, 610 Mullenbach, V. A., 886, 897 Muller, P., 495, 500, 960, 961, 962, 966 Muller, T., 509, 514, 521 Müller, A., 486, 500 Müller, F., 27, 39, 45, 46, 80, 83, 86, 89, 722, 729 Müller, H., 152, 161, 510, 512, 522 Müller, J., 653, 784, 802, 991, 998 Muller-Ruchholtz, W., 319, 320, 323, 341 Müller-Schwarze, C., 356, 380 Müller-Schwarze, D., xvi, xxix, xxxix, xl, 341, 346, 353, 356, 373, 376, 380, 399, 417, 434, 660, 675, 969, 978 Muller-Spahn, F., 508, 509, 521 Mulling, W. J., 857, 859 Mullins, J. J., 645, 649 Mullol, J., 54, 71 Mulvaney, B. D., 35, 43, 46, 120, 136 Mumby, D. G., 414, 433 Mumm, J. S., 96–98, 103, 109, 111, 121, 133 Mummery, C., 101, 113 Muneoka, Y., 661, 674 Munger, S. D., 80–81, 89 Munoz, S. J., 889, 892, 893, 896 Munstedt, R., 284, 285, 294 Munz, H., 1001, 1003, 1023 Murakami, G., 942, 945, 956, 957

1073 Murakami, S., 942, 956, 957, 1003, 1016, 1018, 1023 Muramatsu, H., 664, 677 Muraoka, S., 416, 428 Murchison, C., 205, 218, 228 Murkami, G., 946, 956 Murofushi, T., 485, 496 Murowchick, E., 909, 919, 924 Murphy, C., xxxii, xxxiii, 204, 216, 224, 227, 232–240, 244–247, 256, 269, 271, 329, 342, 461–463, 466–467, 472–473, 473–477, 479–483, 489, 496, 498, 500, 501, 715, 725, 736, 757, 786, 789, 793, 799–802, 832, 844, 884, 889, 896, 900, 982, 987, 994, 996, 999 Murphy, D., 419, 427 Murphy, J. M., 885, 902 Murphy, Jr., G. M., 513, 522 Murphy, K., 267, 269 Murphy, S. P., 26, 28, 33, 41, 145, 155 Murphy, M. R., xxx, xl Murphy-Ellison, T., 816, 822 Murray, A., 661, 662, 674, 909, 930 Murray, A. B., 909, 930 Murray, E. A., 414, 433 Murray, J., 513, 523, 909, 930 Murray, K., 621, 623 Murray, M. J., 909, 930 Murray, N. J., 909, 930 Murray, R., 102, 112 Murray, R. D., 642, 649 Murray, R. G., 661, 662, 663, 674, 773, 779 Murray, T. K., 170, 178, 191, 196, 417, 418, 431 Murrell, W., xxvii, xxxvi Murrell, W. G., xxvii, xxxix, 36, 46, 93, 98, 103, 110, 111 Murren, K., 482, 498, 509, 513, 520 Murtagh, J. A., 439, 441, 458 Murzi, E., 680, 692, 703 Muscat, R., 867, 870, 879 Muske, L., 1001–1003, 1005, 1007, 1023, 1024 Mutel, V., 723, 728 Muto, H., 658, 673 Myer, D. L., 1001, 1002, 1023, 1025 Myers, A. L., 211, 227 Myers, L. J., 471, 475 Myers, M. D., 887, 900 Myers, R. H., 602, 611, 613 Myers, W. E., 783, 790, 794, 795, 801 Mygind, N., 12, 16, 444, 456 Mykytowycz, R., xvi, xxix, xl Näätänen, R., 230, 236, 248 Nachman, M., 732, 756, 875, 877, 911, 916, 918, 930 Naclerio, R.M., 7, 12, 14, 15 Nada, O., 662, 674 Nadai, T., 553, 554, 561, 562, 571 Nadeau, A., 888, 898

Nadi, N. S., 103, 111, 147, 148, 152, 160, 618, 625 Nadimi, H., 658, 676 Nadin, A., 509, 521 Naessen, R., 1, 16, 30, 36, 37, 38, 46, 98, 111, 339, 342 Nafziger, A. N., 887, 900 Nagahara, M., 259, 269, 271 Nagahara, Y., xxvii, xl, 93, 96, 111, 615, 625 Nagai, F., 59, 71 Nagai, H., 709, 710, 729 Nagai, T., 746, 756, 767, 775, 782 Nagai, Y., 553, 568, 666, 676 Nagamachi, S., 601, 611 Nagao, H., 130, 135, 145, 160 Nagao, Y., 887, 889, 900 Nagasaki, N., 359, 380 Nagata, O., 242, 248, 259, 266, 272 Nagato, T., 658, 674, 766, 781 Nagatsu, I., 124, 136, 140, 151, 152, 158, 183, 184, 197, 618, 624 Nagatsu, T., 512, 522 Nagawa, F., 182, 200 Nagel, J. S., 600, 602, 611, 612 Nagel, W.A., 218, 227, 233, 248 Nagilla, R., 549, 550, 561, 563, 570 Nagode, J., 412, 428 Naguro, T., 23, 24, 30, 36, 45, 46 Nagy, J. I., 123, 137 Nagy, Z., 482, 499, 513, 522 Nahon, D. F., 808, 822 Naim, M., xxvi, xliii, 710, 712, 715, 716, 725, 728, 730 Naito, K., 451, 452, 453, 456 Naito, Y., 720, 728 Naito-Hoopes, M., 884, 900 Najlerahim, A., 510, 520 Nakade, S., 81, 87 Nakae, Y., 826, 828, 830, 843 Nakagawa, K., 952, 957 Nakagawa, R., 359, 380 Nakahara, K., 887, 900 Nakai, Y., 102, 113, 585, 590 Nakakuki, K., 551, 557, 570 Nakamura, H., 26, 34, 49, 124, 138, 463, 477, 946, 957 Nakamura, K., 644, 649 Nakamura, O., 644, 649 Nakamura, T., 24, 26, 27, 43, 46, 76, 79, 89 Nakamura, Y., 713, 730 Nakanishi, S., 145, 164, 186, 201, 717, 727 Nakano, T., 367, 376, 439, 456 Nakano, Y., xxxi, xxxvii, 26, 28, 34, 37, 38, 43, 48, 49, 124, 137, 138, 463, 477 Nakao, M., 713, 729 Nakashima, K., 232, 242, 248, 259, 266, 272 Nakashima, M., 187, 198 Nakashima, T., 1, 16, 37, 38, 46, 98, 111, 311, 325, 622, 625, 631, 637

1074 Nakashiro, Y., 942, 956 Nakata, J., 585, 590 Nakaya, K., 713, 730 Nakazawa, M., 789, 800, 960, 962, 965 Nakelski, J. S., 362, 374 Nalbone, V. P., 441, 454 Nalcioglu, O., 601, 610 Naldi, F., 561, 567 Nalwa, V., 680, 701 Nalwalk, T., 406, 408 Namiki, M., 554, 570 Namork, E., 554, 568 Nance, W. E., 332, 342, 848, 860 Nanthakumar, E., 710, 729, 791, 798, 803 Nanto, H., 298, 307 Nappi, G., 236, 247 Narfstrom, K., 552, 558, 569 Narita, M., 551, 570 Narla, R. K., 119, 133 Naruse, I., 517, 522 Naser, M. A., 600, 611 Nash, G., 104, 108, 126, 132 Nash, M. L., 893, 901 Nash, S., 923, 930 Nasr, F., 153, 163 Natelson, B., 526, 529, 538, 544 Nath, A., 511, 515, 520 National Cancer Institute 552, 570 National Dairy Council 883, 900 Natoli, J. H., 364, 381 Natynczuk, S. E., 351, 380, 341 Naus, A., 576, 590 Nauta, W. J. H., 680, 702 Navarrini, A., 78, 87 Navazesh, M., 887, 895, 950, 954 Naylor, G. J., 951, 956 Neale, M. C., 330, 331, 341, 342 Neaton, J. D., 886, 896 Nebert, D. W., 57, 58, 61, 62, 64, 65, 68–71, 552, 559, 567 Neduvelil, J. G., 509, 521 Nee, L. E., 480, 500, 509, 522 Neely, G., 210, 227, 856, 860 Nef, P., 57, 58, 61, 69, 70, 76, 88, 559, 569, 570 Neff, S., 124, 137, 505, 506, 509, 523, 563, 572 Neff, S. R., 145, 158 Neff, W. D., 747, 748, 754 Negishi, M., 57, 62, 64, 65, 68, 69, 552, 559, 567 Negrao, N., 165, 179 Negrin, N. S., 483, 501 Negus, V., 21, 46 Neidermeier, W., 891, 900 Neill, J. D. 976 Neisewander, J. L., 421, 433 Neiswanger, K., 848, 850, 856, 860 Nejad, M. S., 741, 756 Nejat-Bina, D., 192–194, 200, 410, 411, 436 Nelson, A., 662, 663, 672 Nelson, C. J., 532, 546

Author Index Nelson, D. R., 57, 71 Nelson, G., xxv, x, 714, 716, 717, 723, 728 Nelson, L. M., 746, 755 Nelson, R. H., 450, 452, 455 Nelson, R. J., 422, 428, 618, 625 Nelson, S. P., 942, 956 Nemes, A., xxvi, xxxiii, 24, 26, 35–37, 41, 49, 78, 79, 85, 89, 126, 130, 131, 135, 138, 146, 157, 160, 164, 289, 294, 296, 307, 354, 379 Nemetschek-Ganssler, H., 662, 674 Nemitz, J. W., 188, 198 Nemoto, Y., 78, 83, 88, 89 Nespoulous, C., xxiv, xxxiv Nesztler. C., 791, 801 Nethercott, J. R., 528, 545 Neubauer, U., 241, 248 Neugebauer, R., 516, 523 Neumann, H., 830, 844 Neumann, I., 416, 429 Neumann, I. D., 367, 382 Neumann, P., 343 Neumeister, A., 472, 477, 619, 626 Neutra, R. R., 527, 530, 532, 545 Nevin, J. A., 354, 380, 387, 394, 399, 409, 410, 433 Nevitt, G. A., 426, 428, 433 Nevo, E., 394, 395, 398, 404, 407 Newbill, E. T., 596, 611 Newhouse, M. G., 215, 225, 792, 800 Newlin, D. B., 538, 539, 543, 545 Newman, D. R., 837, 842 Newman, H. H., 332, 342 Newman, K. S., 422, 433 Newman, M., xxvii, xl, 102, 103, 111 Newman, M. C., 486, 499 Newman, M. M., 923, 930 Newman, M. P., 36, 46 Newman, N. M., 600, 611 Newman, P. D., 505, 506, 509, 523 Newman, S., 495, 495 Newman, S. W., 361, 380, 422, 432, 619, 626 Newton, C. G., 452, 454 Ng, B. A., 441, 442, 444, 454, 457 Ng, S. H., 940, 957 Ngai, J., xxvi, xxxiv, 24, 26, 27, 32, 35, 36, 41, 44, 48, 76, 78, 80–81, 86, 90, 125, 126, 130, 131, 132, 134, 296, 306, 969, 979 Ngo, H., 413, 432 Nguyen, A., 554, 563, 569 Nguyen, T. 111, 109, 288, 291, 294, 773, 782 Nguyen, V., 187, 197 Niccoli-Waller, C. A., 482, 499 Nickel, A. A., 946, 956 Nickell, W., 102, 103, 110 Nickell, W. T., 149, 152–154, 157, 158, 160, 161, 183, 184, 187, 197, 198, 558, 560, 571, 572 Nicklatsch, J., 789, 799

Nicolaides, N., 339, 342 Nicolaidis, S., 886, 900 Nicolas, P., 554, 572 Nicolaus, L. K., 920–923, 928, 930 Nicolelis, M. A., xvi, xlii, 670, 673 Nicolini, M., 551, 555, 573 Nicoll, R. A., xxiv, xl Nieburgs, A., 1003, 1007, 1012, 1025 Niedetsky, C., 689, 701 Niegowska, J., 891, 901 NIEHS (National Institute of Environmental Health Sciences) 525, 546 Nielsen, G. D., 991, 992, 995, 997, 999 Nielsen, H., 444, 457 Nielsen, L. H., 596, 608 Nielsen, L. P., 444, 456, 457 Nielsen, S. L., 888, 894 Nielsen, S. S., 910, 930 Nielsen, T. G., 444 Nielson, M. H., 12, 16 Nies, A. S., 915, 933 Niesing, J., 923, 926 Nieto-Sampedro, M., xxvii, xli, 105, 107, 111 Nieuw Amerongen, A. V., 645, 646, 647, 650 Nieuwenhuys, R., 139, 161 Niewind, A., 857, 860 Nigam, S. K., 81, 86 Nigrosh, B. J., 354, 380, 387, 392, 394, 399, 400, 409, 410, 433, 436 Niijima, A., 888, 900, 915, 930 Nijjar, R., 832, 844 Nikula, K. J., 54, 56, 57, 64, 70–73, 582, 583, 590 Nillius, S. J., 554, 563, 565 Nilson, L-G., 911, 914, 917, 927 Nilsson, B., 831, 844 Nilsson, C. G., 539, 544 Nilsson, E., 621, 623 Ninomiya, T., 806, 822 Ninomiya, Y., 713, 714, 716, 717, 720, 725–728, 741, 747, 755, 756, 791, 802, 875, 877 Nir, Y., 557, 570 Nisbett, R. E., 828, 844 Nishihira, S., 440, 457 Nishijo, H., 644, 649, 688, 702 Nishikawa, H., 462, 476, 629, 633, 637 Nishimoto, K., 581, 589 Nishimura, M., 507, 512, 521 Nishimura, T., 124, 136, 884, 898 Nishino, H., 174, 178, 182, 197, 680, 688, 689, 701, 702, 704 Nishio, S., 583, 590 Nissenbaum, J. W., 862, 875, 877 Nissenson, R. A., 291, 294 Nitsch, C., 105, 112 Nitschke, K. D., 583, 588 Nitz, S., 299, 307 Njai, D. M., 642, 650 No, C., 472, 477, 619, 626

Author Index Nobel, E. P., xxx, xxxv Noble, A. C., 644, 649, 885, 900 Nochlin, D., 509, 513, 522 Noda, Y., 359, 380 Noé, J., 26, 46, 48, 709, 710, 729 Noel, T., 773, 780 Nofre, C., 713, 730 Nogawa, T., 259, 269, 271 Noguchi, M., 82, 87 Noguchi, S., 643, 649 Noirot, E., 309, 325, 357, 362, 376 Noller, K. L., 923, 928 Nolte, C., 664, 674 Nolte, D. L., 315, 325 Nomura, H., 24, 26, 41, 711, 716, 728 Nomura, S., 661, 674 Nonneman, A. J., 421, 433 Norcia, M. P., 450, 455 Nordborg, C., 93, 108 Nordin, S., 204, 215, 227, 235, 236, 247, 271, 342, 462, 466, 477, 480–482, 489, 495, 500, 525, 544, 786, 791, 801, 802 Norgren, R., xxiv, xl, 644, 649, 667, 674, 679, 680, 682–686, 688, 691, 700–704, 731, 732, 734, 736, 737, 741, 743, 745, 755, 756, 758, 774, 779, 828, 842, 861, 863, 865, 866, 868, 870, 877, 878, 913–914, 916–918, 924, 927, 928, 930–933, 948, 954, 956, 1002, 1003, 1007, 1024 Norita, M., 27, 44 Norlin, E. M., 24, 36, 46 Norman, A., 102, 103, 110 Norman, A. B., 149, 158, 160, 184, 197, 198, 558, 560, 571, 572 Norman, A. W., 662, 673 Norris, D. M., 732, 756 Norris, M. B., 644, 649 Norris, M. L., 365, 380 Norrison, W., xxxi, xl, 867–871, 873, 877 North, A. C. T., 970, 976 North, M., 710, 729, 791, 798, 803 North, W. G., 910, 930 Northcutt, R. G., 165, 168, 179, 670, 671, 767, 778, 1003, 1005, 1022 Northup, J. K., 81, 89 Norton, L., 790, 802 Nosrat, C. A., 101, 111, 670, 674, 763, 765, 766, 771–773, 781 Nosrat, I. V., 670, 674 Nothacker, H. P., 1004, 1022 Notta, A., 629, 638 Novak, R., 56, 64, 70, 71 Novelly, R. A., 268, 270, 494, 496, 499, 949, 954 Novikov, S., 417, 436 Novin, D., 915, 929 Novis, B. H., 887, 900 Novoselov, S. V., 54, 71 Novoselov, V. I., 54, 71

1075 Novotny, M., 351, 361, 362, 377, 380, 381, 970–973, 977, 978 Nowak, R., 320, 323 Nowell, N. W., 352, 361, 333, 379, 380 Nowlis, G. H., 732, 748, 752, 756, 828, 844, 873, 875, 878 Nowycky, M. C., 148, 163, 184, 198 NRC (National Research Council), 525, 527, 528, 530, 536, 546 Nsegbe, E., 390, 392, 397, 399 Nuding, S. C., 746, 756 Nuez, J. M., 99, 113 Numminen, J., 442, 454 Nunes, R. J., 560, 571 Nunez, A. A., 920, 930 Nunez, J. M., 28, 48, 130, 137, 146, 164, 560, 572 Nurcombe, V., 104, 105, 110, 112, 125, 137, 620, 624 Nürnberg, B., 716, 728 Nurse, R. J., 857, 860 Nussbaum, R. L., 514, 522 Nustede, R., 887, 898 Nyakas, C., 170, 177, 359, 380 Nyakas, C. S., 367, 376 Nyby, J., 344, 422, 423, 428, 431, 433, 434 Nysenbaum, A. N., 885, 900 O’Cain, C. F., 441, 458 O’Callaghan, J. P., 583, 589 O’Connell, R. J., 392, 400, 404, 408 O’Connor, E. F., 910, 930 O’Connor, R., 762, 763, 781 O’Connor, E. F., 910, 930 O’Connor, R., 762, 763, 781 O’Doherty, J., 698, 699, 700, 702, 797, 802 O’Rahilly, R., 1002, 1024 O’Rourke, N. A., xxviii, xl Oakley, B., 657, 659, 664, 666, 667, 670, 671, 673, 674, 677, 742, 756, 764, 767–769, 771, 773, 779–782 Obara, N., 32, 48, 666, 676 Obata, E., 192, 198 Obata, K., 121, 135, 560, 570 Ober, C., 339, 341, 342 Oberlander, T. F., 828, 840 Oberling, P., 174, 175, 177, 421, 429 Oberto, M., 101, 111 Obrebowski, A., 960, 962, 963, 965, 966 O’Brien, C. O., 885, 903 O’Brien, J., 365, 366, 375 O’Brien, M. H., 889, 897 O’Brien, P. C., 485, 487, 496, 501, 923, 928 O’Brien, T. F., 621, 625 Obtulowicz, W., 887, 898 Obuta, K., 972, 978 O’Connell, B., 509, 522 O’Connell, J., 241, 246 O’Connell, R. J., 364, 381, 392, 400, 404, 408, 558, 559, 570, 967, 968, 971, 977, 978

O’Connor, M. J. 342, 493, 494, 498, 501 O’Craven, K. M., 258, 269 O’Doherty, D., 268, 270 O’Doherty, J., 193, 198, 259, 264, 266, 271 Odoms, K., 101, 103, 111, 619, 626 Oelkers, R., 238, 245 Oelschläger, H. A., 311, 325, 1002, 1022 Oertel, W. H., 151, 160 Oestreicher, A. B., 36, 46, 125, 138 Oettingen, A. J., xxi, xliv Oftung, F., 554, 568 Ogata, T., 34, 46 Ogawa, H., 101, 102, 108, 446, 457, 661, 663, 672, 673, 736, 740, 741, 745, 755, 756, 757, 783, 797, 801, 916, 918, 930, 933 Ogawa, K., 24, 46, 81, 87, 120, 136 Ogawa, S., xxv, xvii, xxxv, xl, 254, 269, 271 Ogawa, T., 629, 638 Ogawa, Y., 185, 198 Ogden, M. W., 204, 228 Ogden-Ogle, C. K., 662, 672, 713, 726 Ogle, W., xxiii, xl, 629, 638, 959, 966 O’Grady, R. S., 410, 434 Ogren, S. D., 538, 546 Ogura, J. H., 439, 441, 458 Ogura, T., 712, 722, 727, 728 Oh, J. D., 153, 161 Oh, S. H., 55, 71 Ohara, I., 887, 900 Ohara, S., 48 O’Hare, L., 304, 306 Ohashi, Y., 585, 590 Ohba, Y., 643, 649 Ohguro, H., 83, 89 Ohki, M., 450, 456 Ohkubo, K., 54, 71 Ohkura, S., 423, 431 Ohloff, G., 276, 277, 278, 291, 292, 294 Ohlott, P., 211, 227 Ohlson, C. G., 586, 590 Ohlsson, A., 829, 845 Ohm, T. G., 152, 161, 510, 512, 522, 563, 570, 600, 611 Ohmen, J. D., 717, 724 Ohmura, S., 662, 675 Ohnishi, T., 601, 611 Ohno, I., 24, 46, 120, 136 Ohoka, E., 451, 452, 456 Öhrwall, H., xxi, xxii, xl, 805, 816, 822 Ohshima, H., 659, 675 Ohta, M., 232, 248 Ohta, Y., 33, 46 Ohtani, I., 27, 46 Ohtani, M., 712, 729 Ohtsuka, K., 946, 956 Ohyama, M., 24, 46, 55, 71, 120, 136, 581, 589 Oja, S. S., 149, 161 Ojima, H., 142, 153, 160, 161, 168, 179, 188, 198

1076 Ojima, K., 659, 675 Oka, T., 580, 584, 590, 643, 649 Oka, Y., 1002, 1003, 1005, 1024 Okada, H., 54, 71 Okada, K., 266, 267, 272 Okada, Y., 243, 248, 712, 719–722, 728, 729 Okamoto, K., 512, 522 Okamura, H., 27, 46 Okamura, Y., 1003, 1007, 1018, 1022 Okano, M., 30, 46 Okano, S., 204, 225 Okano, T. M., 76, 89 Okazaki, K., 83, 88 O’Keefe, G. B., 865, 878 O’Keefe, J., 419, 427 Okiishi, C., 535, 544 Okinaga, S., 366, 372 Okita, D. K., 553, 569 Okiyama, A., 907, 930 Okoro, E. O., 832, 844 Okoyama, S., 153, 161 Okuda, M., 54, 71 Okuda, Y., 785, 789, 790, 791, 804 Okumura, N., 102, 105, 110 Okuyama, A., 554, 570 Oland, L. A., 129, 130, 136 Olanow, C. W., 555, 567 Olanow, W., 602, 609 Olbe, L., 888, 902 O’Leary, D., 889, 897 O’Leary, D. S. 269, 491, 496 Oles, M., 287, 293 Olesky, J., 887, 898 Oliver, C., 483, 500 Oliver, D. R., 440, 455 Oliver, G., 116, 136 Oliver, K. R., 550, 557, 564, 551, 570 Oliver, R. J., 951, 955 Oliverio, A., 356, 372 Olivier, A., 268, 270, 424, 431 Olivier-Martin, M., 829, 841 Olkkola, K. T., 553, 562, 572 Ollman, B. G., 204, 223, 995, 996 Olmstead, R., 554, 517 Olmsted, J. M. D., 670, 675 Olsen, R., 832, 845 Olson, J. M., 848, 850, 856, 860 Olson, L., 101, 111, 670, 674, 763, 766, 771–773, 781 Olson, R., 893, 902 Olsson, H., 647, 649 Olsson, M. J., 218, 223, 224, 227, 815, 822 Olsson, Y., 552, 557, 558, 560, 569 Olwin, B. B., xxvii, xxxv, 101, 102, 108, 119, 132 O’Mahony, M., 209, 211, 227, 644, 648, 816, 822, 884, 897 Omata, Y., 82, 87 O’Neill, C. A., 54, 67 Ono, S., 266, 271 Ono, T., 688, 702

Author Index Onoda, K., 938, 948, 949, 956 Onoda, N., 145, 161, 174, 180, 182, 183, 185, 192, 197, 198, 263, 273, 622, 625, 972, 979 Onodera, H., 553, 569 Onofri, M., 621, 625 Oomura, Y., 174, 178, 182, 197, 680, 688, 690–692, 696, 701–704, 910, 932 Ooshima, Y., xxiv, xliii, 188, 192, 200, 421, 437 Öpalski, A., xxviii, xl Ophir, D., 31, 46, 124, 136, 341, 445, 450, 457, 467, 473, 475, 477, 491, 492, 498, 891, 900 Ophir, I., 910, 929 Oppenheim, B. A., 305, 306 Oppenheim, R. W., 124, 136 Oppenheimer, M., 827, 842 Oppenheimer, S. M., 686, 702 Oprysk, D., 5, 15, 441, 442, 453 Optican, L. M., 223, 225 Oram, N., 832, 842, 843 Orbach, J., 410, 411, 435 Orci, L., 27, 41, 56, 67 Ordy, J. M., 216, 228, 803 Ore, K., 971, 979 Orgeur P., 311, 312, 315–318, 323, 325, 326, 343, 355, 369, 370, 374, 381, 423, 426, 432, 825, 833, 838, 845 Oriol, R., 123, 131 Orkand, P. M., 34, 47 Orlandi, M., 216, 228 Ormsby, C. E., 918, 930 Orndorff, M. M., 216, 224, 234, 245, 984–987, 991, 997 Orntoft, S., 444, 457 Orola, C. N., 712, 729 Orona, E., 142, 161, 163. 171, 179, 185, 198 Oropeza, V., 765, 780 Orr, G., 129, 136 Orr, M. R., 917, 929 Orringer, D., 887, 897 Orr-Urtreger, A., 101, 111 Orth, K., 24, 44 Ortmann, R., 311, 325 Orvisky, E., 645, 650 Osada, T., 351, 382 Osanai, R., 485, 496 Osawa, K., 62, 68 Osborn, M., 32, 48, 124, 138 Osborne, B., 913, 927 Osborne, D., 603, 611 Osborne, J., 146, 157 Oscar-Berman, M., 492, 500 Osculati, F., 659, 661, 675, 677 Osepian, V. A., 831, 844 Oshima, T., 462, 476 Oshima, T., 629, 633, 637 Oshita, M. H., 187, 201 Osnos, M., 892, 901 Ossebaard, C. A., 788, 794, 802

Ossenkopp, K-P., 910, 915, 917, 920–921, 926, 927, 929, 930 Oster, H., 483, 498, 826, 827, 844, 885, 886, 901 Ostergren, A., 559, 565 Osterhammel, P., 230, 243, 248 Ostlie, N. S., 553, 569 Ostrom, K. M., 784, 787, 799, 949, 954 O’Sullivan, R. L., 472, 477 Oswalt, G. L., 310, 325 Otagawa, T. 306, 308 Otaki, J. M., xxv, xlv, 78, 91, 145, 164, 287, 294, 396, 400, 551, 573 Otsuji, A., 553, 568 Otsuka, M., 910, 932 Otsuka, S. I., 887, 900 Ottenweller, J., 526, 544 Otterness, D. M., 59, 73 Ottersen, O. P., 149, 151, 152, 156, 161, 162 Otto, D., 528, 530, 536, 545 Otto, D. A., 239, 248 Otto, T., 188, 191, 198, 411–415, 417, 425, 426, 428, 429–431, 433, 434, 438, 619, 624 Otto-Bruc, A., 76, 81–84, 89 Ottoson, D., xvi, xxiv, xl, 76, 89, 146, 161, 230, 248 Otvos, Jr., L., 509, 521 Oude Ophuis, P. A. M., 807, 808, 811, 821 Ouellet, J. V., 918, 925 Overbosch, P., 211, 227 Overgaard, C., 829, 844 Overstreet, D. H., 538, 539, 544, 546 Ovesen, L., 786, 802 Owens, D. M., 56, 63, 68 Owens, I. S., 58, 70 Owens, J. G., 392, 399 Oxenboll, B., 554, 563, 568 Ozaki, N., 916, 933 Ozato, K., 620, 624 Ozeki, M., 737, 738, 746, 752, 756 Ozenberger, J. M., 450, 457 Ozirskaya, E. V., 148, 155 Paavilainen, P., 230, 248 Pace, U., xxvi, xl, xliii, 76, 79–80, 89, 712, 715, 729 Paczynski, M., 366, 381 Padilla, G. V., 882, 900 Padilla, M., xxxii, xxxv, 483, 497 Paepke, A. J., 321, 327, 343 Paepke, A. J. Pagano, S. F., 621, 625 Pager, J., 169, 175, 177, 185, 187, 196, 198, 199, 421, 426, 434 Pahor, A. L., 439, 457 Pahwa, R., 484, 486, 495, 496 Paik, S. I., 14, 16, 37, 46, 472, 477 Pain, J. F., 420, 434 Pajno-Ferrara, F., 642, 650 Palacios, J. M., 152, 161

Author Index Palczewski, K., 76, 81–84, 89 Paling, M. R., 594, 597, 611 Palko, M. E., 762, 780 Palladius, R. T. A., xix, xli Pallanch, J. F., 451, 454 Palm, J., 444, 455 Palmateer, S. D., 404, 407 Palmer, C. R., 450, 454 Palmer, L. G., 718, 726, 729, 776, 779 Palmer, R. L., 921, 926 Palmer, T. D., 62, 72, 620, 624 Palmerino, C. C., 419, 420, 434 Palmiter, R. D., 76, 85 Palo, J., 505, 506, 512, 521 Palouzier-Paulignan, B., 188, 195 Paluch, R., 887, 896 Pan, J. B., 620, 624 Pan, L., 762, 779 Panattoni, G., 55, 56, 68, 561, 567 Pandraud, L., 443, 455 Pandya, D. N., 680, 695, 700, 702, 704 Pangborn, R. M., xvi, xli, 206, 227, 644, 649, 784, 787, 802, 807, 819, 822, 882, 884–887, 892, 895, 899, 900–903 Panhuber, H., 183, 194, 218, 220, 223, 226, 228, 391, 393, 394, 399, 400, 471, 476, 892, 899 Paniccia, R., 62, 71 Paniello, R. C., 443, 457 Panizza, B., 652, 666, 675 Pankey, J., 311, 325 Pannen, B. H., 82, 85 Panov, A. V., 564, 565 Pansky, B., 10, 15 Pantel, M. S., 827, 828, 829, 840 Pantelis, C., 491–493, 496, 500 Panter, K. E., 315, 325 Panzanelli, P., 149, 150, 162 Paolesse, R., 299, 306 Paolini, A. G., 148, 153, 161, 170, 179, 418, 434 Papadimitriou, A., 512, 514, 522 Papadopoulos, S., 525, 528, 543 Papapetropoulos, T., 509, 514, 522 Papert, S., 296, 307 Papiernik, E., 340, 341 Papp, E. S., 341 Papp, M., 867, 870, 879 Pappas, D., 946, 955 Pappert, E. J., xxxii, xxxv, 588 Parad, R. B., 343 Parada, L. F., 762, 771, 780 Paradise, J. L., 445, 455, 467, 473, 475 Paradiso, M. A., 347, 372 Paradiso, S. 269, 491, 496 Paran, N., 661, 675 Paranjape, J., 340 Parati, E.A., 621, 625 Pardo, J. V., 173, 174, 180, 204, 227, 255, 258, 259, 261, 264, 266, 271–273, 424, 438, 698, 704, 705, 797, 803, 804

1077 Parent, A., 80, 89 Parfet, K. A. R., 315, 325 Parfitt, D. J., 80, 90 Parhar, I. S., 1002, 1003, 1024 Paris, J., 660, 671 Parise, C. M., 554, 563, 571 Parisi, P., 342 Park, C. I., 450, 458 Park, D., 103, 109 Park, H. J., 466, 476 Park, H. S., 893, 895 Park, J. S., 773, 782 Park, S., 491, 500 Parker, G. H., xvi, xli, 18, 24, 46, 667, 670, 675, 982, 999 Parker, L. A., 736, 756, 865, 878, 907, 910–911, 914–915, 917, 926, 930, 931 Parker, P. E., 359, 380 Parkes, A. S., 346, 365–367, 380, 1003, 1024 Parkinson, D., 184, 195 Parkinson, J., 484, 500 Parmentier, M., xxv, xli Parmigiani, S., 362, 373 Parnavelas, J. G., 144, 157 Parola, S., 509, 514, 518, 521 Parr, L. W., 849, 860 Parrott, D. M. V., xxix, xxxiv, 365, 374 Parson, W., 340 Parsons, D. S., 6, 15 Parsons, P. A., 334, 342 Parys, J. B., 26, 41 Pascual-Leone, A., 600, 611 Paskevich, P. A., 602, 611 Pasquali, A., xxix, xxxix, 320, 324, 349, 356, 378, 404, 408 Passali, D., 443, 457 Passe, D., 392, 399 Passe, D. H., 389, 399 Passey, J., xxix, xli, 403, 407 Passingham, R. E., 682, 700 Passy, J., 386, 399 Pastakia, B., 602, 612 Pasterkamp, R. J., 616, 625 Patanella, J. E., 550, 567 Patarca, R., 538, 543 Patel, S., 553, 563, 566 Patel, T. D., 764, 781 Patel, U., 94, 97, 103, 110 Patel, L. S., 1003, 1006, 1023, 1024 Paterno, G. D., 152, 160 Paternostro, M. A., 98, 111, 143, 161, 620, 625, 626 Patil, M., 190, 196, 413, 430 Patlak, C. S., xxviii, xliii Patneau, D. K., 190, 200 Patra, A. L., 447, 456 Patrikiou, A., 514, 522 Patte, F., 815, 822 Patte, R., 218, 227 Patten, B. M., 291, 293 Patterson, D. L., 54, 55, 71

Patterson, M. Q., 219, 227, 789, 803 Patterson, R. L., 346, 379 Patterson, S. L., 105, 113 Pattison, R. M., 936, 950. 954 Patton, H. D., 861, 878 Paul, C. A., 412, 437 Paul, G., 762, 781 Paul, M., 893, 898 Pauli, E., 212, 215, 226, 232, 234, 236, 238, 239, 244, 244, 246, 247, 444, 445, 455, 462–463, 475, 476, 485, 491, 494, 496, 498 Paulin, F., 341 Paulin, J. M., 886, 902 Pauling, L., xxviii, xli, 254, 271 Paulsen, C. A., 472, 477 Paulsen, J. S., 482, 489, 496, 498, 500 Pause, B. M., 233, 234, 236, 239, 240, 247, 248, 350, 380, 469, 474, 476 Pavirani, A., 551, 567 Pavlov, I. P., xxix, xli Pawlik, G., 241, 248 Paxinos, G., 165–169, 171–173, 176, 179, 180, 254, 258, 259, 261–263, 266, 268, 271, 272, 420, 426, 736, 756, 948, 956 Payne, J. G., 882, 897 Payne, P. A., 300, 305, 307 Payne, T. F., 320, 326 Paysan, J., 24, 46 Paysant J., 950, 954 Paz, D. A., 30, 42 Pazos, A. J., 392, 400 Peabody, C. A 481, 483, 492, 500, 501 Pearce, R. A., 190, 197 Pearce, R. K., 238, 248, 505, 514, 522 Pearl, O., 553, 563, 568 Pearlson, G. D., 481, 489, 499, 601, 611 Pearson, A. A., 1002–1004, 1006–1008, 1010, 1015, 1024 Pearson, P., 828, 840 Pearson, P. B., 830, 831, 845, 883, 884, 903 Pearson, R. C., 491, 498, 510, 512, 520, 522 Pearson, R. C. A., 600, 602, 611 Peck, J. W., 909, 931 Pedal, I., 82, 85 Pedersen, B., 444, 457 Pedersen, M., 12, 16, 554, 563, 568 Pedersen, N. L., 341 Pedersen, O. F., 443, 450, 455, 987, 998 Pedersen, P. E., 145, 157, 315, 325, 355, 360, 380 Pedersen, R., xxvii, xxxiv, 130, 131 Pedersen, R.A., 117, 136 Peeke, H. V. S., 321, 325, 343 Peele, D. B., 60, 68, 392, 399, 553, 567, 583, 589, 590 Peeler, D., 230, 246 Peet, M. M., xxvi, xli, 557, 570 Peeters, B. W. M. M., 920–921, 926, 931 Peiper, A., 826, 844

1078 Pelberg, R., 204, 225, 450, 454, 525, 537, 544, 587, 588 Pelchat, M. L., 856, 860, 885, 900, 908, 914, 931 Pelkonen, O., 57, 70 Pelletier, G., 76, 86 Pellier, V., 36, 46 Pellier-Monnin, V., 105, 107 Pelosi, P., xxiv, xli, 19, 35, 46, 48, 54, 71, 76–78, 86–89, 280, 293, 300, 307, 560, 565, 566 Pelton, G. H., xxxii, xxxv, 483, 497 Pemberton, J. L., 142, 163, 171, 179 Pemberton, M. N., 951, 955 Pencea, V., 515, 522, 620, 627 Pendlebury, W. W., 555, 569 Penfield, W., 948, 950, 956 Peng, H. M., 57, 58, 68, 71 Peng, M., 417, 425, 434 Penn, D., 321, 322, 325, 356, 380 Pennebaker, J. W., 539, 546 Pennell, M. D., 886, 903 Penrose, W. R., 300, 306, 308 Penzoldt, F., xxii, xxxvi Peppel, K., 27, 33, 46, 84, 89 Pepple, J. R., 492, 501 Perbellini, D., 243, 248 Perdue, M. H., 538, 539, 544 Perera, G. M., 262, 265, 266, 271, 491, 492, 499 Peresecenschi, G., 950, 954 Peretto, P., 35, 48 Peretz, S., 481, 498 Perez, C., 921, 931 Perez, T., 440, 456 Perez, W. A., 416, 435 Perez, Y., 413, 436 Perez-Polo, J. R., 105, 112, 619, 620, 626, 920–923, 928 Perez-Tur, J., 488, 496 Perfilieva, E., 93, 108 Perheentupa, J., 887, 902 Perillán, C., 831, 840 Peringa, J., 98, 109 Perio, A., 416, 426, 434 Perkel, D. H., 748, 756 Perl, D. P., 480, 483, 484, 488, 490, 497–500, 551, 555, 563, 567, 569, 570 Perl, E., 481, 500 Perlemuter, L., 893, 898 Perlman, S., 551, 557, 565, 570, 571 Pernollet, J.-C., xxiv, xxxiv Peroutka, S., 553, 573 Perrachio, A., 749, 751, 757 Perras, B., 554, 563, 571 Perren, M. J., 553, 562, 565 Perret, G., 554, 572 Perret, J., xxv, xli Perrett, D. I., 687, 702 Perrier, G., 310, 315, 323, 369, 370, 374, 379, 381 Perrone, A., 486, 501

Author Index Perros, P., 893, 901, 950, 956 Perroteau, I., 101, 111 Perrotto, R. S., 683, 702, 704 Perruchet, P., 390, 392, 397, 399 Perry, C., xxvii, xxxvi, 13, 14, 15, 103, 107, 109, 466, 475, 516, 520 Perry, C. J., 441, 442, 455 Perry, G., 505, 506, 523, 563, 572 Persaud, K. C., 296, 300, 305, 306, 307 Persico, M. G., 1001, 1017, 1022 Persohn, E., 123, 136, 150, 161 Persson, H., 762, 779 Peryam, D. R., 806, 822, 850, 860 Pes, D., 78, 89 Peschel, O., 508, 509, 521 Peshenko, I. V., 54, 71 Peszka, J., 233, 247 Petchpradub, V., 413, 432 Peter, R. E., 61, 72 Peters, G. E., 549, 550, 561, 570 Peters, M. J., 241, 247 Petersen, R. C., 485, 487, 488, 495, 496, 501 Petersen, S. E., 258, 269 Petersen, S. H., 991, 997 Petersik, J. T., 838, 841 Peterson, C., 789, 803, 951, 956 Peterson, D., 93, 108 Peterson, D. A., 620, 621, 624, 626 Peterson, E. L., 556, 564, 568 Peterson, J. M., 535, 538, 543 Peterson, M., 949, 954 Peterson, M. G., 889, 896 Petit, C., 127, 134, 1003, 1012, 1016, 1017, 1022, 1023, 1025 Petito, C. K., 605, 611 Petrides, M., xxix, xliii, 258, 262, 264, 266, 267, 272, 680, 698, 702, 704, 797, 800, 803, 916, 930, 948, 957 Petrie, R. X., 619, 626 Petrie, W. M., xxxii, xliii, 480, 482, 501 Petrovich, G. D., 973, 979 Petruckevitch, A., 238, 249, 488, 501 Petrulis, A., 364, 369, 380, 417, 422, 425, 434 Petruson, B., 440, 458 Petter, A., 169, 178 Pevsner, J., xxiv, xli, 76–78, 86, 89, 125, 136, 280, 293, 560, 571 Pevzner, R. A., 661, 675 Peynegre, R., 893, 898 Pfaff, D. W., xvi, xli, 122, 127, 132, 136, 137, 182, 199, 389, 400, 404, 408, 738, 753, 756. 799, 1002–1007, 1009–1018, 1022, 1024–1026 Pfaffmann, C., xxiv, xxx, xxxi, xxxiv, xli, xlii, 182, 200, 364, 381, 387, 399, 404, 408, 644, 649, 732, 734, 738, 740, 741, 743, 746–748, 750, 752, 755, 756, 758, 805, 820, 862, 864, 875, 876, 878, 908, 933, 999 Pfammatter, J. P., 946, 954 Pfaus, J. G., 357, 378, 423, 431

Pfeffer, R., 489, 498 Pfeiffer, C., 184, 195, 215, 225, 392, 398, 484, 485, 494, 497, 498, 514, 515, 520, 602, 609 Pfeiffer, R. F., 481, 484, 498, 499 Pfennig, D. W., 336, 342 Pfrieger, F. W., 620, 625 Pham, K. L., 145, 146, 158, 182, 197 Pham, L. T., 495, 497, 616, 623, 629, 630, 631, 634, 637 Phelan, E. L., 886, 902 Phelan, S. P., 410, 437 Phelps, M. E., xxviii, xli, 253, 273 Phelps, P. E., xxviii, xliv, 153, 161 Phil, D., 441 Philipot, B. D., 151, 161 Phillips, A. G., 422, 429 Phillips, C., xxxi, xlv, 789, 804 Phillips, C. L., 789, 804 Phillips, G., 867, 870, 879 Phillips, M. L., 687, 702 Phillips, P. P., 596, 612 Phillips, S. E. V., 970, 976 Phillips, W. A., 281, 293 Philopena, J. M., 918, 928 Philpot, B., 103, 111 Philpot, B. D., 618, 626 Philpot, R. M., 57, 58, 66, 280, 293 Picard, M., 315, 325 Picco. C., 287, 293 Piccolino, M., 645, 649 Piccolo, I., 486, 501 Pickens, R. W., 330, 342 Picker, A., 116, 137 Pickett, J., 366, 381 Pickles, J., 101, 109 Picton, T. W., 230, 233, 248 Piel, E., 236, 244, 491, 496 Pieler, T., 116, 133 Piemonte, G., 661, 677 Piemontese, D., 553, 563, 566 Pieper, D. R., 361, 380, 619, 626 Pierce, C. E., 865, 876 Pierce, J. D., Jr., 207, 227, 480, 484, 488, 497, 563, 567 Pieretti, M., 127, 133, 1001, 1017, 1022 Pierri, J., 886, 896 Pierrot-Deseilligny, C., 789, 804 Pierson, M. E., 554, 563, 571 Pierson, S. C., 387, 392, 399 Piesse, G. W. H., xvi, xviii, xli Pietrowsky, R., 554, 563, 571 Pigott, G. H., 583, 589 Pihet, S., 312, 325, 355, 380 Pike, B., 514, 522 Pike, L., 1017, 1021 Piketty, V., 423, 426, 432 Pikielna, N. B., 891, 901 Pikielny, C. W., 56–58, 73 Pilar, G., 124, 136 Pilgrim, F. J., 807, 821 Pilgrim, P. J., 850, 860 Pillias, A. M., 948, 955

Author Index Pillsbury, H. C., 470, 477 Pilot, L. J., 833, 845, 886, 902 Pilpel, Y., 145, 157 Pilz, J., 619, 627 Pinching, A. J., 140, 142, 143, 144, 161, 220, 227, 618, 626 Pinel, J. P. J., 414, 433, 917, 933 Pinkstaff, C. A., 658, 676 Pinkus, F., 1001, 1010, 1024 Pino, M. V., 583, 590 Pinto, S., 62, 71 Piot, O., 416, 417, 432 Piper, C. F., 605, 610 Piras, A. T., 824, 844 Pirie, P. L., 886, 897 Pirker, S., 151, 161 Pisanelli, A. M., 76, 78, 87, 89, 306, 307 Pissonnier, D., 423, 434 Pitcher, R. G., 124, 135 Pitkanen, A., 680, 700 Pitovski, D. Z., 31, 43, 54, 62, 68, 72 Pittman, E. A., 218, 226, 471, 477, 892, 901, 950, 956 Pitts, W. H., xxiv, xxxvi Pixley, S. K., xxvii, xli, 22, 47, 100–105, 109–111, 125, 136, 149, 158, 184, 197, 619, 626 Plahl, D., 723, 728 Plakhov, I. V., 557, 568 Plant, G., 105, 112 Plant, W. D., 889, 897 Plata-Salamán, C. R., 680, 685, 686, 688, 690, 699, 702, 704, 746, 748, 757, 758, 797, 802 Plattig, K.-H., xxiv, xxxviii, xli, 230, 231, 244, 244, 247, 783, 797, 803, 949, 956 Plattner, H., 24, 26, 47 Playford, E. D., 601, 608 Pleasure, D., 894, 901 Plendl, J., 36, 47 Plihal, W., 240, 247 Pliner, P., 909, 931 Ploeger, G. E., 416, 434 Plomin, R., 329, 331, 332, 333, 334, 335, 342, 343 Plopper, C. G., 57, 58, 66, 583, 590 Plowchalk, D. R., 56, 67 Ploye, H., 186, 199 Pluchon, C., 482, 496 Poblenz, A. T., 581, 589 Pochet, R., 26, 41, 83, 85 Podreka, I., 485, 499 Poellinger, A., 260, 271 Poenaru, L., 551, 567 Pohlinger, A., 367, 382 Poindron, P., 312, 318, 325, 326, 343, 423, 431, 432, 434 Polak, E. H., 392, 397, 560, 571, 572 Polak, J. M., 76, 85 Poland, G. A., 342 Polans, A. S., 26, 41, 83, 85

1079 Polet, I. A., 788, 794, 802 Poli, M., 316, 324 Polich, J. M., 234–236, 240, 244, 248, 342 Polivy, J., 887, 898 Polk, M. S., 483, 500 Pollak, A., 960, 965 Pollen, D.A., 509, 522 Polli, G., 62, 71 Pollitt, E., 885, 895 Pollock, G., 104, 111 Polymeropoulos, M. H., 514, 522 Pomerantz, J. R., 816, 817, 821 Pomonis, J. D., 826, 829, 844 Pompeiano, M., 152, 161 Ponto, L. L. 269, 491, 496 Ponzo, M., 658, 675 Poonai, N., 539, 546 Poorkaj, P., 505, 513, 522 Poothullil, J. M., 892, 901 Pope, J. F., 833, 845 Popiela, T., 887, 898 Popik, P., 416, 420, 434 Popov, V. I., 54, 71 Popp, J. A., 582, 591 Poria, Y., 57, 58, 61, 70, 73, 76, 88, 559, 569, 573 Porrini, M., 882, 887, 896 Porter, M. C., 414, 433 Porter, R. H., 216, 227, 310, 311, 313–321, 322–326, 336, 337, 338, 339, 340, 342, 343, 355, 357, 380, 834, 835, 841, 845 Porter, T. D., 57, 68, 71 Portier, M. M., 124, 132 Portin, K., 241, 246, 259, 266, 271 Portmann, M. O., 795, 803 Portugal, L. G., 444, 457 Posadas-Andrews, A., 419, 434 Posner, M. I., 252, 271 Post, E. M., 204, 227 Post, H., 234, 236, 248 Post, M. J. D., 606, 612 Postigo, A., 238, 247 Postolache, T. T., 472, 477, 495, 500, 619, 626 Potapov, A. A., 149, 161 Potkin, S. G., 516, 518 Potter, E. K., 54, 72 Potter, H., 187, 199, 208, 209, 211, 225, 227, 421, 424, 428, 435, 604, 612 Potter, J. D., 858, 860 Potts, C. L., 580, 590 Potts, W., 321, 322, 325, 356, 380 Poueymirou, W. T., 762, 779 Poulin, N., 318, 325, 343 Poulton, E. B., 910, 931 Pound, R. V., 253, 272 Pouplard-Barthelaix, A., 38, 39, 41, 505, 519 Pourtier, L., 335, 343, 314, 325 Poutiainen, E., 606, 609 Powell, J., 714, 715, 725

Powell, M. J. D., 241, 248 Powell, N. J., 313, 324 Powell, T. P. S., 140, 142–145, 147, 148, 161, 162, 220, 227, 600, 611, 618, 626, 680, 701 Powers, A. S., 856, 858 Powers, J. B., xxx, xli, xliv, 746, 756, 967, 970, 978 Powley, T. L., 682, 702, 703 Powlis, W., 910, 929 Prabhakaran, V., xxix, xliii, 174, 180, 255, 259, 260–267, 271–273, 424, 436 Pragliola, A., 127, 133, 1001, 1017, 1021, 1022 Prah, J. D., 204, 227, 231, 232, 234, 248, 255, 271, 387, 392, 399, 583, 584, 590 Prasad, A. S., 893, 899 Prasad, M. R. N., 61, 66 Prasada Rao, P. D., 148, 161 Pratt, H. P., 365, 373 Pratt, N., 58, 69 Pratt, P., 492, 498 Prazma, G., 470, 477 Precht, W., 661, 673 Prescott, C. A., 330, 331, 343 Prescott, J., xxiv, xlii, 788, 803, 818, 822, 884, 901 Prescott, R. J., 451, 455 Press, D. S., 495, 497, 886, 896 Press, G. A., 602, 612 Prestwich, G. D., xxv, xliv, 78, 81, 88, 90 Preti, G., 216, 224, 419, 430 Preussman, R., 63, 72 Preussmann, R., 552, 568 Preyer, W., 313, 325, 826, 844 Prezelin, G., 887, 901 Pribitikin, E. A., 221, 227 Price, B. A., 185, 199 Price, D., 479, 499, 618, 623 Price, D. A., 1004, 1015, 1024 Price, G. W., 183, 194 Price, J. L., 142, 144–148, 157, 159, 161–163, 165, 167–174, 176–179, 188, 192, 194, 196–199, 254, 255, 258, 259, 261–263, 266, 268, 269, 270, 272, 426, 430, 513, 522, 523, 595, 612, 618, 624 680, 693, 700, 702 Price, L. M., 406, 408 Price, R. A., 710, 717, 729, 791, 798, 803, 885, 894, 849, 850, 860 Price, R. W., 605, 612 Price, S., 61, 71, 710, 729 Priest, C. A., 149, 155, 183, 194 Prihoda, T. J., 526, 529, 532, 534, 535, 545 Primeaux, S. D., 619, 624 Principato, J. J., 230, 248, 450, 457 Prineas, R. J., 886, 895, 896 Pritchard, J. A., 311, 323, 825, 844

1080 Pritchard, T. C., 657, 667, 675, 679, 680, 684–686, 702, 703, 861, 863, 878, 948, 956 Pritchard, W. S., 240, 248 Probst, A., 152, 161, 508, 509, 521 Probst, J. C., 416, 432 Procter, A. W., 510, 513, 520 Proctor, D. F., 4, 15, 22, 47, 55, 71, 441, 447, 448, 453, 457, 458, 577, 591, 997 Proctor, G. B., 639, 648 Proetz, A. W., xxiii, xli, 447, 449, 457 Pronchik, D. J., 553, 562, 570 Propper, C. R., 1005, 1023 Prosser, J., 116, 133 Prosser, O., 305, 307 Provenza, F. D., 315, 325, 906, 925 Pruitt, C. H., 968, 977 Pruszewicz, A., 467, 477, 960, 962, 963, 965, 966 Prutkin, J., 853, 854, 855, 860, 961, 965 Prutkin, J. M., 788, 802 Pruzan, A., 334, 343 Pryor, J. S., 893, 902 Pryse-Philips, W., 472, 477 Przuntek, H., 509, 514, 521 Przybyslawski, J., 413, 435 Ptak, J. E., xxxi, xxxiv, 391, 393, 400 Puchalski, R. B., 717, 727 Puche, A., 102, 103, 110, 111, 127, 129, 130, 131, 149, 151, 158, 162, 184, 197, 199, 973, 977 Puerto, A., 915, 917, 927, 929 Puglisi-Allegra, S. Puig, A., 472, 474 Pujol, J. F., 148, 152, 155, 156, 561, 565 Pullara, J. M., 558, 560, 568, 617, 624 Pullen, E. W., 945, 956 Pumariega, A. J., 920–923, 928 Pumplin, D. W., 661, 664, 675, 710, 725 Pun, R. Y. K., 125, 136 Punter, P. H., 204, 215, 227 Puopolo, M., 140, 149, 152, 162 Purcell, A. L., 129, 130, 137 Purcell, E. M., 253, 272 Purcell, M., 882, 900 Purdon, S. E., 491, 492, 493, 500 Purushotham, K., 643, 649 Purvis, K., 368, 374 Puschel, A. W., 116, 136 Püschel, J., 815, 816, 821 Putcha, L., 554, 563, 571 Putnam, P., 788, 802 Puxeddu, R., 658, 675 Pyatkina, G. A., 33, 35, 47, 120, 121, 122, 136, 311, 325 Qasba, P., 24–28, 33, 45, 78, 89 Qiu, M., 117, 136 Qu, W., 61, 72 Qu, Y., 621, 625 Quadagno, D. M., 320, 325, 356, 380 Quanbeck, C., 1012, 1013, 1024, 1025

Author Index Quast, J. F., 582, 589 Quebbeman, J. F., 321, 322 Quek, V., 828, 840 Quencer, R. M., 606, 612 Quenedey, A., 369, 379 Quenedey, B., 369, 379 Quichon, R., 318, 321, 326 Quinn, F. J., Jr., 450, 457 Quinn, N. P., 238, 249, 484, 485, 500 Quinn, T. P., 426, 428, 433 Quiñonez, C., 786, 802 Quint, D. J., 599, 608 Quirion, R., 76, 86, 154, 156, 159, 472, 477, 619, 626 Quraishi, S., 563, 569 Qureshi, U., 419, 427 Qureshy, A., 266, 267, 272 Raatgever, J. W., 559, 568 Rabbitts, T. H., 77, 87 Rabchenuk, S. A., 602, 611 Rabe, E. F., 862, 878 Rabesona, H., 77, 86 Rabi, Y. J., 920, 930 Rabin, B. M., 915, 918, 931 Rabin, M. D., 217, 219, 227, 783, 791, 799, 800 Rabin, R. D., 212, 215, 224 Rabinovitch, A., 511, 522 Rabl, H., 658, 675 Racine, R. J., 191, 195 Raconczay, Z., 154, 158 Radike, M., 388, 394, 396, 398, 578, 583, 589 Radil, T., 484, 500 Radojicic, M., 151, 157 Radtke-Schuller, S., 165, 169, 173, 178, 179 Raff, M., 579, 589, 645, 648, 894, 897 Raffalli, F., 64, 67 Rafols, J. A., 31, 32, 47 Rafter, J. J., 61, 67 Raftogianis, R. B., 59, 73 Ragimova, N. G., 153, 159 Rahaim, J., 892, 896 Rahill, A., 528, 530, 536, 545 Rahm, F., 443, 457 Raichle, M. E., xxviii, xxxvi, 252, 253, 258, 269–272, 686, 702 Raimundo, A. H., 887, 901 Rainer, E. C., 142, 161, 163, 185, 198 Raininko, R., 606, 609 Raisman, G., xxvii, xxxix, 105, 110, 154, 162 Raj, J., 212, 215, 225 Raj, J. M., 786, 803 Raj, P., 642, 647, 648 Rajaram, L., xxxii, xxxvii, 341, 483, 498 Rajecki, D. W., 359, 380 Rajendren, G., 365–367, 380 Rajewski, K., 413, 434 Raju, V. S., 82, 87

Rake, G., 552, 571 Rakic, P., 509, 515, 521, 616, 626 Rakonczay, Z., 512, 521 Rakover, S. S., 220, 227 Rakow, W. 306, 307 Rall, T. W., 915, 933 Rall, W., xxiv, xli, 142, 144, 162, 186, 199 Ram, C. A., 216, 224 Rama Krishna, N. S., 101, 102, 111, 505, 522 Ramanathan, R., 554, 563, 571 Ramasubbu, N., 642, 647, 648 Ramazzini, B., 575, 590 Rambotti, M. G., 27, 32, 47, 48 Ramenghi, L. A., 829, 844 Rameriz, I., 736, 756, 887, 901 Raming, K., xxv, xli, 287, 294 Ramirez-Amaya, V., 918, 930 Ramón-Cueto, A., xxvii, xli, 29, 47, 105, 106, 111–112 Ramoni, R., 19, 48 Rampon, C., 420, 435 Ramsey, J. C., 54, 72 Ramstein, K., 629, 637 Ramus, S. J., 174, 175, 179, 414, 415, 429, 435 Randall, H. W., 52, 54, 56, 67, 71 Randall, J. E., 439, 457 Randall, P. K., 310, 325 Randolph, T. G., 527, 534, 537, 539, 546 Raner, G., 61, 73 Rangga-Tabbu, C., 583, 590 Ranier, E. C., 171, 179 Rankin, K. M., 210, 225, 788, 801, 853, 854, 859, 798, 803, 818, 822 Ransjo-Arvidson, A. B., 313, 327 Ranvier, L., 666, 675 Rao, H., 762, 781 Rao, M., 828, 844 Rao, M. S., 116, 137 Raphael, D., 837, 844 Raphael, G. D., 54, 71 Rapin, C-H., 910, 927 Rapp, M., 299, 307 Raps, E. C., 489, 496 Rashotte, M. E., 873, 877 Rasmus, S., 894, 901 Rasmussen, J. L., 359, 380 Rasmussen, K., 422, 431 Rasmussen, M. H., 554, 563, 568 Rasmussen, T. R., 444, 457 Rasmussen-Conrad, E. L., 892, 897 Rassin, D. K., 920–923, 928 Rathe, A., 532, 544 Ratnasuriya, R. H., 923–924, 931 Rauch, S. L., 686, 702 Raunio, H., 57, 70 Rausch, R., 268, 272, 424, 435, 603, 604, 612 Rauschenberger, J., 911, 930 Rautiainen, M., 442, 454

Author Index Raveh, J., 631, 638 Ravel, N., 154, 162, 164, 174, 175, 176, 187, 188, 190, 191, 195, 198, 199, 415, 423, 426, 432, 435 Ravid, U., 394, 395, 398, 404, 407 Ravstein, P., 713, 727 Rawls, L. H., 316, 322 Rawson, N. E., 37, 38, 47, 221, 227 Ray, J., 620, 621, 624, 626 Ray, J. P., 170, 179, 192, 199 Ray, W. J., 621, 623 Raymond, S. A., 920, 925 Razafimanalina, R., 885, 901 Re, P. K., 291, 293 Rea, W. J., 536, 539, 543, 546, 587, 588 Read, E. A., 13, 14, 16 Read, N. W., 839, 846, 882, 887, 895, 898 Rebeck, G. W., 510, 522 Reber, M., 443, 457 Reddy, C. S., 397, 399 Reddy, G., 552, 571, 582, 590 Reddy, R., 76, 81, 84, 89 Reddy, V. G., 553, 569 Redfern, E., 554, 563, 566 Redman, R., 864, 865, 878 Redman, S. J., 512, 523 Reeb, E., 642, 647, 648 Reed, C. J., 55, 57, 58, 64, 65, 66, 67, 72, 552, 571, 577, 578, 584, 590 Reed, D. M., 487, 500 Reed, D. R., 710, 716, 717, 724, 727, 729 789, 791, 798, 799, 801, 803, 847, 848, 850, 859, 860, 885, 894 Reed, H. C., 346, 379 Reed, M. K., 244, 249 Reed, R. A., 78–81, 85–89 Reed, R. R., xxiv, xxv, xxxvii, xxxviii, xli, 24–28, 33, 41–45, 101, 110, 126, 138, 287, 293, 329, 333, 334, 335, 341, 342, 559, 569 Reedy, F. E., 854, 860, 937, 956 Reedy, F. E., Jr., 655, 656, 658, 674, 963, 965 Reep, R. L., 170, 179 Reese, H. W., 682, 704, 845, 906, 917, 932 Reese, T. S., xxiv, xli, 24, 47, 142, 144, 162, 186, 199 Reeves, A.J., xxviii, xliii Register, R. B., 509, 521 Regnier, F. E., 346, 382 Rehfeld, J. F., 910, 929 Rehn, B., 2, 16, 94–96, 99, 112, 1015, 1021 Rehn, T., 266, 272, 452, 457 Rehnberg, B. G., 644, 649, 714, 721, 729, 785, 803 Reibel, J., 299, 307 Reibenspies, M., 582, 588 Reichardt, L. F., 670, 674, 761, 762, 767, 771–773, 779–781 Reichenberger, H., 241, 244

1081 Reicher, M. A., 914, 931 Reichert, H., 116, 134 Reichl, M. 306, 307 Reichmann, H., 486, 500 Reid, I. C., 354, 380, 410, 435, 602, 612, 619, 626 Reid, K. H., 1, 16 Reid, L., 33, 43 Reid, O., 36, 41 Reifers, F., 116, 137 Reilly, B. M., 828, 840, 843 Reilly, D., 794, 803 Reilly, S., 683, 684, 701–703, 861, 863, 866, 878, 916, 928, 931 Reiman, E. M., 595, 612 Reinarz, D. E., 920, 928 Reinhard, E., 105, 112 Reinhard, G. R., 1018, 1024 Reinhardt, W., 973, 977 Reinhart, P. H., 722, 730 Reinoso-Suarez, F., 170, 176, 192, 195 Reinscheid, R, K., 1004, 1022 Reinshagen, M., 887, 898 Reis, D. J., 564, 565, 618, 623 Reisberg, B., 600, 609 Reiss, C. S., 557, 568 Reitan, R. M., 237, 248 Reiter, E. R., 634, 638 Reiter, J. S., 510, 522 Reivich, M., xxviii, xli, xxxviii, xliii Rejeski, W. J., 359, 380 Remak, R., 669, 675 Remfry, C. J., 366, 381, 971, 976, 978 Remley, N. R., 406, 408 Remunan-Lopez, C., 554, 563, 567 Remy, N., xvii, xli Ren, X. Z., 658, 674 Renkawek, K., 512, 523 Renken, R., 258, 273 Renner, B., 193, 198, 259, 264, 266, 271, 699, 702 Renner, M., xxix, xxxviii Rennert, K., 740, 754 Rennie, M., 736, 756 Renqvist, Y., xxv, xli Rensing, H., 82, 85 Rentfro, L., 97, 108, 118, 133 Rentmeister-Bryant, H., 867, 869, 870, 878 Repérant, J., 149, 153, 159 Resau, J., xxvii, xliv, 98, 113 Rescorla, R. A., 420, 428, 911, 931 Reshef, A., 553, 563, 568 Resko, J. A., 923, 932, 1003, 1007, 1024 Resnick, S. M., 512, 516, 520 Ressier, K. J., 24, 28, 32, 36, 47, 78, 89, 90, 100, 112, 117, 120, 121, 130, 136, 137, 139, 145, 146, 162, 163, 560, 571, 572 Rest, K., 525, 528, 543 Restrepo, D., xxix, xxxvii, 24, 26–28, 33, 37, 38, 40, 42–44, 47, 49, 76, 81, 88–90, 221, 227, 243, 248

Rettig, M., 392, 399 Rettinger, G., 439, 456 Retzius, G., 654, 675 Reul, J. M., 619, 626 Reuter, D., 26, 47 Reutter, K., 659–661, 663–668, 670, 671, 675, 677, 764, 782, 824, 825, 846 Reuzel, P. G. J., 994, 996 Revial, M. F., 170, 171, 173, 179, 188, 194 Revington, M., 54, 72 Revoltella, R., 78, 86, 560, 566 Reyes, P. F., 39, 47, 206, 215, 225, 462, 474, 480, 481, 484, 488, 497, 512, 513, 522, 563, 567, 600, 609 Reyher, C. K. H., 142, 152, 160, 162 Reynierse, J. H., 404, 407 Reynolds, A. R., 689, 705 Reynolds, B. A., 619–621, 623, 626 Reynolds, C., 484, 497 Reynolds, G. S., 359, 380 Reynolds, J., 365, 380 Reynolds, J. B., 551, 554, 555, 565 Reynolds, J. H., 204, 228, 982, 999 Reynolds, M. L., 212, 228 Reynolds, O., 440, 457 Rezek, D. L., 481, 500 Rezek, O., 452, 455 Reznik, G., 19, 22, 23, 24, 44, 52, 72, 582, 590 Reznik-Schüller, H. M., 38, 47 Rha, K. S., 450, 458 Rhee, C. S., 55, 71 Rhein, L. D., 24, 34, 47, 76, 78–79, 90 Rhein, L. D., xxv, xli Rhinehart-Doty, J. A., 864, 878 Rhoades, B. K., 152, 162 Rhoades, C. E., 56, 59, 60, 64, 70 Rhoades, R. W., 760, 782, 983, 998 Rhodes, K., 21, 44 Rhodine, C. N., 745, 757 Rhodus, N., 646, 649 Rhue, M. R., 884, 898 Ribands, M., 686, 700 Ricarte, J., 359, 376 Ricci, J. S., 356, 373 Ricciuti, F., 366, 375 Rice, A. G., 923, 931 Rice, F. L., 764, 781 Rice, M., 346, 382 Richard, J., 480, 481, 495, 500 Richard, N., 102, 103, 110 Richard, P., 423, 432 Richards, B., 330, 343 Richards, D. H., 444, 457 Richards, J. G., 150, 161 Richards, K. S., 660, 675 Richards, L. J., 619, 626 Richardson, A., 81, 90 Richardson, B. J., 991, 997, 998 Richardson, C. T., 887, 896, 901 Richardson, D., 366, 375

1082 Richardson, D.W., 910, 933 Richardson, J. T., 494, 496 Richardson, J. T. E., 424, 435 Richardson, M.A., 942, 955 Richardson, R., 390, 392, 397, 398, 400, 413, 435 Richardson, R. A., 936, 950, 954 Richardson, R. B., 578, 589 Richardson, R. J., 556, 564, 568 Richarz, A. N., 511, 522 Riche, D., 551, 569 Richman, M., 27, 41, 123, 131, 150, 155, 396, 397 Richman, R. A., 204, 227, 839, 844 Richmond, B. R., 223, 225 Richmond, M. E., 365, 382 Richtand, N. M., 149, 158, 184, 197 Richter, C. P., xxix, xxxi, xli, 403, 408, 734, 736, 757, 861, 873, 878, 908, 931 Richter, H. J., xxvi, xli, 557, 570 Richter, J. E., 942, 956 Richter, R. J., 539, 544 Richters, G., 713, 727 Rickmann, M. J., 152, 163 Rico, A. G., 923, 931 Riddiford, L. M., 56, 60, 73 Rideaud, P., 369, 374 Ridgeway, S. H., 1002, 1003, 1022 Ridout J. B., 212, 224 Riechelmann, H., 439, 456 Riederer, A., 508, 509, 521 Rief, M., 305, 306 Riegle, G. D., 366, 376 Riese, U., 305, 306 Rieser, J., 983, 999 Riess, O., 509, 514, 521 Rietze, R. L., 621, 623 Rifkin, B., 211, 227, 848, 856, 858, 790, 793, 799, 812, 822 Rigemar, G., 553, 562, 565 Riggs, L. A., 747, 750, 751, 753, 754 Rignani, J., 581, 588 Riker, D. K., 444, 456 Riklan, M., 484, 497 Riley, A. L., 906, 907, 931 Rim, P., 101–103, 112 Rinehart, K. L., 1004, 1022 Ring, G., 145, 162, 616, 626 Ringstedt, T., 772, 781 Rinkel, H. J., 537, 546 Rinne, J. O., 512, 515, 522 Rio, J. P., 149, 153, 159 Rioux, L., 517, 519, 523 Ripamonti, C., 891, 901 Ripandelli, N., 788, 803 Risberg, J., xxviii, xxxvii, 253, 270 Risky, D. R., 886, 895 Risling, M, 96, 108, 620, 624 Riso, P., 882, 896 Risser, J. M., xxxi, xxxiv, 169, 173–175, 180, 184, 195, 310, 325, 392, 394, 396, 397, 398, 400, 410, 411, 436,

Author Index 470–471, 474, 485, 494, 497, 509, 515, 520, 620, 623 Riti, L., xx, xli Ritter, F. J. 978 Ritter, J. K., 58, 70 Ritter, R. C., 688, 691, 694, 695, 703 Ritter, S., 688, 691, 694, 695, 703, 915, 921, 931 Ritter-Walker, E., 481, 498 Ritz, S., 97, 108, 118, 133 Riva, A., 658, 675, 676 Rivers, A. M., 83, 85 Rivest, R. W., 887, 897 Rivier, J. E., 1005, 1023 Rivlin, R. S., xxxix, 216, 224, 471, 476, 620, 625, 751, 756, 799, 840, 892, 900, 901, 950, 956 Robb, R., 715, 729 Robbins, D. C., 888, 895 Robbins, T. W., 423, 435, 602, 610, 887, 901 Roberts, D. W., 991, 999 Roberts, E., 555, 563, 571 Roberts, E. S., 61, 62, 72 Roberts, G., 554, 563, 566 Roberts, G. W., 76, 85, 510, 520, 603, 612 Roberts, M., 233, 247 Roberts, N., 96, 110, 618, 625 Roberts, R. J., 961, 966 Robertshaw, D., 643, 649 Robertson, A., 472, 477 Robertson, D. H. L., 970, 971, 977, 978 Robertson, E., 621, 623 Robichon, A., 664, 675, 710, 711, 717, 729 Robinette, R. R., 207, 224, 333, 340 Robinson, A. M., 54, 72 Robinson, C. J., xxvi, xlii, 686, 703 Robinson, D. O., 828, 842 Robinson, J. A., 1003, 1009, 1024 Robinson, P. P., 656, 658, 671, 946, 953, 956 Robinson, S. R., 312, 326, 355, 382, 833, 841, 842 Robinson, T., 491, 492, 496 Robinson, T. A., 417, 431 Robroch, L., 888, 903 Roca, E., 890, 895 Roca, S., 165, 168, 171, 179 Rocha, S. M., 305, 306 Rochat, P., 828, 841 Rochefort, C., xxviii, xlii Rochford, J., 367, 379 Rochlin, M. W., 762, 763, 781 Rock, D. L., 551, 557, 572 Rock, J., 349, 382 Rodgers, W. L., 908, 931 Rodin, J., 740, 754, 789, 803, 883, 892, 901, 902, 951, 952, 955, 956 Rodnitzky, R. L., 484, 500 Rodolfo-Masera, T., 388, 400 Rodrigues, V., 340

Rodriguez, A., 511, 523 Rodriguez, I., 126, 138, 350, 380, 972, 973, 978 Rodriguez-Echandia, E. L., 356, 380, 887, 895 Rodriguez-Perez, I., 939, 954 Roeder, K. D., xxiv, xxxvi Roelink, H., 116, 136 Roenbaum, A. E., 594, 613 Roessler, E. B., 206, 227 Rogalski, K. P., 350, 380 Rogers, A. G., 422, 437 Rogers, G. A., 413, 432, 436 Rogers, J., 887, 901 Rogers, J. H., 83, 85 Rogers, M. E., 58, 72 Rogers, P. J., 882, 895, 901 Rogers, Q. R., 884, 897, 899, 900, 906, 927 Rogers, W. R., 538, 546 Roghani, A., 153, 161 Rognon, C., 311, 326, 825, 845 Rogol, A. D., 603, 611 Rohner-Jeanrenaud, F., 887, 897 Rohrbacher, K. D., 57, 72 Rohrer, F., 440, 457 Rohrlein, G., 241, 248 Roisen, F. J., 103, 110 Roitblat, H., 359, 377 Roithmann, R., 443, 444, 452, 457 Roivainen, R., 82, 88 Rojas, A., xxiv, xxxvi Rokem, A. M., 321, 324 Roland, L. P., 253, 271 Roland, P., 174, 179, 258, 262–264, 266–268, 272, 350, 381, 424, 435 Role, L. W., 154, 156 Rolland, R., 554, 563, 571 Rolls, B. J., 472, 475, 690, 691, 696, 703, 836, 844, 882, 883, 901, 923, 931 Rolls, E. T., 192, 193, 195, 198, 199, 259, 263, 264, 266, 269, 271, 272, 421, 425, 428, 435 679–681, 685–699, 700–705, 736, 737, 740–742, 745, 746, 748, 757, 758, 797, 802, 803, 829, 836, 844, 882, 901, 948, 956, 959, 966 Rolls, J. H., 193, 199, 696, 703 Roman, F., 190, 191, 199 Roman, F. S., 170, 179, 410–413, 425, 427, 435 Romanes, G. J., 403, 408 Romani, G. L., 241, 244 Romantschuck, S., 444, 454 Romeijn, S. G., 553, 562, 570 Romeo, G., 244, 244 Romer, D., 211, 223 Romeyer, A., 318, 325, 337, 343 Rommel, C., 658, 672 Romon, M., 495, 500, 960, 961, 962, 966 Ron, M. A., 602, 612 Ronca, A. E., 312, 326 Ronen, T., 715, 728

Author Index Rong, M., 713, 714, 717 Rönnbäck, L., 542, 543 Ronnekleiv, O., 1003, 1007, 1024 Ronnett, G. V., 24, 26, 27, 33, 41, 42, 44, 58, 73, 75–76, 79–84, 86–90, 101–103, 105, 112, 123, 125, 135, 136, 619, 626 Rooney, E., 417, 435 Roos, J., 1003, 1012, 1021 Root, H., 514, 522 Roozen, J. P., 808, 822 Roozendaal, B., 416, 432 Ropartz, P., 361, 381 Roper, S. D., xxv, xlii, 659–664, 671, 673, 675, 686, 690, 700, 704, 719, 721, 723, 725, 726, 731, 737, 738, 740–742, 744, 745, 747, 755–758, 807, 814, 821, 950, 957 Roper, T. J., 419, 434 Roper-Hall, A., 680, 688, 704 Roques, B. P., 416, 417, 432 Rosamond, J. D., 554, 563, 571 Rosario, V., xvi, xxxvii Roscher, S., 234, 236, 238, 246, 248, 538, 545, 587, 589, 990, 998 Rosdahl, D., 525, 528, 543 Rose, C. S., 580, 584, 590 Rose, F. C., 484, 497 Rosellini, R. A., 920, 930 Roseman, J. M., 941, 956 Rosen, B. R., 252, 258, 260, 269, 271, 273 Rosen, T. A., 983, 996 Rosenberg, L., xxxi, xxxv, 113, 108, 213, 216, 225, 340, 461, 474 Rosenblatt, F., 296, 308 Rosenblatt, J. 845, 907, 931 Rosenblith, W. A., 203, 204, 210, 228, 803 Rosenbloom, S., 600, 610 Rosenblum, J. L., 642, 649 Rosenblum, P. M., 61, 72 Rosene, D. L., 165, 167, 169, 173, 177 Rosenstein, D., 826, 827, 844, 885, 886, 901 Rosenthal, N. E., 472, 477, 619, 626 Rosenthal, S., 892, 901 Rosenthal-Zifroni, A., 951, 957 Rosenzweig, S., 710, 711, 726, 729, 730 Roser, B., 320, 322, 356, 381 Rosés, M., 992, 995 Rosin, J. F., 192, 199 Roskams, A. J. I., 2, 75, 82, 90, 101–103, 105, 112, 619, 626 Rosli, Y., 36, 37, 47 Rosolia, D. L., 583, 590 Ross, B. M., 217, 225 Ross, C. A., 81, 86, 514, 520, 523 Ross, C. D., 147, 153, 157, 170, 177 Ross, D. A., 717, 724 Ross, G. H., 539, 546 Ross, G. R. T., xxi, xxxvii Ross, R., xix, xlii Ross, M. A., xxvi, xliv, 557, 572 Rossant, J., 621, 624

1083 Rosselli-Austin, L., 128, 129, 136 Rosser, A. E., 365–367, 377, 380, 381, 971, 975, 976, 977, 978 Rossi, G. H., 536, 546 Rossi, J., 538, 543 Rossie, K. M., 939, 954 Rossier, B. C., 719, 725, 727 Rossini, A. A., 919, 930 Rossiter, K. J., 276, 285, 294, 991, 999 Rossiter, T. R., 908, 925 Rossler, P., 709, 710, 729 Rossner, S., 893, 896 Rosso, I. M., 516, 519 Rossor, M. N., 76, 85, 484, 485, 500 Ross-Russell, R. W., xxviii, xli Rosvold, H. E., 410, 427, 680, 701 Rotatori, D. S., 643, 649 Rotch, T. M., 629, 638, 963, 966 Roth, J., 484, 500 Rothbauer, C., 234, 236, 239, 246, 444, 445, 455, 463, 476 Rothenburg, B. A., 909, 929 Rothkrug, L., xvii, xlii Rothman, A., 972, 978 Rothman, L., 816, 822 Rothman, N., 585, 590 Rothman, R. B., 152, 160 Rothweiler, H., 994, 999 Rotrosen, J., 480, 485, 500, 501, 893, 895 Rott, R., 551, 557, 570 Rotter, A., 154, 162 Rottman, S. J., 405, 408 Rouby, C., 259, 263, 264, 267, 273 Rougon, G., 511, 517, 519 Roulet, E., 553, 562, 569 Roullet, P., 413, 435 Rouquier, S., 17, 42, 291, 294 Rouseff, R. L., 847, 848, 857, 858, 860 Rousmans, S., 358, 372 Rousseaux, M., 495, 500, 960, 961, 962, 966 Rovee, C. K., 210, 227, 834, 844 Rovelli, G., 105, 112 Rowan, P. A., 818, 820 Rowe, E. A., 690, 691, 696, 703, 882, 901 Rowe, E. S., 836, 844 Rowe, F. A., 361, 381 Rowe, M. M., 470–471, 474, 867, 872, 876, 892, 895 Rowland, D., 687, 702 Rowland, L. P., xxviii, xxxviii Rowland, N. E., 862, 866, 877 Rowley, III, J. C., xxxi, xl, 3, 5, 15, 16, 21, 22, 23, 30, 33, 34, 37–39, 44, 45, 46, 47, 121, 122, 135, 140, 160, 466, 476, 631, 638 Roxborough, H., 603, 613 Roy, C. S., xxviii, xlii, xliii, 252, 272 Roy, M., 449, 453 Roy, S., 513, 523 Royal, S. J., 120, 136 Royall, D. R., 483, 500 Royce, G. J., 165, 168, 179

Roycrott, J. H., 583, 590 Roydhouse, N., 579, 580 Royer, S. M., 661–664, 672, 673, 675 Royet, J. P., 145, 153, 162, 174, 179, 186, 193, 199, 213, 226, 258, 261, 264–266, 272, 421, 424, 434, 435, 482, 500 Roytta, M., 515, 522 Rozin, P., 837, 844, 884, 901, 905–908, 910, 914, 917, 928, 931 Rozumek, M., 603, 611 Rub, U., 517, 521 Rubenstein, J., 514, 522 Rubenstein, J. L., xxvii, xxxiv, 116, 117, 133, 136 Rubenstein, J. L. R., 130, 131, 760, 778 Rubin, B., 1003, 1006, 1023, 1024 Rubin, B. D., 145, 146, 162, 182, 199, 259, 272 Rubin, I., 17, 43, 145, 157 Rubinraut, S., 553, 563, 568 Rubinstein, M., 58, 70 Rubio, J., 334, 340 Ruch, T. C., 861, 876, 878 Ruck-Keene, E. A., 305, 307 Rudel, R., 124, 137, 505, 506, 509, 523 Rudel, R. A., 563, 572 Rudy, J. W., 414, 436, 906, 933 Ruel, J., 620, 623 Ruff, L. R., 321, 322 Ruffin M. T., 858, 859 Rugarli, E. I., 127, 136 Ruis, H., 554, 563, 571 Ruiz, C. C., 83, 89 Ruiz-Avila, L., xxvi, xlii, 664, 675, 677, 710, 711, 717, 728, 729 Rumelhart, D. E., 301, 308, 681, 701 Rumsey, J. A., 863, 864, 867, 870, 871, 878 Rumsey, T. S., 923, 931 Runge, V. S., 593, 594, 609 Runner, M. N., 366, 381 Ruscheinsky, D., 511, 517, 523 Rush, R. A., 101, 102, 113 Rusiniak, K. W., xxxi, xxxvi, 396, 398, 419, 420, 427, 434, 838, 843, 913, 928, 931 Ruskin, J., 587, 590 Russchen, F. T., 170, 177, 179, 261, 263, 272 Russell, G. F. M., 922–924, 931 Russell, M. J., 313, 318, 321, 325, 338, 343, 560, 571 Russell, R. M., 893, 897 Russell, R. N., 885, 902 Russell, V. I., 52, 67 Russo, A. F., xv, xlii Russo, J., 585, 588 Rust, N. C., 81, 89 Ruther, U., 117, 136 Rutishauser, U., 123, 128, 134, 136, 1016, 1023, 1025 Rutka, J., 629, 638

1084 Rutowski, R. L., 348, 381 Rutter, M., 343 Ruzicka, E., 484, 500 Ryabchikova, E. I., 551, 571 Ryan, B. A., 894, 897 Ryan, C. M., 525, 546 Ryba, N. J., 351, 382, 798, 798 Ryba, N. J. P., xxv, xxxvii, xl, 709–711, 714, 716, 717, 723, 724–728, 848, 858, 972, 978 Rybczynski, R., 78, 90 Rybicki, B. A., 556, 564, 568 Rydberg, E., xxviii, xlii Rydel, T., 622, 624 Rydzewski, B., 467, 477, 580, 584, 585, 591 Ryo, Y., 971, 978 Rytzner, C., xxv, xxxvi, 661, 672 Ryugo, D. K., 33, 42, 81, 86, 564, 565, 617, 623 Ryymin, P., 442, 454 Ryzhikov, A. B., 551, 571 Rzoska, J., 910, 931 Saad, M. J. A., 888, 901 Saad, S. T. O., 888, 901 Saar, D., 412, 425, 435 Sabatini, L. M., 658, 671 Sabnani, J. B., 444, 457 Saccardi, C., 32, 47 Sacchetta, P., 58, 66 Sachs, B. D., 364, 381 Sachs, C. J., 553, 562, 571 Sachs, S. R., 422, 427 Sachser, N. 381 Sackin, D. A., 893, 895 Sadovsky, E., 280, 289, 293 Saeki, S., 85, 87 Safari, L., 142, 160 Saggese, G., 54, 68, 101–103, 109 Sahakian, B. J., 887, 901 Sahamethapat, A., 562, 572 Sahara, Y., 151, 162, 185, 199 Sahu, S. C., 366, 367, 381 Sai, K., 61, 70 Saiki, C., 659, 675 Sailasuta, A., 562, 572 Saini, K. D., 31, 32, 47 Saino, S., 511, 515, 519 Saint-Pierre, S., 76, 86 Sainz, E., 713, 714, 717, 729 Saishin, Y., 721, 730 Saito, H., 120, 123, 136, 973, 977 Saito, I., 661, 675 Saito, S., 221, 226, 235, 244, 244, 783, 787, 793, 797, 801, 813, 821 Saito, T., 509, 523 Saiz, G., 305, 306 Sajjadian, A., 490, 500 Sakai, F., 963, 966 Sakai, M., 36, 43, 124, 136 Sakai, N., 683, 704, 705, 916–918, 931, 933

Author Index Sakai, S. C., 962, 966 Sakakibara, R., 952, 955 Sakane, T., 553, 561, 562, 571 Sakano, H., xxv, xliv, 182, 200, 287, 294 Sakashita, G., 152, 160 Sako, N., 683, 705, 720, 728, 916, 933 Sakuma, K., 232, 242, 248, 259, 266, 272 Sakuma, Y., 364, 378 Sakumoto, M., 232, 246 Sakurada, T., 471, 476 Sakurai, S., 906, 929 Salas, M., 310, 326 Salata, J. A., 786, 803 Salazar, I., 968, 978 Salcman, M., 551, 556, 558, 565 Salehi-Ashtiani, K., 101, 112, 126, 136 Salem, N., Jr., 394, 398 Sales, M., 58, 69 Saletu, B., 367, 381 Salimbeni, D., 299, 306 Salin, P.-A., 151, 162 Salisbury, P. L., 939, 940, 954, 956 Sallam, F. S., 962, 965 Sallaz, M., 145, 153, 162, 172, 179, 182, 184, 199, 622, 626 Salmelin, R., 241, 246, 259, 266, 271 Salmoiraghi, G. C., 153, 162 Salmon, D. P., xxxii, xxxiii, 480, 481, 483, 496, 500 Salmon, L. R., 847, 848, 850, 856, 858 Salomon, Y., xxvi, xl, 76, 80, 89 Salter, M. W., 712, 729 Saltman, A., 190, 200, 256, 273 Salvador, A., 359, 376, 382 Salvador, R. A., 553, 563, 566 Salvetti, A., 64, 65, 70 Sam, M., 351, 381 Samaan, H., 552, 558, 569 Samaha, A.-N., 357, 378 Samanen, D. W., 94, 96, 112, 129, 130, 133, 616, 626 Samet, J. 996 Sampson, C. T., 786, 803 Sams, J. M., 557, 571 Sams-Dodd, F., 417, 427 Sananes, C. B., 413, 433 Sanchez, L. E., 505, 509, 516, 519 Sánchez Quinteiro, P., 968, 978 Sanchis, C., 359, 382 Sande, K., 892, 896 Sanders, M. S., 167, 168, 172, 176 Sandick, B., 784, 794, 803 Sandmark, B., 586, 590 Sandner, G., 420, 429 Sandre-Banon, D., 554, 572 Sandrini, G., 236, 247 Saneyoshi, M., 553, 562, 573 Sanger. D., 790, 802 Sangha, G. K., 583, 589 Sanghera, M. K., 680, 688, 704 Sanides-Kohlrausch, C., 171, 179 Sano, M., 826, 828, 830, 843 Sano, N., xxviii, xli

Santacana, M., 104, 112, 129, 130, 137 Santangelo, A., 882, 896 Santibanez, G., 410, 435 Santini, M., 167, 172, 177 Santorelli, G., 494, 500 Santos-Benito, F., xxvii, xli, 105, 107, 111 Santucci, A., 56, 57, 61, 70 Sapain, R., 886, 902 Saper, C. B., 563, 564, 571 Saper, J., 553, 573 Saphier, D., 173, 179 Sara, S. J., 192, 194, 413, 435 Sara, V., 101, 113 Sarai, P. A., 767, 771, 779 Saranen, A., 56, 57, 70 Sarangapani, R., 56, 67 Saransaari, P., 149, 161 Sardana, M. K., 509, 521 Saria, A., 784, 800 Sarkar, M. A., 561, 571 Sarles, H., 887, 901 Sarnat, H. B., 355, 381 Sarnat, H. B., 312, 325 Saroli, A., 709, 729 Sarris, E. G., 403, 408 Sarvas, J., 241, 245 Sasai, Y., 116, 136 Sasaki, C. T., 4, 16 Sasaki, T., 232, 248 Sasaki, Y., 471, 476 Sasaki, Y. F., 145, 158, 185, 186, 197 Saski, H., 938, 949, 957 Sass, K. J., 494, 499 Sassoè-Pognetto, M., 149–152, 155, 157, 162 Sato, M., 232, 248, 690, 691, 696, 703, 713, 729, 737, 738, 740–742, 745, 752, 756, 757, 894, 897, 898 Sato, N., 553, 562, 571 Sato, T., xxv, xxxix, xliv, 62, 68, 76, 78, 88, 182, 185, 198, 199, 287, 288, 293, 294, 711, 712, 714, 717, 719–723, 727–730 737, 757 Satoh, M., 102–104, 106, 112, 190, 199 Sattely-Miller, E. A., xxxi, xlii, 635, 638, 884, 902, 951, 956, 961, 966 Saucier, D., 36, 41, 46, 100, 110, 123, 130, 131, 145, 155, 388, 398, 551, 557, 565, 569 Sauders, S. H., 439, 441, 458 Sauer, F. W., 118, 136 Saul, L. B., 713, 729 Saunders, G. F., 116, 133 Saunders, N.A., 441, 458 Saunders, R. C., 511, 517, 518, 521 Sautter, A., 80, 90 Savage, C. R., 686, 702 Saveliev, N. A., xxiv, xlii Savic, I., 174, 179, 258, 259, 262–264, 266–268, 272, 350, 381, 424, 435 Savoy, L. D., 746, 756 Savoy, R. L., 258, 269

Author Index Sawada, H., 968, 978 Sawyer, L., 77, 86 Sawyer, T. F., 416, 435 Saxen, L., 887, 902 Sayer, L. M., 553, 566 Saykin, A. J., 516, 520 Saynor, D. A., 553, 562, 565 Sbarbati, A., 659, 661, 675, 677 Scadding, G. K., 467–468, 475, 476 Scalera, G., 683, 684, 701, 704, 868, 870, 878, 916, 931 Scalia, F., 165, 167, 168, 171, 173, 179, 973, 974, 978 Scalzi, H. A., 663, 676 Scanlan, R. A. 997 Scanlon, L. W., 537, 545 Scapagnini, U., 367, 375 Scarborough, P. E., 61, 71 Scarpa, A., xxii, xlii, 1, 17, 47 Schaal, B., 310–312, 314–319, 321, 323–326, 355, 369, 370, 374, 379, 380, 381, 825, 833–835, 838, 843, 845 Schachner, M., 105, 111, 123, 125, 135–137 Schachter, H., 58, 70 Schacterle, R. S., 536, 545 Schaefer, D., 231, 247 Schafe, G. E., 915–916, 931, 932 Schafer, J., 22, 48 Schaffer, J. P., 825, 845 Schalit, C., 789, 801 Schall, B., 343 Schandar, M., 76, 90 Schank, D., 789, 803, 951, 956 Schank, J. C., 345, 381 Schapiro, S., 310, 326 Scharf, B., 807, 822 Schattmann, L., 423, 431 Schaumburg, H. H., 894, 898, 901 Schechter, P. J., 579, 589, 604, 612, 645, 648, 894, 897, 963, 965 Schecterson, L. C., 762, 781 Scheffler, B., 93, 112, 621, 625 Schellenberg, G. D., xxxii, xxxvii, 341, 483, 498 Schellinck, H. M., xxix, xxxi, xliii, 318, 326, 356, 381, 410, 413, 417, 427, 435, 972, 977 Schellinger, D., 257, 266, 269, 271, 424, 432, 593, 604, 612 Schemmel, R. A., 893, 895 Schenk, F., 418, 432 Scherer, P. W., 55, 69, 266, 270, 448–450, 453, 455–457 Scherr, S., 882, 901 Scherrer, J. L., 447, 458 Scheurink, A. J. W., 887, 902 Schieberl, M. J., 889, 890, 896 Schieferstein, G. J., 552, 571, 582, 590 Schieken, R., 342 Schiet, F.T., 218, 219, 224, 228 Schiff, J. M., xxii, xlii

1085 Schifferstein, H. N. J., 795, 800, 806, 807, 810–820, 821, 822 Schiffman, S. S., xxv, xxxi, xli, xlii, xlv, 212, 216, 228, 461, 477, 480, 500, 501, 559, 571, 605, 610, 635, 638, 714, 715, 716, 718, 729, 734, 757, 788–790, 793–795, 803, 804, 813, 816, 817, 822, 884, 885, 891–894, 895, 901–903, 936, 950–953, 954, 956, 961, 966 Schild, D., 24, 26, 27, 47 Schild, L., 719, 725 Schiller, D., 906–907, 931 Schiller, M., 417, 425, 434 Schimmang, T., 117, 136 Schinkele, O., 658, 676 Schirra, J., 887, 898 Schlaepfer, W. M., 789, 802 Schlatter, C., 994, 999 Schleicher, S., 27, 47, 84, 90 Schleidt, M., 321, 323, 326, 331, 339, 343 Schleidt, W. M., 309, 319, 327 Schlesinger, K., 347, 376 Schley, P., 310, 326 Schlienger, J. L., 886, 902 Schlitzer, J. L., 889, 890, 896 Schlosser, S., 535, 544 Schlotfeldt, C., 463, 473, 476 Schlotfeldt, C. R., 238, 245 Schluesener, H. J., 82, 85 Schmajuk, N. A., 411, 435 Schmale, H., xxiv, xlii, 644, 645, 648, 649, 650, 658, 664, 676, 698, 701 Schmezer, P., 64, 67 Schmidt, C., 2, 16, 99, 112, 356, 374 Schmidt, C. E., 987, 999 Schmidt, F., 386, 400 Schmidt, H. J., 61, 68, 304, 308, 317, 321, 322, 322, 326, 825, 830, 838, 839, 840, 845 Schmidt, M. L., 509, 513, 514, 521, 522, 523 Schmidt, R., 582, 588 Schmidt, U., 2, 16, 99, 112, 356, 374 Schmitt, A., 972, 977 Schmitt, F. A., 507, 510, 521 Schmitt, F. O., 751, 754 Schmitt, P., 618, 625 Schmitz, S., 423, 434 Schmutzler, C., 1004, 1022 Schnabel, K.-O., 991, 999 Schnarch, A., 947, 957 Schneider, C. V., xxii, xlii Schneider, D., xxiv, xlii Schneider, F., 261, 269 Schneider, G. E., xxx, xl Schneider, J. E., 920, 933 Schneider, N. G., 554, 571 Schneider, R. A., 445, 457, 471, 476, 603, 611, 987, 999 Schneider, R. J., 686, 704 Schneider, R. W. S., 185, 199 Schneider, S., 241, 244, 248

Schneider, S. H., 884, 902 Schneider, S. P., 142, 160, 162, 165, 167–169, 173, 177, 179, 188, 200 Schneider, W., 411, 429 Schneider-Axmann, T., 517, 520 Schneyer, C. A., 646, 648, 640, 649 Schneyer, L. H., 640, 649 Schnieden, H., 619, 626 Schnieder, R. A., 11, 16 Schnuerer, J., 305, 306 Schoeller, D. A., 883, 902 Schoenbaum, G., 173–175, 179, 182, 188, 191–193, 199, 412, 421, 425, 426, 426, 435 Schoenfeld, T. A., 141, 142, 146, 147, 160, 162, 168, 171, 179, 185, 198 Schoenfeld, W. A., 127, 134, 603, 610, 1016, 1023 Schoenwolf, G. C., 116, 137 Scholfield, C. N., 190, 199 Schols, L., 509, 514, 521 Scholz, A. T., 426, 430 Schonfeldt-Bausch, R., 518, 519 Schoonover, F. W., 169, 175, 180, 194, 200, 391–394, 400, 410, 426, 436 Schoppa, N. E., 151, 155, 162, 185, 195, 199 Schoppe, S., 491, 500 Schormann, T., 266, 267, 272 Schott, P., 233, 236, 240, 247 Schottenfeld, R. S., 538, 542, 546 Schottler, F., 188, 191–194, 198, 200, 410–412, 414, 426, 433, 434, 436 Schou, M., 910, 914, 932 Schrader, K. A., 24, 42, 80, 82, 87, 89 Schreibman, M. P., 1001, 1003, 1022 Schreiver, W., 645, 649 Schröder, W. H., 26, 47 Schroeder, H. E., 784, 801 Schubert, C., 461, 477 Schubert, M. T., 826, 845, 883, 902, 990, 997 Schuckit, M., 886, 896 Schuckman, H., 410, 411, 435 Schul, R., 421, 435 Schuler, P., 238, 246, 494, 498 Schulkin, J., 683, 700, 907, 924 Schuller D.E., 942, 955 Schultz, C. A., 1313, 1025 Schultz, E., 93, 96, 112 Schultz, E. W., xxvi, xxvii, xlii, 1, 2, 16, 60, 72, 556, 557, 571, 615, 626 Schultz, G., 76, 90, 643, 649, 972, 977 Schultz, G. S., 643, 649 Schultz, H. G., 882, 901 Schultz, J., 788, 801 Schultz, M., 1, 981, 999 Schultz, R. L., 167, 180 Schultz, S., 392, 399 Schultze, M., xxii, xlii, 18, 24, 37, 47 Schultze, V., 444, 458 Schulze, F. E., 652, 676 Schumacher, M. A., 983, 996

1086 Schumm, J., 864, 865, 878 Schurmans, S., xxv, xli Schurmeyer, T., 359, 377 Schuster, I., 55, 56, 68 Schusterman, D. J., 538, 546 Schutz, G., 420, 432 Schutz, S., 304, 308 Schwab, J. M., 82, 85 Schwalbe, G., xx, xxi, xlii, 652, 655, 661, 662, 676 Schwann, T., 652, 676 Schwanzel-Fukuda, M., 122, 127, 132, 136, 137, 389, 400, 1002–1007, 1009–1018, 1021, 1022–1026 Schwarting, G. A., xxvii, xxxix, 102, 103, 106, 110, 119, 121, 135, 972, 978, 1003, 1004, 1013, 1014, 1016, 1025 Schwartz, B., xxvii, xxxvi, 64, 68, 608, 612 Schwartz, B. S., 214, 216, 217, 224, 228, 463, 473, 475, 576, 580, 581, 584, 585, 588, 590, 823, 842 Schwartz, D. N., 452, 457 Schwartz, G. E., 244, 247, 342, 526, 535, 538, 543, 544, 587, 588 Schwartz, G. J., 683, 700, 732, 757, 862, 866–868, 877, 878 Schwartz, H. G., 667, 676 Schwartz, J., 581, 588 Schwartz, J. H. 998 Schwartz, L. B., 443, 451, 459 Schwartz, L. K., 646, 650 Schwartz, M. D., 838, 845 Schwartz, M. S., 542, 543 Schwartz, S., 646, 650 Schwartz, T. W., 888, 902 Schwartz Levey, M. A., 118, 119, 137, 616, 626 Schwartzbaum, J. S., 913, 932 Schwartzkroin, P. A., 76, 85 Schwarzer, C., 151, 161 Schweikert, H. U., 472, 476 Schweitzer, C., 723, 728 Schweitzer, V. G., 600, 612 Schwob, J. E., 14, 15, 33, 35, 37, 38, 43, 44, 47, 53, 62, 65, 69, 72, 76, 87, 96, 98, 100–102, 106, 109, 110, 112, 118–125, 127, 133, 134, 137, 138, 145, 147, 162, 163, 168, 179, 229, 230, 233, 243, 247, 388, 392, 394, 396, 400, 463, 472, 476, 477, 616, 617, 621, 624, 626, 1017, 1025 Sclafani, A., 683, 701, 744, 755, 794, 795, 800, 803, 862, 875, 877, 892, 902, 906, 921, 924, 927, 929, 932 Scoggin, J. A., 553, 562, 570 Scolari, R., 243, 247 Scott, A. E., 466, 477 Scott, C. L., 683, 701 Scott, D. K., 422, 437 Scott, J. W., xxiv, xlii, 27, 32, 36, 41, 47, 123, 131, 139, 142, 150, 155,

Author Index 161, 163, 168, 171, 179, 182–188, 194, 196, 198–200, 289, 294, 396, 397 Scott, K. C., 564, 565 Scott, L., 774, 781 Scott, T. R., 680, 681, 683–686, 688, 690–692, 699, 700–702, 704, 705, 731, 734–736, 740, 744, 745–748, 751, 752, 754, 755–758, 783, 797, 802–804, 830, 845, 873, 878, 882, 902, 918, 926 Scott, W. R., 448, 458 Scotti, A., 105, 112 Scrivani, S. J., 946, 947, 956, 957 Scriven, R., 285, 293 Seago, J. D., 406, 408 Sears, S. B., 204, 227, 231, 248, 255, 271, 387, 399 Sebens, J. B., 512, 522 Seebeck, T., 321, 327, 343 Seeburg, P. H., 150, 159 Seeley, R. J., 680, 683, 701, 703 Seery, C. S., 480, 481, 500 Seffrin, L., 386, 400 Segal, N. L., 329, 330, 331, 332, 333, 336, 338, 343 Segal, S. Jr., 4, 15 Segerson, T. P., 149, 151, 158, 162, 185, 199 Segerstad, A. T., 713, 725 Sego, R., 642, 649, 670, 674, 939, 954 Segovia, C., 825, 842 Segovia, S., 975, 978 Segrest, J. 342 Seguin, S., 148, 155 Sehgal, A., 61, 66 Sehlmeyer, F., 57, 69 Seibel, J., 552, 568 Seibly, W. S., 167, 180 Seidemann, E., 280, 289, 293, 294 Seiden, A. M., 14, 16, 37, 46, 445, 458, 463, 468, 472–473, 475, 477, 630, 637, 788, 790, 800, 803 Seidman, B. C., 585, 589 Seidman, L. J., 491, 492, 500, 501 Seifert, G., 659, 674 Seifert, K., 17, 18, 19, 23, 24, 30, 35, 36, 47 Seifert, R., 76, 87, 716, 722, 728, 729 Seiger, A., 101, 113, 670, 674 Seilheimer, B., 123, 137 Seitz, M., 305, 306 Seixas, N. S., 564, 567 Seki, K., 507, 509, 523 Seki, M., 552, 557, 571 Seki, T., 553, 562, 571, 1003, 1016, 1023 Sekiguchi, R., 415, 416, 436 Sekine, I., 507, 510, 512, 521 Sekinger, B., 212, 215, 226, 462, 475, 485, 496 Seldner, A. C., 893, 902 Selfridge, O. G., 296, 308 Sell, R., 276, 294

Sellers, A. H., xxvi, xliv, 557, 572 Selliseth, N. J., 660, 676 Selner, J. L., 539, 540, 542, 546 Selner, M. E., 539, 540, 542, 546 Seltzer, B., 680, 695, 704 Selvig, K. A., 660, 676 Semb, G., 204, 209, 228 Semb, H., 482, 499 Semenov, H., 439, 441, 458 Semenov, L. V., 915, 925 Semira, C., 960, 963, 966 Semke, E., 370, 381 Sémon, E., 370, 381 Senba, E., 123, 137 Sendera, T. J., 143, 160 Sengoku, S., xxv, xliv, 287, 294 Sengstake, C. B., 918–919, 925, 932 Sengupta, P., 76, 78, 87, 90 Senior, C., 687, 702 Sennewald, K., 795, 803, 884, 902 Seo, R., 242, 249, 259, 269, 271 Seppala, T., 553, 562, 572 Septa, L. N., 34, 35, 48 Serabjit-Singh, C. J., 57, 58, 66 Serafetinides, E. A., 268, 272, 424, 435, 603, 604, 612 Serby, M. J., xvi, xxxvii, 121, 137, 216, 228, 424, 436, 479–481, 483, 485, 491, 492, 495, 496, 500, 501, 600, 605, 608, 612, 891, 893, 895, 996 Serclerat, D., 482, 500 Sergeev, A. N., 551, 571 Series, F., 440, 457, 458 Serin, J., 890, 902 Serizawa, S., 182, 200 Seroogy, K. B., 151, 152, 156, 164, 183, 196 Serrano, M. A., 359, 376 Sessle, B. J., 786, 788, 790, 791, 802952, 955 Sesti, F., 27, 46, 80, 86 Seta, Y., 661, 664, 672, 676 Seto, K., 973, 977 Settle, R. G., xxxi, xxxv, 204, 216, 224, 225, 266, 270, 440, 454, 463, 471, 474, 480, 495, 496, 497, 595, 604, 609, 629, 635, 637, 721, 729, 787, 788, 790, 791, 795, 799, 803, 886, 890, 892, 894, 896, 902, 935, 938, 945, 950, 951, 954, 957, 959, 961, 963, 965, 966 Settles, A. M., 776, 781 Setzer, A. K., 394, 400 Severin, E., 300, 307 Sezaki, H., 553, 561, 571 Shaalan, H., 7, 15 Shaber, G. S., 863, 864, 867, 870, 871, 878 Shafer, D., 790, 802 Shafer, D. M., 789, 803, 946, 957 Shafer, J. A., 509, 521 Shaffer, G., 210, 225, 788, 801 Shaffer, G. S., 853, 854, 859

Author Index Shafton, A. D., 153, 159 Shah, A., 829, 841 Shah, U., 508, 510, 514, 520 Shaio, E., xxxi, xlii, 635, 638, 961, 966 Shair, H., 310, 323 Shair, H. N., 310, 326 Shakib, S., 444, 453, 458 Shalchiantabrizi, C., 971, 979 Shallenberger, R. S., 714, 729 Shaman, P., xxiv, xxxv, xxxix, 107, 108, 204, 206, 212, 213, 215–217, 220, 224, 225, 227, 332, 341, 440, 454, 461–463, 471, 474, 480, 489, 495, 496, 497, 593, 608, 629, 633, 635, 637, 783, 787, 788, 790, 791, 795, 799, 800, 890, 894, 896, 935, 938, 945, 950, 951, 954, 959, 961, 963, 965 Shammah-Lagnado, S. J., 165, 179 Shang, H., 698, 702 Shanker, Y. G., 710, 713, 714, 717, 726, 728 Shanmugalingam, S., 116, 137 Shannahoff-Khalsa, D. S., 450, 456 Shannon, C. E., 738, 757 Shannon, D., 289, 294 Shannon, D. E., 289, 294 Shannon, I. L., 641, 649 Shapera, M. R., 832, 845, 893, 902 Shapiro, A., 204, 225, 450, 454, 525, 537, 544, 587, 588 Shapiro, C., 883, 900 Shapiro, L. S., 972, 977 Shapiro, M., 425, 429 Shapiro, M. D., 593, 594, 598, 612 Shapiro, R. E., 916, 932 Shapiro, R. M., 512, 517, 519 Sharav, Y., 947, 957 Sharif, Z., 516, 521 Sharma, K. N., 882, 900 Sharma, S. D., 882, 900 Sharon, D., 145, 157 Sharp, A. H., 33, 42, 81, 86, 512, 514, 520 Sharp, F., xxviii, xxxviii Sharp, F. R., xxviii, xxix, xlii, 145, 163, 190, 200 Sharp, K. A., 291, 293 Shatzman, A. R., 645, 649, 664, 676, 891, 900 Shaw, A., xxi, xlii, 981, 999 Shaw, E. 844, 907, 931 Shaw, G. K., 602, 612 Shaw, J. L., 831, 845 Shaw, N., 873, 878 Shaw, U., 510, 514, 520 Shay, U., 481, 500 Shear, P. K., 886, 896 Sheares, B. T., 644, 649 Shearman, M. S., 509, 521 Shedlack, K. J., 192, 195, 411, 421, 425, 428 Sheedy, J. E., 576, 579, 590

1087 Sheehe, P. R., 118, 138, 185, 201, 204, 227, 267, 273, 388, 392, 394, 396, 398, 400, 442, 445, 452, 455, 457, 795, 804, 839, 844 Sheelar, S., 867, 869, 870, 878 Shehata, O., 962, 965 Shehin-Johnson, S. E., 56, 72 Sheikh, S. P., 291, 294 Sheinberg, D. L., 193, 198 Sheldon, P. W., 170, 179 Sheldon, R. E 1001, 1025 Sheldon, W. G., 552, 571, 582, 590 Shelhamer, J. H., 54, 71 Shelley, E. D., 495, 501 Shelley, G., 359, 373 Shelley, W. B., 495, 501 Shemen, L., 443, 458 Shen, C. Y., 581, 591 Shen, F. M., 855, 859 Shen, M. M., 116, 138 Shen, X.-L., 971, 978 Sheng, J., 55, 57, 62, 65, 68, 72 Shephard, B. C., 238, 245, 479, 482, 485, 486, 488, 490, 498, 501, 514, 520 Shepherd, G. M., xxiv, xxvi, xxviii, xxxvi, xxxviii, xli, xlii, xliii, 27, 42, 76, 81, 84, 87, 88, 91, 139, 141–146, 148–153, 155–157, 159, 161–164, 167, 169, 172, 173, 177, 181–186, 190, 193, 194, 195, 198–201, 220, 228, 258, 273, 276, 280, 289, 293, 294, 296, 351, 355, 377, 380, 396, 399, 552, 558, 561, 569, 621, 624 Shepherd, R., 696, 703, 893, 902 Sheppard, N. F. Jr., 149, 158 Sher, L., 472, 477 Sherer, T. B., 564, 565 Sherins, R. S., 1017, 1021 Sherman, B. M., 922–923, 928 Sherman, P. W., xix, xxxiv, 336, 342, 343 Sherman, T. A., 662, 673 Sherrington, C. S., xxviii, xlii, 252, 272 Sherry, D. F., 355, 376, 908, 927 Sherwood, M. R., 923, 925 Sherwood, N. M., 1004, 1012, 1024, 1025 Shevrygin, B. V., 445, 458 Shi, J., 971, 978 Shi, X. P., 509, 521 Shiba, Y., 661, 674 Shibuya, T., xxiv, xxxviii, 83, 88, 124, 137 Shick, T. R 797, 802 Shide, D. J., 829, 841 Shields, C. B., 103, 110 Shields, J., 332, 343 Shigematsu, K., 555, 571 Shih, C. T., 940, 957 Shih, D. M., 539, 544 Shikata, H., 874, 878 Shimada, A., 36, 43, 553, 569 Shimada, K., 259, 269, 271 Shimada, S., 721, 730

Shimada, T., 232, 246 Shimada, Y., 717, 727 Shimamura, A., 660, 664, 676 Shimamura, K., 117, 136 Shimazaki, K., 713, 729 Shimizu, E., 420, 435 Shimoda, N., 554, 561, 571 Shimomura, F., 170, 179 Shimura, T., 682, 683, 701, 704, 705, 916–918, 927, 928, 933 Shin, H. W., 305, 307 Shingai, T., 888, 894 Shinohara, M. M., 149, 151, 158 Shiosaka, S., 102, 105, 110 Shiotsuka, R. N., 583, 589 Shipley, M. T., 60, 71, 72, 100, 102, 103, 105, 109–111, 119, 120, 122, 123, 125, 127, 129, 130, 131, 133, 139, 142, 143, 146–149, 151–154, 155–163, 165, 167–169, 171, 172, 176, 180, 181, 183–185, 187, 191, 192, 194–201, 268, 272, 409, 413, 424, 431, 436, 550, 551, 556, 558, 560, 571, 572, 664, 676, 948, 957, 973, 977 Shiraishi, S., 102, 105, 110 Shirley, S. G., xxvi, xlii, 560, 572 Shiroky, J., 647, 648 Shklair, I. L., 643, 648 Shlapack, W., 210, 227 Shoji, M., 510, 512, 522 Shore, A. B., 848, 849, 851–853, 856–858, 858, 859 Shore, D. I., 220, 225 Shore, F. M., 204, 228 Shorey, H. H., 348, 381 Shors, T. J., 366, 381 Shou, J., 97, 101–103, 105, 112 Shpirer, I., 443, 457 Shrager, E. E., 892, 896 Shrestha, R., 656, 673 Shrott, M., 857, 860 Shryne, J. E., 923, 932 Shu, X., 736, 754 Shuangshoti, S., 515, 520 Shuber, A. P., 343 Shuford, E. Jr., 862, 878 Shuler, M. G., 766, 782 Shulman, R. G., 258, 273 Shultz, G., 716, 728 Shumrick, D. A. Shusterman, D. J., 576, 579, 586, 590, 634, 637 Shusterman, Z., 557, 568 Shuvaeva, T. M., 54, 71 Shvets, N. M., 991, 999 Shyu, W. C., 553, 562, 572 Sibley, D. R., 84, 90 Sicard, G., 102, 103, 108, 145, 162, 182, 200, 258, 264, 265, 266, 272, 314, 325, 335, 343, 424, 435 Sicotte, N., 480, 481, 491, 496 Siddeek, M., 258, 273

1088 Siddiqi, F., 259, 264, 265, 266, 273, 340, 344 Siddiqi, M., 552, 568 Siddiqi, O., 340, 343 Siddiqui, W. H., 583, 589 Sidky, M., xxii, xlii Sidman, J., 982, 994, 996, 999 Sidman, J. D., 982, 999 Sidman, R. L., 621, 624 Sidtis, J., 605, 612 Siegel, A. M., 268, 270, 424, 431 Siegel, M. I., 3, 16, 17, 48, 969, 979 Siegel, R. E., 651, 676, 951, 957 Siegel, S., 538, 546, 586, 590 Siegelbaum, S. A., 80, 90 Sieghart, W., 151, 161 Sienaert, I., 26, 41 Sienkiewicz, Z. J., 680, 681, 685–689, 691, 692, 703–705, 736, 746, 748, 757, 758 Sigman, M., 190, 196 Sigman-Grant, M., 832, 843 Sigmundi, R. A., 909, 924 Signoret, G. P., 348, 381 Siklos, L., 152, 163 Siklosi, G. 341 Sikorsky, L., xxxi, xxxv, 107, 108, 213, 216, 225, 340, 461, 474 Silberberg, A. M., 354, 381, 394, 399, 400, 410, 414, 433, 436 Silbersweig, D., 516, 520 Silkoff, P. E., 442, 458 Sillers, M. J., 445, 455 Silos-Santiago, I., 767, 771, 779 Silva, A. J., 417, 420, 432, 436 Silva, D. A., 945, 957 Silver, F. L., 516, 520 Silver, J., 105, 109, 130, 133 Silver, W. L., xxix, xxxvii, 24, 37, 38, 40, 44, 47, 103, 112, 165, 170, 179, 204, 227, 232, 248, 351, 383, 388, 400, 715, 718, 720, 725, 746, 757, 802, 982, 983, 985, 996, 998, 999 Silverman, A. J., 122, 132, 136, 1003, 1004, 1007, 1009, 1024, 1025 Silverman, F. S., 539, 546 Silverman, K., 535, 546 Silverman, S. J., 952, 955 Silverman, S. L., 554, 563, 572 Silverstein, R. M., xxix, xxxix, xl, 346, 373 Silvoniemi, P., 451, 458 Silvotti, L., xv, xvi, xxxiii Simbayi, L. C., 916, 918, 932 Simbruner, G., 826, 845, 883, 902 Simeone, A., 116, 131, 137 Simmen, B., 847, 859 Simmen, D., 447, 458, 462, 477 Simmons, A., xxix, xlv, 256, 259, 266, 267, 273 Simmons, J. T., 602, 612 Simmons, R. D., 554, 563, 571 Simmons, S. R., 662, 672, 713, 726

Author Index Simms, I., 483, 484, 499, 500 Simola, M., 445, 458, 467, 477 Simon, C., 886, 902 Simon, G. E., 532, 533, 535, 539, 541, 546 Simon, M. I., 710, 727 Simon, S. A., xvi, xlii, 659–661, 663, 664, 670, 673, 674, 675, 715, 720–722, 726, 727–730, 740–742, 744, 745, 747, 757, 758 Simon, T. R., 536, 539, 546 Simon, T. W., 220, 228 Simon, V. M., 359, 382 Simonetto, I., 170, 179, 413, 435 Simonian, S. X., 921, 932 Simonin, C., 621, 624 Simpkins, J. W., 920, 927, 932 Simpson, D., 786, 791, 801 Simpson, F. O., 886, 902 Simpson, R. W., 472, 477 Simpson, S. J., 680, 692, 703 Sincali, A. J. 306, 308 Singer, A., 318, 327 Singer, A. G., 346, 351, 356, 364, 369, 381, 373, 381, 383, 970, 971, 977, 978, 1003, 1025 Singer, A. W., 582, 591 Singer, M. S., 146, 163, 181, 200, 276, 289, 293, 294 Singh, A., 486, 493, 496, 497 Singh, B., 492, 496 Singh, N., 470, 477 Singh, P., 310, 323 Singh, P. B., 320, 322, 356, 381 Singh, P. J., 310, 326 Sinnarajah, S., 26, 27, 48 Sinowatz, F., 36, 47 Sipila, J., 451, 458 Sirota, P., 491, 492, 493, 498, 501 Siskos, P., 525, 528, 543 Sisley, A. C., 721, 729, 788, 803 Sisson, G. A. Sr., 597, 612 Sitcheran, R., 28, 48, 99, 113, 130, 137, 146, 164, 560, 572 Sito, E., 887, 898 Sivak, M., 490, 500 Sjöberg, L., 204, 225 Skakura, Y., 37, 49 Skarda, C. A., 186, 196 Skarnes, W. C., 621, 624 Skeen, L. C., 165, 180 Skierkowski, P., 170, 180 Skinhoj, E., xxviii, xxxviii, 253, 271 Skinner, B. F., xxix, xlii Skinner, J. D., 833, 845 Skinner, J. E., 170, 177, 413, 430 Sklar, P. B., xxiv, xxvi, xli, xlii, 76, 77, 79, 80, 89, 90, 560, 571 Sklavounou, A., 941, 955 Skoda, A., 855, 860 Skoletsky, J., 343 Skoufos, E., 291, 294 Skudlarski, P., 259, 264–266, 270

Skyberg, K., 608, 612 Slack, W. V., 886, 903 Sladek, J. R., 130, 135 Slangen, J. L., 923, 926 Slater, J. M., 300, 308 Slaughter, C., 24, 44 Sleiffer, D. T., 891, 900 Sleigh, M. A., 24, 48 Sleight, S. D., 553, 569, 583, 590 Slev, P. R., 321, 322 Sloan, H. E., 773, 781 Sloan, H. R., 642, 649 Slob, A. K., 923, 932 Slootweg, P. J., 598, 610 Slot, W. B., 554, 563, 572 Slotkin, T. A., 320, 324 Slotnick, B. M., xxix, xxxi, xxxiv, xliii, 145, 159, 163, 169–175, 179, 180, 192, 194, 198, 199, 200, 218, 228, 310, 325, 354, 358, 379, 380, 381, 387, 389, 390–394, 397, 397, 398, 399, 400, 404, 408, 409–411, 412–414, 420–422, 425, 426, 428, 432, 433, 435–437, 863, 867–870, 874, 876, 878 Slutsker, L., 883, 900 Slutsky, A. S., 441, 458 Small, D. M., 258, 262, 264, 266, 267, 272, 698, 704, 797, 803, 948, 957 Small, S. L., xxix, xliii, 616, 623 Smallman, R. L., xxvii, xxxiii, 663, 671, 769, 778, 939, 954 Smargiassi, A., 581, 589 Smart, I., 620, 626 Smart, I. H. M., 93, 112, 118, 137 Smart, J. C., 885, 900 Smeets, M. A. M., 923, 926 Smeland, E. B., 62, 69 Smets, R. J. M., 920, 931 Smirnov, Y. V., 116, 138 Smit, D., 356, 374 Smit, J. M., 891, 900 Smit Vanderkooy, P. D., 894, 897 Smith, A. D., 482, 499, 513, 522 Smith, A. L., 509, 521 Smith, B. A., 826, 828, 829, 841, 845 Smith, B. E., 444, 457 Smith, B. H., 185, 186, 200, 394–396, 399 Smith, B. P., xxxi, xl, 216, 227 Smith, C., 644, 649 Smith, C. G., 1, 16, 93, 96, 112, 512, 523 Smith, D. A. R., 214, 225 Smith, D. B., 230, 248 Smith, D. R., 394, 396, 400 Smith, D. U., 893, 903 Smith, D. V., xvi, xliii, 14, 16, 37, 46, 445, 458, 468, 472–473, 473, 477, 658, 661, 664, 670, 672, 674–676, 710, 720, 725, 726, 729, 732, 733, 735–737, 739–753, 754–758, 767, 778, 783–785, 788, 790, 791, 794, 797, 800, 802–804, 866, 876, 948, 957, 962, 963, 965

Author Index Smith, F. J., 888, 902 Smith, F. R., 471, 476, 620, 625, 892, 893, 900, 902, 950, 956 Smith, G., 923, 928 Smith, G. N., 516, 521 Smith, G. P., 864, 876, 878, 918, 928 Smith, I., 553, 562, 565 Smith. J., xxiv, xliii Smith, J. C., xvi, xxix, xliii, 360, 382, 392, 399, 741, 756, 863–865, 867, 869–871, 873, 877–879, 910, 918, 932, 987, 998, 999 Smith, K. G., 946, 953, 956 Smith, L., 539, 544 Smith, M., 343 Smith, M. A., 419, 429 Smith, M. B., 256, 257, 273 Smith, M. H., 404, 408 Smith, P., 713, 727 Smith, P. C., 766, 782 Smith, P. E., xxxi, xl, 216, 227 Smith, P. K., 915, 926 Smith, R., 212, 215, 225 Smith, R. A., 36, 37, 41, 47, 64, 69 Smith, R. L., 29, 48, 151, 152, 163, 514, 523 Smith, T., xxx, xliii, 124, 135 Smith, T. D., 3, 16, 17, 48, 969, 979 Smith, T. J., 57, 72 Smith, V. L., 748, 757 Smithana, J. M., 735, 756 Smith-Crafts, L. K., 184, 194 Smithson, K. G., 169, 170, 180 Smith-Swintosky, V. L., 690, 702, 746, 758, 797, 802 Smotherman, W. P., xxxi, xliii, 123, 137, 312, 315, 326, 355, 382, 833, 838, 841, 842, 845, 907, 918, 929, 932 Smout, A. J. P. M., 686, 704 Smutzer, G. S., 153, 159, 505, 509, 511, 516, 517, 519, 523 Sneath, R. W., 300, 305, 307 Sneddon, H., 315, 326 Sneddon, L., 946, 954 Sneesby, K., 101, 109 Snell, T. C., 741, 747, 748, 756 Snider, W. D., 761, 762, 764, 782 Sniffen, J., 832, 844 Snow, D. T., 366, 382 Snow, J. B., Jr., xxxi, xxxv, 2, 16, 35, 37, 42, 98, 111, 119, 132, 172, 178, 207, 208, 210–212, 216, 224, 311, 325, 339, 340, 440, 454, 479, 480, 497, 588, 600, 602, 604, 609, 622, 625, 629, 635, 637, 639, 648, 656, 660, 674, 709, 728, 732, 755, 759, 765, 768, 773, 780, 787, 788, 790, 791, 795, 799–803, 840, 890, 894, 896, 918, 924, 935, 937, 938, 945, 950, 951, 954, 955, 959, 961, 963, 965, 982, 997–999 Snow, R. B., 604, 610 Snowdon, C. T., 405, 408, 735, 756

1089 Snowman, A. M., 76, 89 Snutch, T. P., 79, 87 Snyder, D. J., 853–855, 857, 859, 860 Snyder, E. Y., 621, 624, 627 Snyder, L. H., 847, 848, 860 Snyder, P., 209, 217, 224, 228, 234, 245 Snyder, P. I., 984–987, 991, 997 Snyder, P. J., 127, 138, 469, 472, 474, 478 Snyder, R. L., 366, 382 Snyder, S., 709, 727 Snyder, S. H., xxiv, xxvi, xxxiii, xli, xlii, 33, 41, 42, 58, 73, 76–82, 86, 89, 90, 125, 136, 280, 293, 560, 571, 645, 649, 658, 673, 999 Sobel, N., xxix, xliii, 174, 180, 190, 200, 255, 256, 259–267, 271–273, 424, 436, 452, 458, 486, 501 Sobotta, J., 669, 676 Soderstrom, D., 586, 589 Soemmering, S. T., 654, 658, 664, 670, 676 Soergel K. H., 644, 648 Sofferman, P. A., 960, 966 Soffie, M., 416, 436 Sojka, B., 233, 234, 236, 239, 240, 247, 248, 350, 380, 469, 474 Sokoloff, L., xxviii, xxxvi, xxxviii, xli, xliii, 253, 271 Sokoloff, R. Sokolov, V. E., 422, 437 Solberg, B., xxx, xxxiv Solero, C. L., 621, 625 Sollars, S. I., 766, 776, 782 Solomon, F., 950, 954 Solomon, G. S., xxxii, xxxxix, xliii, 480, 482, 499, 501 Som, J., 664, 670, 676 Som, P. M., 593, 594, 596–598, 612 Somlyo, A. V., 81, 90 Sommers, P., 873, 877 Sommerville, B. A., 318, 324, 326, 342 Son, J. H., 511, 515, 519 Song, B. H., 468, 476 Song, Q., 101, 103, 113 Song, Z. Y., 538, 543 Songsanand, P., 481, 498 Sonnenburg, W. K., 82, 85, 716, 730 Soong, C. W., 553, 562, 572 Soong, T. K., 449, 459 Sooy, K., 511, 522 Sorensen, M., 786, 802 Sorensen, P. W., 61, 72, 351, 353, 382, 389, 399, 417, 434, 660, 675, 969, 979, 1005, 1023 Sorensen, S. C., xxviii, xxxviii Sorg, B. A., 537, 538, 543, 546 Soria, E. D., 947, 957 Soriano, E., 143, 157 Soriano, E., 166, 178 Sorokin, S. P., 52, 72 Sorri, M., 57, 70 Sosa, I. R., 343 Sotelo, C., 143, 157, 166, 178

Soteres, B. J., 620, 627 Sotiropoulos, V., 357, 378 Soucheier, C., 186, 199 Souchier, C., 145, 162 Soucy, J., 888, 898, 902 Soukup, T., 764, 781 Soulier, N., 388, 397 Soumireu-Mourat, B., 170, 179, 411–413, 425, 427, 435 Soussignan, R., 314–316, 324, 326, 355, 381, 833, 834, 843, 845 Soussi-Yanicostas, N., 1017, 1022 Southwick, C. H., 336, 342, 320, 324, 356, 379 Souville, C., 887, 901 Sower, S., 1003, 1004, 1013, 1014, 1022, 1025 Spaeth, J., 444, 458 Spalding, P. M., 441, 453 Spangler, K. M., 667, 674 Spano, P. F., 513, 514, 518, 521 Sparkes, R. S., 472, 477 Sparks, D. L., 54, 56–58, 69, 71, 507, 510, 520 Sparks, J., 392, 399 Sparks, L., 483, 498, 560, 570, 577, 589, 843 Sparks, P. J., 532, 533, 535, 539, 541, 546 Spear, L. P., 361, 377 Spear, N. 843 Spear, N. E., 319, 322, 361, 377, 908, 932 Spearman, C. R., 585, 590 Speca, D. J., 969, 979 Spector, A. C., xxxi, xliii, 732, 734, 738, 741, 751, 756, 758, 766, 782, 861–874, 875, 876–879, 910, 916, 918, 931, 932 Speedie, L. J., 481, 489, 499, 602, 611 Speelman, H., 597, 610 Speert, D., 98, 108 Spence, C., 236, 248 Spence, M. J., 833, 841 Spencer, D. D., 29, 48, 152, 163, 494, 499, 514, 523 Spencer, J., 368, 382 Spencer, K. M., 233, 240, 248 Spencer, M. M., 404, 407 Spencer, P. S., 894, 898 Spencer, R. F., 551, 556, 560, 561, 565, 572 Spencer, W. A., 243, 248 Spengler, J. D. 996 Sperk, G., 151, 161 Sperling, H. G., 750, 751, 755 Sperling, M. R., 493, 494, 498, 501 Sperry, R. W., 387, 399, 410, 432 Spickofsky, N., 664, 675, 710, 711, 716, 717, 728, 729 Spiegel, A. M., 471, 477 Spiegel, D. K., 356, 373 Spiegel, R., 480, 481, 495, 498 Spiegelhalder, B., 552, 568 Spieker, C., 891, 903

1090 Spielman, A. I., 20, 36, 38, 45, 48, 639, 645, 649, 650, 658, 676, 709–712, 726, 729, 730 Spiers, W., 788, 802 Spies, H. G., 923, 932 Spies, H. S., 356, 374 Spies, T. D., 891, 900 Spigelman, M. N., 887, 898 Spillantini, M. G., 510, 513, 514, 523 Spinelli, S., 19, 48 Spink, B. C., 61, 69 Spink, D. C., 57, 61–63, 68, 69, 73 Spironello, E., 367, 382 Spironello-Vella, E., 365–367, 375, 382 Spitzer, L., 892, 902 Splendiani, A., 602–603, 609 Spoor, A., 439, 458 Sprague, J. M., 747, 756 Spreca, A., 27, 32, 47, 48 Spreekmeester, E., 367, 379 Sprich, W. W., 167, 168, 171, 179, 255, 272 Springer, K., 204, 206, 228 Sprissler, C., 661, 676 Spruijt, B. M., 417, 433 Spurgeon, A., 526, 546 Spyker, D. A., 538, 539, 542, 544, 546 Squier, C. A. 800 Squinto, L. M., 104, 108, 119, 122, 133 Squire, L. R., 414, 438, 602, 612 Squires, A., 101–103, 110 Squires-Wheeler, E., 511, 516, 521 Sreebny, L. M., 646, 647, 650 Sreenivasan, K. V., 492, 501 Srikumar, D., 26, 27, 48 Srinivas, N. R., 553, 562, 572 Sripanidkulchai, K., 167, 169, 170, 172, 180 Srivastava, L., 101, 108, 151, 156, 184, 194, 619, 626 St. John, J. A., 126, 137 St. John, S. J., xvi, xliii, 664, 670, 676, 734, 738, 740, 741, 751, 753, 754, 756, 758, 766, 782, 864, 865, 867, 868, 871, 874, 875, 877, 879 Stacey, N. E., 61, 72, 353, 382, 389, 399, 1005, 1023 Stackenwalt, G., 153, 163 Stadlan, E. M., 479, 499 Stahlbom, P.-A., 101, 107 Stahlman, M. T., 366, 382 Stain, E. V., 535, 546 Stallings, V. A., 883, 902 Stammberger, H., 6–10, 15, 16 Stanley, R.S., 123, 135 Stanton, B. A., 719, 721, 725 Staples, V., 482, 498, 505, 510, 513, 520 Stappen, I., 486, 501 Starcevic, S. L., 56, 58, 65, 72 Stare, F. J., 886, 903 Stark, B., 356, 382 Stark, M. R., 116, 137 Starkstein, S. E., 602, 612

Author Index Starr, A., 234, 236, 245 Stasky, A. A., 96, 112, 120, 125, 137, 616, 626 Staszewski, L., 723, 727 Staubli, U., 190–194, 199, 200, 410–415, 434, 435–437 Staudenmayer, H. J., 539, 540, 542, 546, 586, 589 Staunton, H., 268, 270, 424, 431 Steadman, J. W., 745, 757 Stebbins, W. C., xxix, xliii, 389, 400, 869, 876 Steckler, T., 619, 626 Stedman, H. M., 658, 671 Stedman, T. J., 492, 501 Steel, E., 970, 979 Steele, J. C., 480, 483, 488, 497, 563, 567 Steenland, K., 585, 588 Stefan, H., 235, 236, 238, 241, 246, 248, 259, 271, 424, 431, 494, 498 Steffens, A. B., 887, 902 Stegeman, J. J., 57, 71 Stehn, R. A., 365, 382 Stein, D. J., 472, 477 Stein, G. W., 405, 408 Stein, J. F., 266, 273 Stein, L. J., 830, 833, 840., 845, 886, 902 Stein, M., 419, 429, 430 Steinberg, C., 554, 571 Steinbrecht, R. A., 26, 35, 48 Steindler, D. A., 93, 112, 621, 625 Steiner, J. E., 481, 500, 682, 704, 826–828, 834, 841, 842, 845, 866, 879, 883, 884, 902, 906, 917, 927, 932, 950, 951, 952, 954, 955, 957 Steiner, R. A., 923, 932 Steiner M., 539, 544 Steiniger, von, F., 910–911, 932 Steinmetz, K. A., 858, 860 Stell, W. K., 1004, 1025 Stellar, E., 862, 879, 892, 896 Stellar, S., 238, 245, 484, 497, 513, 520, 563, 567 Stellman, S. D., 882, 902 Stem, M. B., 215, 225 Stenn, P. G., 539, 542, 546 Stenquist, B., 888, 902 Stenroos, E. S., 510, 514, 522 Stensaas, L. J., 311, 324, 969, 979 Stephens, L., 221, 226 Stephens, R., 526, 546 Stephens, R. E., 24, 48 Steranka, L. R. 999 Stern, I. B., 666, 671, 764, 778, 824, 841 Stern, J., 885, 892, 901, 903 Stern, J. J., 404, 408 Stern, K., 350, 382 Stern, M. B., 484–486, 497, 501, 514, 520, 521, 602, 609 Stern, R. C. 343 Stern, Y., xxxii, xxxv Stetson, S., 495, 498 Stetter, J. R., 299, 300, 306, 308

Stevens, B., 829, 845 Stevens, B. J., 829, 842 Stevens, C. N., 445, 458, 468, 477 Stevens, D. A., xxix, xxxi, xliii, 404, 408, 713, 729, 813, 821 Stevens, D. R., 722, 729 Stevens, J. C., 207, 211, 215, 216, 219, 227, 228, 388, 400, 441, 445, 450, 452, 455, 456, 709, 726, 740, 754, 787, 789, 803, 807, 812, 818, 819, 822, 856, 859 Stevens, M. H., 468, 477 Stevens, R., 920, 926 Stevens, S. S., 203, 204, 210, 228, 787, 803, 811, 822, 854, 860, 873, 879 Stevens, T. J., 602, 613 Stevenson, R. J., 818, 822 Steward, D. J., 838, 841 Stewart, C., 643, 649 Stewart, C. A., 619, 626 Stewart, C. L., 27, 41, 123, 131, 150, 155, 396, 397 Stewart, D., 587, 590 Stewart, F. W., 1002, 1003, 1025 Stewart, J. M. 999 Stewart, K. K. 802 Stewart, R. A., 886, 903 Stewart, R. B., 885, 902 Stewart, R. E., 722, 729, 759, 775, 776, 779, 782 Stewart, T. J., 621, 625 Stewart, W., 581, 588, 590 Stewart, W. B., xxix, xliii, 141, 145, 157, 163, 182, 200, 220, 228, 355, 380, 551, 556, 558, 572 Stickrod, G., 123, 137, 315, 326, 355, 382, 838, 845, 907, 932 Stiernburg, C. M., 450, 457 Stiersstirfer, B., 36, 47 Stillman, J. A., 786, 791, 803 Stinson, S. F., 19, 22, 23, 24, 44, 582, 590 Stirnimann, F., 826, 828, 845 Stock, M. J., 888, 902 Stocklin, W., 713, 729 Stockmayer, M., 37, 48, 504, 523 Stoessl, A. J., 602, 610 Stoffers, D. A., xxxiii Stoler, M., 491, 492, 498 Stolerman, I.P., 915, 932 Stoll, R., 887, 898 Stoll, W., 468, 474 Stone, C. P., 387, 400 Stone, D. M., 103, 107, 184, 194, 564, 565, 572, 617, 618, 623, 626 Stone, E. H., 917, 921, 925 Stone, H., 982, 999 Stone, L. M., 670, 676, 709, 725, 769, 782 Stone, L. S., 670, 676, 767, 782 Stone, R. E., 891, 900 Stone, S. C., 560, 573 Stone, V. K., 211, 227

Author Index Stone, W. C., 84, 85 Stone-Elander, S., 413, 436 Stoner, S. A., 472, 475 Stoof, J. C., 48l, 486, 501, 514, 519, 523 Stopa, E. G., 1003, 1006, 1023–1025 Stopfer, M., 185, 186, 200, 261, 273 Storch, V., 660, 677 Storer, S., 262, 265, 266, 271, 491, 492, 499 Storey, A. E., 366, 375, 382 Storlien, L. H., 888, 895 Storm, D. R., xxvi, xliv, 24, 26, 27, 37, 49, 76, 80, 85, 86, 90, 397, 400, 426, 428 Storm-Mathisen, J., 149, 151, 152, 156, 161 Stortkuhl, K. F. xxv, xliv Stornaiuolo, A., 116, 137 Stott, W. T., 54, 72, 582, 589, 590 Stout, R. P., 127, 130, 137 Stovall, J. R., 440, 456 Stowe, R., 553, 562, 565 Stowers, L., 971, 979 Stoye, J. P., 709, 710, 717, 728 Strack, A. M., 557, 572 Straddon, J. E. R., 869, 876 Strahan, R. C., 463, 476 Strahan, R. E., xxxi, xxxvii, 38, 43 Straits, K., 832, 844 Strasser, A., 98, 113 Strassmann, B. I., 345, 382 Strathmann, S., 300, 308 Stratigos, J., 941, 955 Stratton, G. M., xx, xliii Straughan, D. W., 124, 135 Strauss, K. E., 910, 929 Strauss, M., 595, 608 Strauss, M. E., 481, 489, 499 Streeter, G. L., 983, 999 Strehle, G., 233, 234, 246 Streim, B. J., xxvi, xliii, Streit, P., 140, 151, 158, 159 Stremler, R. L., 829, 842 Stresser, D. M., 56, 72 Stricker, E. M., 643, 648, 682, 701, 910, 915, 921, 926, 927, 930, 932, 933 Strickland, D. K., 510, 522 Strickler, A., 660, 673 Striegel-Moore, R., 952, 955 Striem, B. J., 712, 715, 716, 728, 730 Striplin, C. D., 394, 396, 400 Stripling, J. S., 190, 200 Strittmatter, S. S., 76 Strittmatter, W. J., 483, 496 Strohl, K. P., 441, 450, 455, 458 Ström, L., 667, 671, 885, 896 Stromberg, I., 101, 111 Strome, S., 469, 476 Stroop, W. G., 551, 556, 557, 572 Strotmann, J., xxv, xli, 32, 36, 41, 48, 83, 86, 100, 110, 112, 126, 134, 137, 287, 293, 294, 340 Strott, C. A., 366, 382

1091 Strowbridge, B. W., 151, 156, 157, 185, 196 Strubbe, J. H., 887, 902 Struben, C., 554, 563, 571 Struble, R. G., 142, 163, 512, 513, 523 Strupp, B. J., 419, 420, 436 Struve, M. F., 392, 399 Stryker, J. A., 891, 895 Strynadka, K., 511, 522 Stuart, R. O., 81, 86 Studler, J-M., xxv, xxxvii Stuiver, M., 217, 228, 446, 458 Stumpf, W. E., 1003, 1023 Stunkard, A. J., 883, 902, 905, 910, 918, 927 Sturart, B. P., 583, 589 Stürckow, B., xxix, xliii Stylopoulos, L. A., 601, 609 Su, T., 56–58, 61, 64, 65, 69, 72 Su, Z., 54, 69 Suarez, M. G., 39, 47 Suarez-Pinzon, W. L., 511, 522 Suay, F., 359, 382 Subbaraya, I., 83, 89 Subiaco, L., 230, 232, 244 Suddath, R. L., 603, 612 Suddick, R. P., 641, 649 Sudo, M., 551, 557, 570 Sugai, N., 27, 46 Sugai, T., 972, 979 Suggs, M. S., xxxi, xlii, 635, 638, 788, 803, 951, 956, 961, 966 Sugimoto, K., 715, 730 Sugitani, M., 972, 979 Sugiura, M., 266, 267, 272 Sugiyasu, K., 553, 568 Suh, B. D., 443, 453 Sühnel, J., 807, 822 Suhonen, J. O., 621, 626 Sukhov, R. R., 618, 623 Sulkava, R., 505, 506, 521 Sulkowski, J. W., 580, 584, 585, 591 Sulkowski, W. J., 467, 477 Sullivan, C., 367, 375 Sullivan, E. V., xxix, xliii, 174, 180, 190, 200, 255, 256, 259–267, 271–273, 424, 436, 452, 458, 486, 501 Sullivan, J. F., 893, 895 Sullivan, K., 1003, 1025 Sullivan, N., 482, 499, 513, 522 Sullivan, R. L. Jr., 216, 227 Sullivan, R. M., 152, 153, 160, 163, 164, 170, 172, 173, 175, 177, 180, 184, 185, 187, 190, 191, 196, 198, 200, 201, 314, 318, 320, 326, 410, 430, 835, 845 Sullivan, S., 831, 838, 845, 882, 895 Sullivan, S. L., 24, 28, 32, 36, 47, 48, 78, 89, 90, 100, 112, 117, 120, 121, 130, 136, 137, 139, 145, 146, 162, 163, 560, 571, 572, 713, 714, 717, 729 Sult, T. A., 526, 538, 544

Sulzer, M., xxi, xliii Summers, M., xvii, xliii Sumndeel, J., 204, 223 Sumner, D., 212, 228, 604, 612, 629, 630, 632, 634, 638, 945, 954, 959, 960, 963, 966 Sun, H., 342 Sun, J. D., 62, 72, 582, 590 Sun, T. J., 392, 400, 581, 582, 591 Sunada, I., 961, 966 Sunavala, G., 710, 729, 730 Sunday, S. R., 794, 800 Sundberg, H., 417, 436 Sundel, H., 366, 382 Sunderland, T., xxvii, xliv, 98, 113, 119, 121, 124, 137 Sunderman, F. W., 38, 48 Sundgren, H., 305, 306 Sundler, F., 907, 932 Sung, M. W., 55, 71 Sung, Y., 76, 81, 84, 89 Sung, Y. W., 450, 458 Suo, L., 153, 159 Suonpaa, J., 451, 458 Suoya, Y., 232, 245 Surrey, J., 538, 546 Suruda, A. J., 536, 545, 577, 580, 585, 588, 591 Suskind, D., xxix, xlv, 256, 259, 266, 267, 273 Suslick 291, 294 Suslov, O., 621, 625 Susser, E., 516, 523 Sutherland, R. J., 173, 180, 414, 436 Sutherling, W. W., 241, 244, 686, 704 Sutter, F., 631, 638 Sutter, J., 299, 308 Sutter, T. R., 61, 69 Sutton, P., 419, 427 Sutton, S., 239, 248 Suverkropp, C., 583, 590 Suzuki, M., 4, 16, 594, 612 Suzuki, N., 32, 48, 101, 102, 108, 552, 557, 572, 661, 663, 672, 673, 916, 918, 933 Suzuki, T., 31, 33, 44 Suzuki, Y., 22, 31, 32, 33, 48, 98, 108, 666, 676 Svare, B., 920, 930 Sved, A. F., 915, 926 Svedson, K. H., 886, 896 Svejda, J., 656, 676 Svensson, L. T., 218, 223, 807, 820 Svensson, M., 621, 624 Svikis, D. S., 330, 342 Swain, R., 554, 563, 572 Swan, G. E., 483, 501 Swan, S., 359, 376 Swandulla, D., 232, 248 Swank, M. W., 916, 918, 932 Swann, H. G., 387, 400, 403, 404, 408, 410, 413, 436 Swanson, H. H., 369, 382

1092 Swanson, L. W., 261, 272, 973, 979 Swanson-Hyland, E., 486, 499 Swaroop, A., 716, 730 Sweazey, R. D., 741, 758 Sweeney, K., 690, 691, 696, 703 Sweeney, T. P., 97, 98, 110 Swenberg, J., 553, 565 Swenberg, J. A., 582, 588 Swender, P., xxxi, xl, 216, 227 Swerdlow, R., xxvii, xliv, 98, 113 Swets, J. A., 204, 208, 209, 225, 228, 869, 877 Swett, C., 538, 546 Swierczek. J., 887, 898 Swift, D., 467, 476 Swift, D. L., 7, 16, 442, 447–449, 451, 453, 456–458, 577, 591 Swinson, R. P., 539, 546 Swoboda, P., 35, 48 Sydow, M., xxx, xxxv Sykes, A. P., 444, 457 Sylven, C., 210, 225 Sylvester, D. M., 60, 70 Syme, G. J., 910, 932 Syme, L. A., 910, 932 Symington, L. E., 853, 859 Syniar, G. M., 942, 956 Sytsma, D., 406, 408 Szabados, T., 341 Szabo, S., 785, 789, 791, 799 Szalai, J. P., 443, 444, 452, 457 Szallasi, A., 983, 999 Szczesiul, R., 211, 227 Szmeja, Z., 963, 966 Szmukler, G. I., 923–924, 931 Sznajderman, M., 891, 901 Szolcsanyi, I., 983, 998 Szolcsanyi, J., 907, 932 Szucs, E., 443, 458 Szumowski, K. E., 472, 477, 616, 626 Szumowski, K. E. M., 96, 100, 102, 106, 109, 112, 120, 125, 137 Szyszko, S. 306, 307 Tabak, A., 647, 649 Tabak, L., 647, 648 Tabata, K., 462, 476, 629, 633, 637 Tabata, M. J., 664, 677 Tabaton, M., 505, 506, 523, 563, 572 Taborsky-Barba, S., 320, 326 Taddio, A., 829, 845 Taft, R. W., 992, 995 Taggart, N. E., 366, 382 Tago, H., 153, 161 Taguchi, A., 887, 889, 900 Tai, C. F., 451, 459 Taillibert, S., 789, 804 Taillon-Miller, P., 127, 133, 1017, 1022 Takagi, H., 553, 569, 910, 932 Takagi, K., 952, 955 Takagi, S., 615, 626 Takagi, S. F., xv, xvi, xviii, xxiv, xxxvi, xliii, 170, 173, 174, 180, 182, 187,

Author Index 188, 190, 192, 198–200, 204, 228, 255, 263, 264, 266, 273, 412, 421, 436, 437, 462, 469, 477, 604, 612, 633, 638, 690–692, 696, 703, 704 Takahashi, H. 48, 514, 523, 942, 946, 957 Takahashi, J., 62, 72 Takahashi, K., 232, 248, 655, 677 Takahashi, S., 28, 48, 124, 137, 138 Takahashi, T., 659, 675 Takahashi, Y., 28, 48, 124, 137 Takahashi, Y. K., 145, 164, 182, 200 Takala, A., 553, 562, 572 Takamatsu, K., 83, 89 Takami, S., 508, 510, 511, 523, 969, 972, 979 Takasaka, T., 462, 471, 476, 629, 633, 637 Takashima, T., 594, 612 Takata, K., 31, 33, 44 Takatama, M., 512, 522 Takatsuji, K., 721, 730 Takebayashi, M., 182, 185, 199 Takeda, K., 620, 624 Takeda, M., 31, 32, 33, 48, 659, 666, 675, 676 Takeda, S., 661, 676 Takeda, T., 235, 244 Takeichi, N., 32, 48 Takemoto, D. J., 81, 90 Takenaka, T., 968, 978 Takenouchi, K., 691, 701, 736, 755 Takeshima, Y., 242, 248, 259, 266, 272 Takeuchi, H., 767, 782 Takeuchi, K., 551, 557, 570 Takeuchi, M., 102, 103, 106, 112, 451, 456 Takeyama, M., 54, 67 Taki, Y., 561, 571 Takigami, S., 351, 382 Tal, N., 58, 70 Talal, N., 646, 648 Talamo, B. R., 22, 37, 42, 48, 124, 137, 504–506, 509, 523, 563, 572 Talbert, E. I., 994, 998 Talbot, N. L., 492, 501 Talianakis, S., 423, 431 Talley, F. A., 578, 583, 591 Tallkvist, J., 551, 554, 555, 556, 572 Talmain, G., 17, 48 Tam, P. P. L., 670, 676, 769, 782 Tamada, A., 121, 123, 125, 138 Tamari, I., 366, 383 Tamura, H., 56, 59, 65, 71, 72 Tamura, H.-O., 32, 36, 45 Tan, S. S., 670, 676, 769, 782 Tanabe, T., xxiv, xliii, 173, 174, 180, 182, 188, 192, 200, 255, 263, 264, 273, 412, 421, 436, 437, 604, 612, 692, 704 Tanaka, H., 683, 704 Tanaka, M., 37, 38, 46, 359, 380 Tanaka, P., 882, 895 Tanaka, S., 31, 33, 44

Tanaka, Y., 440, 458 Tanapat, P., xxviii, xxxvii, xliii, 622, 624 Tanase, D., 684, 701 Tandeciarz, S., xxxi, xxxiv Tandler, B., 658, 662, 674, 676 Tanemura, K., 709, 711, 723, 727 Tang, A. C., 190, 200 Tang, W.-J., 24, 44, 80, 90 Tang, Y. P., 420, 435 Tangel, D.J., 441, 456 Tani, A., 186, 197 Tanifuji, M., 145, 164, 182, 200 Taniguchi, M., 37, 49, 971, 979 Tanikawa, H., 444, 458 Tanila, H., 425, 429 Tanimizu, T., 916, 933 Tanimura, K., 48 Tanioka, H., 766, 781 Tank, D. W., xxviii, xl, 254, 269, 271 Tanner, C. M., 486, 501, 564, 571 Tanner, W. P., Jr., 204, 208, 228 Tanyeri, H., 468, 475 Tanyolaç, N. N., 297, 308 Tao, W., 116, 137 Tapp, J. T., 405, 408, 413, 433 Tapper, D. N., 873, 875, 879 Tardif, R., 581, 589 Tardy, E., 4, 5, 16 Tareilus, E., 26, 46, 76, 79, 81, 83, 86, 710, 729 Tarlo, S. M., 539, 546 Tarozzo, C., 35, 48 Tarttelin, M. F., 919, 923, 932 Tashiro, K., 938, 949, 957 Tashiro, T., 961, 966 Tate, L. G., 606, 612 Tateda, H., xxiv, xliii Tatemoto, K., 76, 85 Tateyama, T., 234, 236, 248 Tatzer, E., 826, 845, 883, 902 Taudorf, E. 998 Taukulis, H., 910, 913, 932 Taulbee, D.B., 448, 458 Tauxe, R., 883, 900 Taverner, D., 444, 453, 458, 942, 957 Taxy, J. B., 38, 48 Taylor, B., 644, 648 Taylor, B. A., 335, 343 Taylor, B. J., 661–663, 673 Taylor, C. W., 81, 90 Taylor, E. M., 915, 924 Taylor, G., 561, 562, 567 Taylor, G. T., 416, 430 Taylor, J. A., 564, 570, 616, 617, 625, 970, 978 Taylor, P., 915, 933 Taylor, P. J., 992, 995 Taylor, R., 661, 664, 671 Tazawa, Y., 174, 180, 190, 199, 263, 273 Tazi, A., 416, 428 Teague, R., 101, 102, 104, 105, 108 Tecce, J. J., 233, 248 Tedeschi, G., 553, 563, 566

Author Index Teeter, J. H., xxv, xxvi, xxxiv, xli, 76, 89, 243, 248, 660, 672, 715, 718, 720, 723, 725 Teff, K. L., 888, 902 Teghtsoonian, M., 267, 273 Teghtsoonian, R., 267, 273 Tegoni, M., 19, 48 Tehnisch, P. Teicher, M. H., xxix, xliii, 141, 157, 163, 310, 315, 322, 326, 355, 382, 538, 546 Teichner, W. H., xxix, xliii, 406, 408 Teillac, A., 449, 453 Teitelbaum, P., 867, 869, 870, 871, 877 Tejedor, J., 170, 176, 192, 195 Tekula, F. D., 24, 27, 33, 35, 45 Telitsina, A. Y., 356, 374 Telle, H. J., 362, 382 Tellegen, A., 342 Temple, E. C., 825, 845 Ten Dijke, P., 101, 113 Tengamnuay, P., 562, 572 Tennent, R., 104, 108, 126, 132, 137 Tennissen, A. M., 790, 804, 882, 900 Teow, B. H., 886, 896 Tephly, T. R., 58, 70 Tepper, B. J., 855, 857, 860, 883, 884, 893, 902 Terada, N., 444, 458 Teraki, Y., 662, 663, 677 Teramoto, K., 585, 590 Teranishi, R., 276, 293 Terasawa, E., 1012, 1013, 1024, 1025 Terkildsen, K., 230, 243, 248 TerKonda, R. P., 469, 476 terLaak, H. J., 512, 523 Terman, C. R., 365, 367, 382 Ter-Pogossian, M. M., xxviii, xxxvii, xliv, 253, 273 Ter-Pogossian, M. M. Terr, A. I., 533, 539, 541, 546 Terrace, H., 359, 377 Terranova, J. P., 416, 426, 434 Terry, L. M., 837, 845 Tessarollo, L., 762, 780 Testa, M., 949, 954 Testa Riva, F., 658, 675 Tetrud, J. W., 486, 497, 501 Teucher, B., 220, 227 Thal, L. J., 619, 625 Thaler, B. I., 886, 902 Thaler, H. T., 320, 327, 356, 383 Than, T., 723, 725 Thangavel, R., 510, 511, 512, 523 Thanos, P. K., 174, 180, 394, 400, 410, 411, 413, 437 Tharp, C. D., 797, 799 Tharp, G., 359, 373 Thatcher, B. J., 645, 650 Thaw, A. K., 863, 867, 870, 871, 879 Theerasilp, S., 713, 730 Theisen, J. C., 884, 897, 906, 927 Theodore, R. M., 752, 757

1093 Theodorsson, E., 887, 903 Theodosis, D. T., 128, 131 Theologides, A., 910, 930 Therianos, S., 116, 134 Thesleff, I., 765, 780, 887, 902 Thibault, J., 172, 178 Thibault, O., 412, 436 Thibodeau, S. N., 487, 501 Thiele, C., 103, 108 Thiele, T. E., 916, 931 Thiele, V., 234, 248 Thiery, J. C., 423, 434 Thiessen, D. D., 346, 382, 404, 408, 417, 422, 430, 434 Thirlby, R. C., 887, 902 Thoenen, H., 619, 626 Thom, R., 296, 308 Thoman, E. B., 367, 375 Thomas, D., 442, 445, 455 Thomas, D. A., 38, 43, 99, 109 Thomas, J. G., 891, 898 Thomas, J. H., 35, 48 Thomas, L., xxxii, xliv, 318, 320, 323, 327, 344, 356, 383, 410, 437 Thomas, L. B., 621, 625 Thomas, R., 260, 271 Thomason, M. E., 486, 501 Thompson, A. C., 356, 378 Thompson, B., 209, 227 Thompson, D. R., 690, 704 Thompson, D.G., 686, 700 Thompson, G., 341 Thompson, H.A., 220, 223 Thompson, P., 494, 496 Thompson, R., 413, 437 Thompson, R. F., 243, 248, 916, 929 Thompson, S. N., 909, 933 Thomsen, E. S., 991, 999 Thomson, G. M., 527, 546 Thor, D. H., 415, 416, 437 Thor, P., 887, 898 Thorens, B., 719, 725 Thorndike, E. L., 385, 400 Thorne, R. G., 551, 553, 556, 567, 572 Thorner, J., 1004, 1023 Thorner, M., 367, 375 Thornhill, R., xxvii, xliv, 93, 112, 329, 341 Thornton, J. E., 363, 376 Thornton-Manning, J. R., 51, 57, 64, 72, 73, 559, 560, 561, 572 Thorpe, S. J., 680, 687, 688, 692, 694, 695, 703, 704 Threattle, R. M., 936, 955 Thürauf, N., 243, 249, 983, 999 Thurstone, L.L., 203, 228 Thyssen, J., 63, 72 Thyssen, J. H., 583, 589 Tibboel, D., 130, 138 Tichy, J., 484, 500 Tie, K., 853–854, 855, 860 Tietze, K. J., 554, 563, 571 Tigges, J., xxii, xxxvii, 550, 569

Tiitinen, H., 230, 248 Tikhonova, M. A., 661, 675 Tillet, Y., 172, 178, 423, 432 Tilley, B. C., 923, 930 Timberlake, W., 914, 929 Timischl, W., 826, 845, 883, 902 Timmes, A., 773, 782 Timms, D., 280, 293 Tinklenberg, J. R., 481, 500, 501 Tinston, D. J., 583, 589 Tinti, J. M., 713, 730 Tintner, R., 601, 608 Tipton, K. F., 58, 70 Tirindelli, R., xxv, xxvi, xxxiii, 351, 382, 972, 978 Tisay, K., 105, 112 Tisay, K. T., 126, 137 Tisdall, F. F., xxvi, xliv, 557, 572 Tissingh, G., 480, 486, 501, 514, 523 Tjälve, H., 56, 60, 65, 69, 70, 72, 551, 554, 555, 556, 566, 568, 572 Tkacheva, N. V., 551, 571 Toates, F., 862, 866, 877 Tobach, E., 310, 326 Tobet, S. A., 1003, 1004, 1006, 1013, 1014, 1023, 1025 Tochon-Danguy, H. J., 267, 269 Todd, R. B., xxii, xliv, 18, 48, 981, 999 Todrank, J., 318, 320, 323, 326, 341, 789, 804 Togiyama, T., 888, 900 Toh, H., 658, 674, 766, 781 Tohyama, M., 721, 730 Toida, K., 140, 144, 151, 158, 163 Tokarz, F., 963, 966 Tokumaru, Y., 952, 955 Tolbert, L. P., 17, 48, 129, 130, 136 Tolis, G., 554, 572 Tolkamp, B. J., 909, 929 Toll, K., 444, 455 Tolley, N. S., 450, 452, 454 Tomac, A., 101, 111 Tomarev, S., 660, 673 Tomasiewicz, H., 128, 134 Tomie, J. A., 418, 437 Tomihara, K., 364, 378 Tominaga, M., 983, 996 Tominaga, Y., 26, 35, 48 Tomita, H., 785, 789–791, 804, 945, 946, 956, 957 Tomkinson, A., 442, 458 Tomlinson, A. H., 60, 72, 551, 557, 567 Tomooka, L. T., 473, 477 Toms, M., 223, 228 Ton, C. C., 116, 133 Tonkin, A., 444, 458 Tonlorenzi, R., 127, 133, 1017, 1022 Tonndorf, J., 439, 441, 458 Tonnel, A. B., 440, 456 Tonoike, M., 182, 185, 199, 233, 242, 249 Tonon, M. C., 76, 86

1094 Tonosaki, K., 124, 137, 714, 730, 737, 758 Tonra, J. R., 773, 782 Topazzini, A., 560, 565 Topolski, T. D., 329, 331, 333, 343 Tordoff, M. G., 682, 702, 717, 724, 727, 882, 885, 894, 902, 907, 930 Toriello, A., 553, 563, 566 Torii, K., 720, 728, 907, 930 Torii, S., 233, 249 Toriumi, D. M., 597, 612 Torner, L., 367, 382 Torrey, E. F. 271, 342, 492, 499 Torry, H. C., 253, 272 Torvik, A., 913, 933 Tos, M., 14, 16 Toschi, N., 367, 382 Tosun, T., 634, 637 Toth, J., 444, 455 Toto, P. D., 658, 676 Toubas, P., 314, 318, 326, 835, 845 Toubeau, G., 661, 676 Touhara, K., xxv, xliv, 287, 294 Toulouse, E., xiii, xliv Tourne L.P., 952, 957 Townsend, J. L., 603, 611 Townsend, M. J., 204, 224 Toyono, T., 661, 676 Toyoshima, K., 660–662, 664, 676 Toyota, B., 204, 228 Traber, J., 170, 177 Trabue, I. M., xvi, xli, 807, 822 Tracewell, T., 449, 456 Trafton, C. L., 865, 879 Tramezzani, J. H., 34, 35, 48 Tranel, D., 513, 519 Trant, A. S., 890, 902 Trapido-Rosenthal, H. G., 56, 60, 72, 807, 821 Trask, B. J., xxv, xxxiv, 17, 42 Traub, O., 105, 111 Travers, J. B., 691, 700, 732, 734, 736, 737, 739, 740, 742, 743, 747–752, 754, 757, 758, 913, 932, 933 Travers, S. P., 691, 700, 736, 740–745, 758, 865, 878, 913, 933 Traverzo, A., 819, 822 Travis, M. L., 392, 397, 400 Traynor, D., 81, 90 Treherne, S., 831, 840 Treisman, R., 84, 87 Treloar, H., 104, 105, 110, 112 Treloar, H. B., 125, 129, 130, 137, 620, 624 Tremaine, M. J., 784, 798 Treneer, C. M., 909, 919, 924 Treves, A., 192, 199, 263, 272, 421, 435, 681, 695, 699, 703 Tricker, A. R., 63, 72 Trifiletti, R. R., 76, 89 Trifunovic, R., 684, 703, 866, 878 Trinh, K., xxvi, xliv, 24, 26, 37, 49, 76, 80, 90, 397, 400

Author Index Trinkaus, E., 439, 454 Troemel, E. R., 76, 78, 87 Trojanowski, J. Q., 5, 15, 37, 44, 98, 109, 505–510, 512–517, 519–523, 583, 590 Trombley, P. Q., 139, 149–151, 153, 155, 163, 164, 183–185, 194, 200 Troncoso, J. C., 514, 520 Tronier, B., 888, 899 Tropepe, V., 620, 623 Trotier, D., xxiv, xxxiv, 17, 48, 967, 971, 977, 979 Trotter, P.J., 297 Truax, B. T., 947, 957 Truchet, B., 412, 427 Trunck, F., 884, 885, 900 Truwitt, C. L., 38, 48 Tsai, J. R., 601, 609 Tsai, M. S., 487, 501 Tsai, S. Y., 581, 588 Tschopp, A., 586, 589 Tseng, G. F., 167, 173, 180, 191, 200 Tseng, H., 491, 493, 501 Tsien, J. Z., 420, 435 Tsou, S. K., 513, 522 Tsuang, M. T., 491, 492, 501 Tsuboi, A., xxv, xliv, 182, 200, 287, 294 Tsubone, H., 452, 458 Tsuchiya, K., 580, 591 Tsudo, N., 789, 800, 960, 962, 965 Tsuji, S., 514, 523 Tsuji, Y., 717 Tsukada, M., 968, 978 Tsukamoto, G., 915, 924 Tsukamoto, T., 716, 727 Tsukamoto, Y., 663, 673 Tsukatani, T., 152, 160, 634, 638 Tsunenari, T., 712, 730 Tsuruta, M., 884, 903 Tucker, A. M., 310, 326 Tucker, D., xxxiii, 344, 388, 400, 982, 987, 998, 999, 1002, 1025 Tucker, D. K., 356, 381 Tucker, K. L., 763, 782 Tuckerman, F., 665, 676, 824, 825, 845 Tumanova, N. L., 148, 155 Tumeth, S. S., 600, 608, 612 Tung, C., 417, 431 Tunsuriyawong, P., 789, 804 Tuorila, H., 784, 804 Turetsky, B. I., xxxii, xliv, 269, 271, 479, 491–493, 498, 499, 515, 516, 519, 522, 523, 603, 612 Turin, L., 280, 283, 285, 286, 289, 291, 294 Turing, A., 296, 308 Turk, A., xvi, xxix, xxxvii, xliii, 204, 206, 213, 226, 228, 801, 841 Turk, M. A., 553, 572, 583, 591 Turk, M. A. M., 60, 63, 72 Turner, B. H., 165, 168, 171–173, 180 Turner, C. P., 105, 112, 619, 620, 626 Turner, E. H., 472, 477, 492, 495, 500, 501, 619, 626

Turner, L. S., 221, 226 Turner, R., 258, 270 Turner, R. E., 828, 841, 883, 886, 896 Turner, W. J., 127, 138, 472, 478 Turner, W. J. D., 594, 599, 603, 613 Turner, W. M., 491, 501 Turvey, T. A., 789, 801 Tvedt, B., 608, 612 Tweedle, D. E., 890, 897 Tym, A., 855, 857, 859 Tytgat, G. N., 554, 563, 572 Tyukody, V., 267, 271 Tzoumaka, E., 394, 400 U. S. Environmental Protection Agency 552, 572 Uchenna Agu, R., 562, 572 Uchida, N., 145, 158, 164, 182, 185, 186, 197, 200 Uchida, T., 661, 676 Uchida, Y., 714, 730 Uchimura, A., 551, 570 Uchiyama, T., 952, 957 Udovichenko, I. P., 81, 90 Udry, J. R., 363, 379 Ueda, H., 662, 664, 672 Ueda, K., 509, 523, 746, 756 Uemura, T., 37, 38, 46, 631, 637 Ueno, Y., 212, 228 Ugawa, S., 721, 730 Ugolini, G., 557, 572 Ugurbil, K., 254, 269, 271 Ujike, H., 941, 957 Ukmar, M., 489, 501 Ulbrich, L., 806, 812, 818, 821 Ulfig, N., 152, 161 Ullrich, N., 855, 860 Ulmar, G., 517, 521 Ulpinar, E., 764, 782 Uma-Pradeep, K., 698, 704 Umeda, R., 215, 225, 392, 397, 399 Umemura, T., 36, 43 Unger, J., 508, 509, 521 Uraih, L. C., 52, 72, 578, 583, 591 Uramoto, N., 101, 102, 111 Ursick, J. A., 893, 895 Ursin, H., 417, 436 Ursino, F., 55, 56, 68 Ursino, F., 561, 567 Uryu, K., 514, 520 Ussing, H. H., 718, 727 Uttal, W. R., 296, 308 Uvnas-Moberg, K., 313, 314, 322, 327 Vaccarezza, O. L., 34, 35, 48 Vaidehi, N., 276, 289, 293 Valdez, I., 647, 648 Valensi, P., 554, 572 Valenti, A., 63, 70 Valentin, G., xxii, xliv Valentincic, T., 723, 725 Valerio, M. G., 583, 590 Valkov, I. M., 952, 957

Author Index Vallante, M. A., 411, 427 Valsecchi, P., 316, 324 Valsecchu, P., 370, 377 Valverde, F., 104, 112, 129, 130, 137, 146, 164, 166, 167, 169, 171, 173, 177, 180 van Abeelen, J. H. F., 336, 343 van Brouweshave, J., 713, 727 van Buskirk, R. L., 732, 735, 739–744, 746–751, 757, 758, 864, 879 van Critters, R. L., 888, 903 van de Moortele, P. F., 797, 800, 948, 954, 960, 962, 965 van de Poll, N. E., 369, 382, 923, 926 van den Brink, T. H. C., 416, 432 van den Broeke, L. T., 552, 571 van den Eijnden-Van Raaij, J., 101, 113 van den Mooter, G., 562, 572 van den Noort, S., 481, 485, 489, 498 van den Pol, A. N., 149, 151, 152, 158, 164 van der Boot, L. M., 970, 977 van der Heijden, C. A., 582, 591 van der Klaauw, N. J., 788, 794, 795, 800, 803, 804, 812, 817–819, 821 van der Kooy, D., 128, 136, 616, 620, 623, 625, 916, 933 van der Kwaak, H., 647, 650 van der Ouderaa, F., 713, 727 van der Reijden, W. A., 646, 647, 650 van der Schueren, B., 55, 69 van der Starre, H., 350, 378 van der Vliet, A., 54, 67 van der Weele, D. A., 683, 684, 691, 701, 703 van der Wel, H., 662, 672, 713, 725–727, 730 van der Werff ten Bosch, J. J., 923, 932 van Deventer, S. J., 554, 563, 572 van Dishoeck, H., 441, 458 van Duijvenvoorde, P. M., 696, 703, 882, 901 van Garderen-Hoetmer, A., 582, 591 van Gemert, L. J., 280, 282, 286, 290, 293, 294 van Groen, T., 171, 180 van Haaren, F., 918, 933 van Hausen, G. W., 556, 568 van Heerden, B 472, 477 van Heertum, R. L., 262, 265, 266, 271 van Hest, A., 918, 933 van Heyningen, V., 116, 133, 134 van Hoesen, G. W., 268, 270, 600, 610 van Houten, J., xv, xliv van Itallie, T. B., 828, 832, 842 van Lunteren, E., 450, 455 van Nie, C. J., 130, 138 van Oostrom, C. G., 115, 138 van Ree, J. M., 415, 416, 420, 434, 436, 914, 933 van Riezen, H., 619, 626 van Setten, G., 643, 649 van Staveren, W. A., 340

1095 van Tassel, P., 598, 612 van Toller, C., 244, 249 van Toller, S., 244, 249, 329, 340, 635, 638, 987, 998 van Trijp, H. C. M., 807, 809–811, 821 van Wassenaar, P. D., 713, 727 van Wimersma Greidanus, T. B., 416, 437 vande Spiegle, K., 101, 113 Vandenbergh, J. G., xxix, xxxviii, xliv, 348, 368, 377, 382, 970, 977, 979 Vanderbelt, D. J., 713, 725 VanHoesen, G. W., 509, 512, 513, 516, 517, 519, 521, 523 Vanlingen, S., 26, 41 Vanne, M., 784, 804 Vannelli, B., 103, 108 Vannelli, G. B., 62, 66, 73 Vanscheeuwijck, P., 101, 113 Van’t Hof, W., 645, 650 VanWorkum, F. P., 512, 523 Vardon, G., 390, 392, 397, 399 Varendi, H., 313, 314, 316, 326, 835, 845 Vargas, G., 103, 112 Varkevisser, B., 714, 717, 730 Varney, N. R., 635, 638, 961, 966 Varro, M. T., xix, xliv Vartiainen, N., 82, 88 Vaschide, N., xxiii, xliv Vasey, F. B., 541, 546 Vasiliev, O. L., 116, 138 Vasilieva, N. Y., 422, 437 Vasquez, M., 830, 831, 845 Vassar, R., 24, 28, 32, 36, 48, 78, 82, 87, 90, 99, 113, 130, 137, 146, 164, 296, 307, 560, 572 Vassart, G., xxv, xli Vassella, F., 946, 954 Vatner, S. F., 888, 903 Vatza, E. J., 423, 431 Vaudry, H., 76, 86 Vaughn, J. E., 153, 161 Vaux, D., 98, 113 Vavrek, R. J. 999 Vawter, M. P., 119, 121, 124, 137 Vayssettes-Courchay, C., 714, 726 Vaz, A. D., 57, 61, 62, 68, 72 Vazquez, M., 883, 884, 903 Veeger, W., 891, 900 Veerman, E. C. I., 645, 646, 647, 650 Veith, M., 284, 294 Vejsada, R., 762, 764, 779, 781 Velakoulis, D., 492, 493, 496 Veletza, V., 514, 522 Velin, R. A., 495, 497, 886, 896 Velley, L., 885, 901 Velonakis, E., 525, 528, 543 Venable, J. C., xxiv, xli, 77, 89 Venkatachalam, S., 98, 109 Venneman, W., 130, 138 Verbalis, J. G., 910, 915, 921, 926, 927, 930, 932, 933 Verbeke, N., 562, 572

Verdun di Cantogno, L., 149, 150, 162 Vereijken, P. F. G., 211, 225 Veress, B., 621, 623 Verghese, A., 492, 501 Verhaagen, J., 125, 138, 616, 625, 745, 758, 762, 763, 781 Verhagen, J. V., 684, 704 Verhoef, J. C., 553, 562, 570 Verkman, A. S., 31, 48 Verleger, R., 240, 249 Verma, A., 58, 73, 76, 81–82, 90, 709, 727 Vernet-Maury, E., 358, 372, 373, 388, 397 Verney, C., 1017, 1022 Verrill, A. H., xviii, xliv Versace, R., 193, 199, 261, 264, 272 Verscheuren, K., 101, 113 Verson, E., 658, 676 Verwoerd, C. D., 115, 138 Vesalius, A., xx, xliv Vescovi, A. L., 621, 623 Vesselkin, N. P., 153, 159 Vetter, H. 89, 903 Vetter, N., 891, 898 Vetulani, J., 416, 434 Viader, F., 488, 496 Viana DiPrisco, G., 186, 187, 200 Viaud, M. D., 414, 437 Vicino, M., 554, 566 Vick, N., 894, 901 Vickers, J. C., 511, 518 Vickers, N. J., 186, 193, 200 Vickers, Z. M., 910, 930 Vickland, H., 105, 113 Victor, M., 602, 612 Vida, I., 689, 701 Vidal, M., 981, 999 Vierling, J. S., 349, 382 Vig, P. S., 441, 453 Vigorito, M., 906, 924 Vigouroux, M., 174, 175, 176, 182, 187, 188, 190, 191, 195, 198, 258, 264–266, 272, 358, 373410, 415, 424, 433, 435 Viinikka, L., 887, 902 Vijande, M., 831, 840 Vijayan, V., 560, 571 Vila-Jato, J. L., 554, 563, 567 Vilanova, X., 305, 306 Vilardarga, J. P., 291, 294 Villa, S., 891, 901 Villanueva, L., 685, 700 Villar, H. O., 291, 293 Vince, M. A., 315, 327 Vincent, A., 101–103, 109 Vincent, F., 19, 48 Vincent, G., 914, 927 Vincent, J. D., xxviii, xxxvi, xli, 143, 149, 151, 152, 154, 155–157, 162, 184, 196, 514, 521 Vincent, S., 484, 497 Vincent, S. R., 79, 87

1096 Vintschgau, M., xxi, xliv Virtanen, I., 505, 506, 521 Vishney, A., 390, 392, 397, 400, 413, 435 Visser, T. J., 140, 151, 158 Vissink, A., 646, 647, 650 Vitello, F., 621, 625 Vivino, A. E., xviii, xliv Vodanovic, M., 791, 799 Vodyanoy, V., 26, 27, 48 Voet, H., 341 Vogel, B., 553, 573 Vogl, A., 26, 48 Vogl, T., 593, 594, 612 Vogt, M. B., 736, 742, 757, 758, 778, 782, 906, 933 Vogt, R. G., xxv, xliv, 56, 58, 60, 72, 73, 78, 90 Vogtle, F., 304, 308 Voigt, J. M., 57, 58, 63, 64, 66, 73 Voigt, R., 185, 196 Volkow, N. D., 252, 273 Vollert, H., 1004, 1022 Vollmecke, T. A., 424, 433 Vollrath, M., 32, 48, 124, 138 Volpe, B., 511, 515, 519 Volta, A., xxi, xliv Vom Saal, F. S., 411, 437 von Bartheld, C. S., 1001, 1002, 1023, 1025 von Békésy, G., 785, 804 von Bèkesy, G., 987, 999 von Brunn, A., xxii, xliv, 1, 13, 14, 16, 37, 48 von Campenhausen, H., 28, 32, 36, 46, 145, 160, 972, 973, 977, 979 von Doersten, P., 946, 955 von Düring, M. V., 660, 676 von Euler, G., 538, 546 von Frisch, K., xxix, xliv von Haller, A., xix, xliv, 655, 657, 658, 672, 784, 801 von Helmholtz, C., 241, 245 von Helmholtz, H., xxxii, xxxvii von Hönigschmied, J., xxi, xliv von Kölliker, R. A., 19, 48 von Marxow, F., 229, 230 von Möllendorf, W., 658, 677 von Ranson, C., 991, 999 von Skramlik, E., xvi, xliv, 805, 822, 984, 985, 987, 999 von Stralendorff, F., 310, 315, 324 von Sydow, D., 209, 225 von Uexküll, J., 403, 408 von Vintschgau, M., 666, 676, 773, 781 Vonsattel, J. P., 602, 611, 613 Voorhees, R. E., 530, 532, 546 Vora, S., 351, 381 Vories, A. A., 946, 957 Vorkink, G. L., 886, 903 Vortkamp, A., xxv, zliv, 117, 136 Vosshall, L. B., xxv, xliv, 28, 48, 99, 113, 130, 137, 146, 164, 560, 572

Author Index Voss-Wermbter, G., 24, 45 Vostrowsky, O., 319, 323, 341 Vuillemin, T., 631, 638 Wachenheim, F. L., 837, 846 Waddell, W. J., 525, 546 Waddington, C.H., 296, 308 Wade, D., 285, 294 Wade, G., 919, 924 Wade, G. N., 919, 920, 930, 933 Wade, M. J., 513, 522 Wagner, R., 667, 677 Wahle, P., 171, 179 Wahlund, L. O., 480–482, 495, 499, 500 Wainer, B. H., 564, 564, 571 Waite, P., xxvii, xxxix, 107, 110 Waite, P. M. E., 551, 555, 568 Waite, S., 367, 375 Wakabayashi, K., 514, 523, 1003, 1023 Wakasugi, C., 662, 663, 677 Wakeling, I., 788, 803 Wakeman, E. A., 193, 199, 421, 435, 688, 692, 694, 695, 704 Wakisaka, H., 942, 957 Wakisaka, S., 664, 677, 916, 933 Walburn, J. N., 831, 840 Walchak, C., 887, 897 Waldman, B., 315, 323 Waldmann, R., 721, 730 Waldrop, B. R. 195 Waldton, S., 479, 481, 501 Walker, A. E., 861, 876, 878 Walker, D. M., 53, 62, 64, 65, 69, 552, 568, 582, 589 Walker, J. C., 204, 227, 228, 231, 248, 255, 256, 271, 273, 387, 389, 391, 392, 399, 400, 982, 983, 987, 990, 998, 999 Walker, N. J., 61, 69 Walker, S. E., 1004, 1025 Walker, V., 553, 565 Walker, V. E., 53, 62, 64, 65, 69, 552, 568, 582, 589 Wallace, K., 839, 844 Wallace, L. A., 528, 530, 532, 536, 545, 546 Wallace, P., 318, 327, 338, 343, 404, 408, 422, 434 Wallace, S. J., 553, 573 Wallaert, B., 440, 456 Waller, A., 652, 660, 677 Waller, G., 445, 456 Waller, K., 359, 376 Wallis, G., 680, 704 Wallstedt, L., 621, 624 Walser, E. S., 309, 327 Walsh, K. A., 83, 89 Walsh, T. D., 890, 903 Walsoe de Reca, E. N. 306 Walt, D. R., 299, 306–308 Walter, W. G., 233, 249 Walters, C. P., 142, 163 Walters, D. E., 714, 726, 730

Walters, E., 27, 41, 64, 65, 73, 98, 113, 123, 125, 132, 309, 327, 550, 551 557, 566 Walz, B., 81, 90 Wanaka, A., 664, 677 Wanamaker, J. R., 597, 611 Wang, C., 230, 246 Wang, D., 970, 971, 979 Wang, F., xxvii, xxxiv, xxxix, 36, 44, 49, 78, 89, 126, 130, 131, 134, 135, 138, 146, 160, 164, 289, 294, 296, 307, 354, 379 Wang, H., 116, 138 Wang, H. W., 355, 382, 426, 437, 622, 626 Wang, J. C., 440, 457 Wang, J. D., 581, 588 Wang, L., 736, 754, 755, 758 Wang, Q., 56–58, 73 Wang, S., 698, 702 Wang, V. S., 285, 293 Wang, X. R., 298, 307 Wang, Y., 855, 859, 907, 915–916, 921, 926, 933 Wang, Z., 422, 430, 437, 438 Wangerek, L., 104, 105, 110, 620, 624 Wanichanon, C., 661, 673 Wannagat, U., 277, 284, 285, 294 Wanner, G. A., 82, 85 Wanner, I., 32, 36, 48, 100, 112, 126, 137, 340 Wantanabe, K., 945, 957 Wantzin, J., 554, 564 Ward, C. D., 331, 343, 485, 501 Ward, C. W., 216, 224 Ward, J. D., 629, 637 Ward, J. M., 582, 589 Ward, T. M., 315, 327 Warden, M. K., 151, 164 Wardlaw, S. A., 64, 73 Warheit, D. B., 582, 591 Warick, Z. S., 885, 892, 894, 901, 903 Waring, S. C., 469, 475, 485, 487, 488, 495, 501 Warner, M. D., 481, 483, 491, 501 Warner, T. F., 658, 671 Warr, C. G., xxv, xxxv Warren, D. W., 256, 273, 982, 999 Warren, J. M., 680, 702 Warren, R. P., 908, 933 Warrenburg, S., 244, 246, 259, 264, 265, 266, 270 Warrick, R., 392, 399 Warshaw, R., 585, 589 Waruszewski, B. A., 59, 67 Warwick, R., 665, 677 Warwick, T. T., 140, 143, 145, 154, 156 Warwick, Z. S., 480, 500, 501, 793, 803 Wassef, M., 17, 48 Wasserstein, J., 949, 954 Watabane, I., 450, 456 Watanabe, H., 419, 432 Watanabe, K., 561, 573 Watanabe, N., 54, 67

Author Index Watanabe, S., 149, 164, 580, 591, 620, 624 Watchus, J., 423, 432 Waterman, D., 853, 859 Waterman, M. R., 57, 71 Watkins, G. L. 269, 491, 496 Watkins, J. C., 413, 420, 421, 433 Watras, J., 511, 519 Watson, C., 166, 167, 173, 179 Watson, G., 647, 648 Watson, J. B., 385, 400 Watson, M., 365, 382 Watson, R. L., 891, 898 Watson, S. D., 862, 879 Watson, S. J., 560, 570 Watt, E. J., 300, 308 Watt, L., 480, 498 Watt, L. B., 828, 829, 841 Watt, W. C., 85, 90 Waxman, A. O., 536, 547 Waxman, D. J., 57, 61, 71, 73 Waxman, S., 105, 109 Wayman, G. A., 80, 90 Wayne, M. L., 342 Wayner, B. H., 190, 198 Wayner, M., 187, 196 Wayner, M. I., 982, 997 Weaver, V. M., 536, 541, 547 Weaver, W., 738, 757 Webb, A. D., 206, 227 Webb, C. J., 947, 957 Weber, A. F., 30, 46 Weber, E. H., 203, 208, 228 Weber, K., 32, 48, 124, 138 Webster, H. H., 619, 626 Webster, M. M., 910, 924 Webster’s Third New International Dictionary of the English Language (Unabridged) 528, 547 Weddell, G., 667, 676 Wedege, E., 554, 568 Wedekind, C., 319, 321, 327, 339, 340, 343 Wedral, E., 936, 952, 356 Weesner, G., 1013, 1025 Wegert, S., 723, 725 Wehr, M., 186, 200 Wehr, R., 116, 133, 136 Wehr, T. A., 472, 477, 495, 500, 619, 626 Wei, J., 27, 49 Weidong, M., 970, 978 Weiffenbach, J. M., 216, 228, 481, 495, 499, 646, 650, 784–788, 792–794, 804, 828, 841, 846, 853, 854, 855, 860, 866, 879, 952, 957 Weigel, M. T., 470, 477 Weijnen, J. A. W. M., 864, 879 Weiler, E., 33, 36, 49, 96–98, 102, 107, 113, 119, 125, 138, 392, 397, 582, 588, 968, 979 Weinberger, N. W., 191, 195, 200 Weiner, M. F., 601, 608

1097 Weingarten, H. P., 857, 859, 862, 879, 882, 894, 899 Weinshilboum, R. M., 59, 73 Weinstein, H., 713, 714, 717, 728 Weinstein, V., 394, 395, 398, 404, 407 Weinstock, R. S., 471, 477, 893, 903 Weintraub, Z., 833, 843, 886, 898 Weir, M. W., 366, 382 Weiskrantz, L., 908, 933 Weismann, A. M., 31, 41 Weismann, K., 893, 903 Weiss, D. G., 552, 558, 560, 561, 573 Weiss, L., 52, 72 Weiss, M. L., 169, 170, 180 Weiss, P., 552, 558, 561, 573 Weiss, R. A., 891, 894 Weiss, S., 619, 620, 626 Weiss, S. R., 551, 561, 569 Weiss, U., 261, 269 Weisse, S., 885, 900 Weissenbach, J., 1004, 1017, 1023 Weissman, S. M., 716, 730 Weitkamp, L., 341, 342 Wekesa, K. S., xxvi, xliv Welch, J. M., 722, 730 Welch, W. J., 510, 523 Weldon, D. A., 392, 397, 400 Welge Lüssen, A., 238, 249 Welker, W.I., 406, 408 Weller, M. P. I., 492, 496 Wellerdieck, C., 287, 293 Wellington, J. L., 357, 378, 417, 427 Wellis, D. P., 183, 184, 185, 196, 200 Wells, A. S., 839, 846 Wells, M. A., 891, 895 Wells, P., 336, 343 Wells, U. M., 13, 16 Wells, R. G., xxiv, xxxviii Welsch, U., 660, 677 Wenig, B. L., 448, 454 Wenisch, S., 26, 49 Wennberg, A., 542, 543, 585, 587 Wenning, G. K., 238, 249, 488, 501 Wensley, C. H., 560, 573 Wenzel, B. M., 148, 164, 204, 228 Werblin, F. S., xxiv, xxxviii, 76, 79, 87 Wermuth, B., xxxi, xxxiv Werner, D. I., 336, 343 Werntz, D. A., 450, 458 Wertheim, L., 829, 840 Wertkin, A., 513, 523 Wesley, D. S., 56, 68, 553, 559, 567 Wessel, T., 564, 572, 617, 626 Wessel, T. C., 918, 928 Wesselingh, S. L., 557, 565 Wessels, H. 306–307 West, A., 93, 101, 104, 105, 108, 113 West, A. K., 126, 132, 138, 511, 518 West, C. H., 423, 437 West, D. B., 717, 724 West, J. R., 129, 131 West, S. E., xix, xliv, 489, 496, 501, 950, 957

Westbrook, F., 420, 436 Westbrook, G. L., 149, 151, 155, 158, 162, 185, 195, 199, 200 Westby, E., 891, 900 Westenholme, G. E. W., 551, 555, 566 Westerfield, M., 35, 49, 116, 121, 136, 138 Westerman, R., 93, 113 Westhofen, M., 960, 965 Westphal, E., 319, 323, 341 Westphal, H., 116, 132 Westrate, J. A., 888, 903 Westrum, L. E., 105, 113 Wetter, S., 233, 235, 236, 239, 247, 462, 476, 483, 501 Wetzel, C. H., xxv, xliv, 287, 294 Wetzel, N., 230, 245, 246 Weusten, B. L. A. M., 686, 704 Wexler, B. E., 259, 264, 265, 266, 270 Wheeler, T. T., 643, 649 Whelan, J., 36, 42 Whishaw, I. Q., 418, 437 Whiskin, E. E., 357, 358, 376, 419, 430 Whissell-Buechy, D., 277, 294, 314, 327, 333, 344 Whitaker, J. R. 802 White, A., 451, 455 White, B. J., 603, 611 White, C. M., 60, 62, 63, 66 White, D. D., 831, 840 White, D. J., 419, 430 White, D. P., 441, 456 White, E. L., 143, 164, 220, 228 White, F. A., 760, 782 White, J., 145, 157, 181, 195, 299, 306–308, 1005, 1025 White, K. D., 887, 903 White, K. S., 734, 751, 755 White, M. V., 54, 70 White, N. M., 414, 437, 916, 929 White, T. L., 213, 226, 424, 437 Whitear, M., 659, 660, 673, 677 Whitehead, M. C., 664, 677, 737, 746, 754, 756, 854, 860 Whitehouse, P. J., 513, 523 Whitesides, J. G. III, 125, 138 Whitesides, G., 103, 108 Whitesides, J., 62, 73, 105, 113 Whiting, G. S., 992, 995 Whiting, M. R., 420, 427 Whitlock, C. P., 893, 895 Whitlock, K. E., 35, 49, 115, 121, 138 Whitmore, C., 913, 927 Whitney, G., 344, 423, 428, 433, 434, 709, 710, 712, 725, 726, 729, 730, 873, 877 Whitsett, J. M., 970, 979 Whitten, C. F., 886, 903 Whitten, W. K., xxix, xliv, 345, 346, 348, 350, 352, 366, 374, 383, 970, 979 Whitter, T. B., 57, 66 Whittington, R., 910, 929 Wiberg, A., 789, 804 Wichterle, H., 620, 624

1098 Wictorin, K., 515, 520 Widdicombe, I. G., 982, 999 Widdicombe, W. G., 13, 16 Wide, L., 554, 563, 565 Widstrom, A. M., 313, 327 Wiebe, S. P., 415, 437 Wiegand, S. J., 620, 627, 762, 779 Wieneke, J. H., 686, 704 Wiener, N., 295, 308 Wiener, S. I., 412, 437 Wierda, J. M. K. H., 945, 955 Wierson, M., 369, 382 Wieselthaler, A., 151, 161 Wieser, H. G., 268, 270, 424, 431 Wiesler, D., 970, 978 Wigand, M. E., 468, 473, 475 Wight, R. G., 12, 15, 441, 443, 452, 455 Wigmore, S. W., 419, 430 Wikner, K., 983, 999 Wilbertz, J., 621, 623 Wilcock, G. K., 510, 512, 520, 563, 567, 600, 609 Wilcoxon, H., 913, 933 Wildman, D., 664, 675, 710, 711, 717, 729 Wildman, H. E., 888, 897 Wiley, R., 551, 556, 573 Wilhelmsson, B., 585, 589 Wilkens, W.F., 297 Wilkie, D. M., 917, 933 Wilkin, L. D., 910, 917, 933 Wilkinson, G. A., 762, 780 Willard, D. M., 920, 925 Willard, H. F., 1017, 1022 Willatt, D. J., 452, 455, 458 Willcox, M.E., 218, 220, 226 Willemen, A. P., 416, 434 Willems, T., 55, 69, 562, 572 Willey, T. J., 167, 180 Willi, J., 923, 933 William, C. S., 388, 400 Williams, A., 789, 799 Williams, B., 982, 999 Williams, C. F., 890, 903 Williams, D., 360, 374 Williams, D. E., 56, 72 Williams, G. R., 721, 729 Williams, G. W., 788, 803 Williams, H. L., 439, 441, 458 Williams, J. L., 422, 437 Williams, J. M., 422, 428 Williams, J. R., 422, 432, 437 Williams, L., 483, 501 Williams, M., 310, 326 Williams, M. B., 564, 570, 616, 617, 625 Williams, P. L., 665, 677 Williams, R., 95, 101, 102, 113 Williams, R. J., 301, 308 Williams, R. J. P., 281, 293 Williams, R. W., 266, 273 Williams, S., 365, 375 Williams, S. C., xxix, xlv, 687, 702 Williams, S. C. R., 256, 258, 259, 266, 267, 270, 273, 342, 593, 613

Author Index Williams, T. H., 124, 135 Williamson, E. C., 915, 928 Williamson, S. J., 241, 244 Williamson-Vasta, R., 482, 500 Willis, T., xxi, xliv Willner, J. A., 405, 407 Willner, P., 867, 870, 879 Wills, A. M., 621, 624 Wills, C., 849, 850, 855, 860 Willson, C. A., 620, 625 Wilson, A. J., 550, 557, 565, 567 Wilson, C. S., 424, 433 Wilson, C. W. M., 885, 903 Wilson, D. A., 153, 163, 164, 170, 172–170, 176, 177, 180, 184–193, 195, 196, 200, 201, 410, 418, 426, 430, 437, 617, 618, 627 Wilson, D. B., 658, 677 Wilson, E. O., 346, 351, 383 Wilson, F. A. W., 688, 704 Wilson, H. C., 345, 383 Wilson, M., 193, 201 Wilson, M. A., 188, 196, 201, 410, 425, 430 Wilson, R. D., 167, 172, 177 Wilson, R. J., 538, 542, 544 Wilson, R. S., 332, 344 Wilson, S. M., 343 Wilson, S. W., 116, 135, 137 Winans, S. S., xxx, xli, xliv, 165, 167, 170, 173, 178, 179, 967, 970, 973, 974, 978 Winberg, J., 313, 314, 316, 320, 322, 325–327, 355, 380, 835, 845 Winblad, B., 482, 499 Windebank, A., 894, 901 Windham, G. C., 359, 376 Winkelstein, A., 887, 897 Winkler, J., 619, 625 Winne, G. I., 888, 903 Winocur, G., 419, 420, 437 Winograd, S., 753, 758 Winogrodzka, A., 486, 501, 514, 523 Winokur, A., 495, 496, 951, 954 Winslow, J. T., 416, 418, 426, 437 Winter, A. L., 233, 249 Winterbauer, S. H., 542, 547 Winters, B., 394, 400 Wirsig, C. R., 1001–1005, 1022, 1025 Wirsig-Wiechmann, C. R., 389, 400, 1002, 1004, 1005, 1006, 1025 Wirth, S., 174, 175, 177, 180, 421, 429 Wisden, W., 150, 159 Wise, D. R., 368, 375 Wise, P. M., 212, 228 Wise, R., 914, 933 Wisen, O., 887, 903 Wisiewski, L., 887, 903 Wissel-Buechy, D., 849, 850, 855, 860 Witek, T. J., 444, 456 Withee, J., 884, 900 Witherly, S. A., 887, 892, 900–903 Witkin, J. W., 1003, 1025

Witschi, H., 583, 590 Witschi, J. C., 886, 903 Witt, D., 330, 343 Witt, M., 659–668, 670, 675, 677, 764, 782, 824, 825, 846 Witter, M. P., 168, 173, 176 Wobst, B., 318, 319, 323, 324, 341, 342 Wohlleben, R., 468, 473, 475 Wöhrmannj-Repenning, A., 345, 383 Woitalla, D., 509, 514, 521 Wolf, A., xxviii, xli Wolf, B., 82, 85 Wolf, D. C., 582, 589, 591 Wolf, G., 684, 705, 908, 929 Wolf, R. O., 784–786, 792, 804 Wolf, S., 11, 16, 445, 457, 462, 468, 473, 475 Wolf, S. R., 212, 215, 226 Wolfe, J. H., 621, 624 Wolfe, S. R., 462, 475 Wolfensberger, M., 238, 249 Wolff, A., 153, 158, 647, 648 Wolff, J. R., 152–154, 158, 163 Wolff, N. J., 153, 161 Wolfsdorf, J., 7, 16 Wolkoff, P. 998, 994, 999 Wolman, L., 513, 523 Wolozin, B. L., xxvii, xliv, 98, 101, 102, 109, 113, 118, 119, 121, 124, 133, 137 Wolstenholme, G. E. W. Wolterink, G., 415, 416, 434, 436 Wolters, E. C., 480, 486, 501, 514, 519, 523 Won, J., 3, 16 Won, Y. S., 443, 453 Wong, A.M., xxv, xliv Wong, D. M., 773, 774, 780 Wong, E. C., 258, 269 Wong, G. T., xxvi, xlii, 664, 677, 710, 711, 729, 730 Wong, L., 664, 677 Wong, S. L., 762, 780 Wong, S. T., xxvi, xliv, 24, 26, 37, 49, 76, 80, 86, 90, 397, 400 Wong, V., 773, 782 Woo, C. C., 145, 146, 153, 158, 164, 182, 187, 197, 201, 618, 627 Wood, C., 828, 842 Wood, C. M., 644, 648, 829, 844 Wood, E. R., 414, 415, 425, 428, 429, 433, 437 Wood, G., 366, 381 Wood, J. B., 212, 228 Wood, J. G., 617, 618, 627 Wood, J. J., 184, 201 Wood, J. N., 722, 725 Wood, N., 244, 249 Wood, R. W., 538, 543 Wood, S. F., 76, 80–81, 90 Wood, T. C., 59, 73 Wood, T. F 292, 294

Author Index Woodhall, E., 93, 101, 104, 105, 108, 113, 126, 132, 138 Woods, A., 105, 109 Woods, J. H., 861, 863, 876 Woods, M. P., 936, 957 Woodward, C., 342 Woodward, K. M., 342 Woolf, T. B., 144, 164 Woolridge, M. W., 885, 903 Woolston, D. C., 748, 758 Worden, F. G., 751, 754 Working, P. K., 38, 43, 99, 109 World Health Organization, 579, 591 Worley, P. F., 514, 520 Worms, P., 416, 426, 434 Woskow, M.H., 218, 226, 784, 804 Wotjak, C. T., 416, 429, 432 Wouterflood, F. F., 151, 160 Wouters, A., 361, 380 Woutersen, R. A., 582, 588, 591 Wratten, S. J., 550, 567 Wray, A. D., 262, 265, 266, 271, 493, 499 Wray, S., 122, 138, 1003, 1007, 1012, 1013, 1022, 1025 Wren, A. F., 619, 626 Wright, C. E., 970, 976 Wright, D. E., 152, 164, 1003, 1022, 1025 Wright, H. N., 204, 212, 213, 227, 228, 394, 400, 462, 471, 477, 783, 795, 804 893, 903 Wright, J., xvi, xxii, xliv, xx Wright, J. H., 827–829, 831, 840, 842 Wright, J. W., 616, 624 Wright, R. H., 280, 294 Wright, S. T., 64, 69 Wrobel, D., 277, 284, 294 Wrynn, A. S., 149, 158 Wszolek, E. K., 484, 499 Wszolek, Z. K., 329, 344, 484, 499 Wu, C., 717, 724 Wu, D., 710, 727 Wu, H., 762, 767, 780 Wu, J., 183, 197, 480, 481, 491, 493, 496, 501 Wu, J. Y., 151, 152, 158, 184, 197, 618, 624 Wu, T. N., 581, 591 Wunderle, V., 127, 134. 1017, 1023 Wurtman, J. J., 919, 933 Wurtman, R. J., 481, 498, 689, 705 Wurzburger, R., 81, 85 Wustenberg, E. G., 472, 477 Wyatt, J. K., 538, 544 Wyatt, L. M., 149, 160. 184, 198 Wyble, B. P., 191, 192, 197, 413, 432 Wylie, N. R., 352, 374 Wyngaarden, P., 365, 375 Wyrick, M., 311, 325 Wyse, B. W., 950, 955 Wysocki, C. J., xxv, xxix, xxx, xxxiii, xxxiv, xliv, 61, 68, 217, 228, 234, 245, 329, 331, 333, 335, 336, 343, 344, 351, 355, 382, 383, 426, 437,

1099 469, 477, 622, 626, 838, 846, 891, 899, 970, 978, 982, 998 Wyss, H. I., 367, 373 Wyss, H. v., 656, 677 Wyss, J. M., 167, 169–174, 180 Xia, Y., 509, 523 Xia, Z., xxvi, xliv, 24, 26, 37, 49, 76, 80, 86, 90, 397, 400 Xie, T.M., 361, 377 Xiong, W., 151, 155, 185, 195 Xu, F. B., 413, 436 Xu, H., 723, 727 Xu, M., 509, 521 Xu, P., 145, 146, 164 Xu, Q., 116, 135 Xu, W., 389, 394, 400 Xu, Y., 191, 195 Xu, Z., 762, 781 Yabe, I., 938, 949, 957 Yagodin, S., 149, 158 Yajima, T., 553, 562, 573 Yakabow, A., 554, 572 Yale, S., 493, 499 Yamada, H., 713, 730 Yamada, K., 266, 271, 664, 672 Yamada, M., 48 Yamada, S., 32, 37, 49 Yamada, T., 775, 777, 782 Yamagishi, M., xxxi, xxxvii, 26, 34, 37, 38, 43, 49, 124, 138, 463, 477, 507, 508, 509, 510, 511, 523 Yamagiwa, M., 37, 49 Yamaguchi, E., 709, 711, 727 Yamaguchi, K., 764, 767, 768, 779 Yamaguchi, M., 242, 249, 318, 327 Yamaguchi, S., 689, 705, 722, 730, 734, 758, 783, 804, 806, 812, 813, 822, 884, 897 Yamaguchi, T., 266, 271, 889, 899, 900 Yamaguchi, Y., 789, 800, 960, 962, 965 Yamakoshi, T., 444, 458 Yamamoto, K., 101, 102, 111 Yamamoto, M., 85, 87, 717, 727, 941, 957 Yamamoto, S., 961, 966 Yamamoto, T., 149, 164, 644, 649, 683, 702, 704, 705, 716, 721–723, 730, 745, 746, 758, 8887, 899, 905, 915–918, 925, 930, 931, 933 Yamamura, M., 244, 249 Yamasaki, F., 655, 677 Yamasaki, T., 153, 161 Yamashita, H., 713, 730 Yamashita, S., 553, 561, 562, 571, 740, 741, 745, 756, 757 Yamashita, T., 712, 729 Yamashita, Y., 745, 756 Yamauchi, Y., 887, 899, 946, 956, 963, 966 Yamazaki, K., 318, 320, 322, 323, 327, 337, 340, 344, 356, 372, 383, 394, 398, 410, 437, 835, 840, 884, 901

Yamazaki, N., 182, 200 Yan, C., 27, 49, 82, 90, 716, 730 Yan, W., 710, 711, 726, 729, 730 Yanagihara, N., 942, 956, 1003, 1007, 1018, 1022 Yanagisawa, K., xxxi, xlv, 854, 860, 938, 957 Yanai, I., 17, 43, 145, 157, 692, 705, 797, 804 Yanai, J., 129, 136 Yanaihara, N., 152, 158 Yancey, R. A., 35, 42, 123, 133 Yancopoulos, G. D., 762, 779 Yandava, B. D., 621, 627 Yang, C., 621, 624 Yang, G., 983, 998 Yang, H., 723, 725 Yang, L., 116, 138 Yang, P. Y., 299, 308 Yang, Q. X., 256, 257, 273 Yang, R.-B., 82, 87 Yang, X., 258, 273 Yang, Y. M., 299, 308 Yankell, S., 216, 224 Yankova, M. P., 27, 30–33, 38, 41, 45, 664, 674 Yano, B. L., 582, 589 Yano, H., 259, 269, 271 Yao, B., 664, 677 Yao, M., 621, 623 Yaremko, J., 831, 840 Yarita, H., xxiv, xliii, 188, 192, 200, 263, 264, 273, 421, 437, 692, 704 Yasoshima, Y., 683, 705, 916, 933 Yasuda, N., 170, 179 Yasuhara, K., 553, 569 Yasuoka, A., 709, 711, 716, 727 Yata, N., 553, 571 Yatim-Dhiba, N., 557, 568 Yau, K. W., xxv, xxxviii, 24, 27, 41, 42, 78, 80, 82–84, 86–90, 287, 293 Yaxley, S., 680, 681, 685–689, 691, 692, 703–705, 736, 746, 748, 757, 758 Yayuma, N., 746, 758 Ye, Q., 720, 730, 776, 782 Ye, X., 116, 133 Yee, K. K., 395, 396, 400, 616, 627, 631, 634, 638 Yee, L., 357, 362, 374 Yen, D. M., 790, 795, 799, 953, 954 Yen, K. C., 940, 957 Yeterian, E. H., 680, 702 Yeung, D. L., 886, 903 Yilma, S., 26, 27, 48 Yin, K. C., 509, 521 Yin-Zeng, L., 580, 591 Yirmiya, R., 420, 431 Yokel, P., 914, 933 Yokoi, M., 145, 164, 186, 201 Yonas, A., 983, 999 Yoneda, Y., 359, 383 Yonezawa, T., 887, 889, 900 Yonkers, J. D., 660, 672

1100 Yoon, H. R., 443, 453 Yoshida, H., xxiv, xxxvii, 230, 245 Yoshida, K., 1016, 1025 Yoshida, M., 207, 228, 553, 561, 562, 571, 884, 901 Yoshida, S., 102, 105, 110, 659, 675 Yoshida, T., 104, 112 Yoshida-Matsuoka, J., 27, 44, 351, 382 Yoshidan, T., 244, 249 Yoshie, S., 662, 663, 677 Yoshihara, S., 182, 200 Yoshihara, Y., 28, 32, 36, 46, 78, 83, 88, 89121, 123, 125, 130, 135, 138, 139, 145, 160, 186, 193, 198, 972, 977 Yoshii, F., 276, 292, 294 Yoshii, H., 553, 568 Yoshikawa, T., 806, 822 Yoshimoto, M., 509, 510, 511, 514, 523 Yoshimura, H., 368, 383 Yoshimura, I., 963, 966 Yoshioka, I., 658, 673 Yoshioka, S., 266, 271, 713, 730 Young, A. H., 603, 613 Young, A. W., 687, 702 Young, B. J., 415, 438 Young, F. W., 212, 228 Young, H. F., 629, 637 Young, I. M., xxxiii, 783, 788, 790, 795, 799 Young, J. A., 640, 649 Young, J. K., 920–923, 928, 933 Young, J. M., xxv, xxxiv Young, J. T., 582, 583, 590 Young, K. E., 116, 132 Young, L. J., 422, 438 Young, P. T., 865, 879, 883, 903 Young, S. N., 827–829, 831, 833, 840, 842, 844 Young, T. A., 185, 201 Young, W. S., III., 151, 164 Youngentob, S. L., 33, 35, 38, 43, 47, 53, 62, 65, 69, 72, 76, 87, 96, 98, 106, 112, 118, 119, 122, 134, 137, 138, 185, 201, 266, 267, 271, 273, 355, 383, 388, 392–396, 398, 400, 418, 438, 442, 445, 452, 455, 457, 463, 476 616, 617, 621, 624, 626 Younglai, E. V., 366, 378 Youngs, W. M., 147, 148, 152, 160, 165, 167, 169, 173, 177 Yousem, D. M., xxix, xxxii, xxxv, xliv, xlv, 37, 49, 127, 138, 256, 259, 264–267, 269, 273, 329, 344, 470, 472, 478, 489, 490, 495, 497, 501, 503, 512, 516, 520, 523, 593, 594, 596, 597, 599, 603–604, 606–608, 609, 611–613, 616, 623, 629–631, 633, 634, 637, 638 Yu, C., 661, 664, 670, 675, 676, 710, 725 Yu, C. P., 448, 449, 458, 459, 971, 977 Yu, X., 147, 149, 153, 159 Yu, Z. X., 620, 624

Author Index Yuan, D. L., 907, 926, 920–921, 933 Yuen, J., 26, 27, 48 Yugar, Y., 887, 900 Yui, M., 946, 957 Yun, J. B., 55, 71 Yun, K. S., 468, 476 Yuyama, N., 745, 758 Záborszky, L., 147, 148, 153, 164, 170, 180, 413, 438 Zacharias, R., 367, 383 Zack, S., 410, 431 Zagotta, W. N., 80, 90 Zagraniski, R. T., 790, 791, 800 Zald, D. H., 173, 174, 179, 180, 193, 199, 204, 227, 255, 258, 259, 261, 263–266, 271–273, 424, 438, 698, 704, 705, 797, 803, 804 Zambarbieri, D., 236, 247 Zambito, C. M., 865, 876 Zamel, N., 442–444, 452, 457, 458 Zamostiano, R., 553, 563, 568 Zampighi, G. A., xxv, xxxvii Zancanaro, C., 661, 677 Zandstra, E. H., 832, 841 Zang, K., 762, 780 Zangar, R. C., 64, 71 Zanier, F., 489, 501 Zarahn, E., 258, 269, 273 Zaraisky, A. G., 116, 138 Zaromb, S. 306, 308 Zarrow, M. X., 310, 327, 366, 369, 376, 379 Zasler, N. D., 473, 474, 604, 608, 609, 629, 631–633, 637, 638, 789, 799, 959, 960, 965 Zatorre, R. J., xxix, xliii, xlv, 190, 192, 201, 214, 226, 258, 259, 262–268, 270, 272, 273, 424, 431, 698, 699, 704, 705, 797, 803, 948, 957 Zatta, P., 551, 555, 573 Zawakzka, L., 891, 901 Zayas, Z. A., 320, 327, 356, 383 Zecca, E., 891, 901 Zehavi, U., xxvi, xliii, 710, 712, 715, 716, 725, 728–730 Zeifman, D., 828, 846 Zeiger, J. D., 797, 799 Zeiss, K., 653 Zeldin, D. C., 61, 70, 72 Zelena, J., 764, 781 Zell, A., 304, 308 Zeller, M., 537, 546 Zeng, Q., 664, 677, 768, 769, 782 Zeni, M. T., 486, 501 Zervakis, J., xxxi, xlii, 635, 638, 788, 803, 951, 956, 961, 966 Zhang, C., 28, 49, 771, 781, 782 Zhang, C. X., 660, 664, 666, 670, 674, 677 Zhang, D., 101, 103, 113 Zhang, H., 116, 138, 718–721, 728, 737, 752, 755

Zhang, J. H., 58, 73 Zhang, L., 683, 701 Zhang, Q. Y., 56–58, 61–64, 69, 72, 73 Zhang, Y., xxv, xl, 174, 180, 194, 201, 394, 400, 411, 415, 426, 438, 714, 716, 717, 728 Zhao, A. Z., 27, 43, 49, 82, 83, 86, 88, 90, 716, 730 Zhao, G., 723, 728 Zhao, H., xxv, xlv, 78, 91, 126, 138, 145, 164, 287, 294, 396, 400, 551, 573 Zhao, J. C., 54, 62, 68 Zhao, X., 83, 89, 553, 566 Zhao, Z., xxix, xliii, 259–267, 269, 272, 273, 424, 436 Zheng, B.-B., 98, 113 Zheng, C., xxvii, xliv, 126, 138 Zheng, C. J., 855, 859 Zheng, D. R., 24, 41, 120, 123, 128, 132, 825, 841 Zheng, J. Z., 710, 726 Zheng, L. M., 154, 164, 1003, 1011–1014, 1025, 1026 Zhou, D., 619, 627 Zhou, F. M., 149, 155, 183, 194 Zhou, Y. L., 58, 73 Zhukovsky, S., 553, 563, 568 Zhuo, X., 61–63, 70, 73 Zide, B. M., 9, 16 Zidek, L., 970, 978 Ziegler, A., 444, 455 Ziegler, C., 304, 308 Ziegler, D., 553, 573 Ziegler, M. G., 450, 456 Zielinski, B. S., 34, 49, 53, 56, 58, 65, 69, 72, 73 Ziem, G., 587, 591 Zierold, K., 26, 47 Ziff, E. B., 124, 133 Zigova, T., 620, 627 Zilles, K., 266, 267, 272 Zilstorff, K., 230, 243, 248, 960, 966 Zilversmith, D. B., 887, 903 Zimering, R., 495, 498 Zimmer, J., 173, 180 Zimmer, L. A., 149, 153, 156–158, 183, 184, 187, 191, 192, 195, 197, 201, 413, 424, 431 Zimmerman, H., 33, 34, 41 Zimmerman, I. A., 884, 902 Zimmerman, J. E., 511, 523 Zimmerman, R. A., 604, 613 Zimmerman, R. D., 604, 610 Zimmermann, B., 299, 307 Zimmermann, K. W., 658, 677 Zinn, K., 80, 86 Zinreich, S. J., 7, 9, 15, 594, 613 Zinsmeister, A. R., 887, 898 Zippel, H. P., xvi, xlv, 1, 14, 16, 18, 49, 981, 999 Zippelius, H. M., 309, 327 Zirinis, P., 554, 572

Author Index Zirkle, R. E., 643, 648 Zivadinov, R., 489, 501 Zmartzy, S. A., 839, 846 Zohar, J., 493, 499 Zola-Morgan, S., 414, 438 Zoller, M., 723, 727 Zondek, B., 366, 383 Zong, X., 80, 90 Zongrone, J., 554, 563, 571 Zopi, G., 642, 650 Zorrilla, J., 423, 431 Zorzon, M., 489, 501 Zotterman, Y., xxiv, xlv, 667, 671, 713, 725, 732, 754, 885, 896, 999 Zoumas-Morese, C., 887, 897

1101 Zozulya, S., 288, 291, 294 Zubin, J., 239, 248 Zucco, G. M., 424, 435, 483, 486, 501 Zucker, I., 618, 625, 923–924 Zuckerkandl, E., xxiv, xiv Zuckerman, A., 919, 929 Zuckerman, S., 369, 378 Zufall, F., xxv, xxvi, xxxv, xxxvii, 26, 49, 76, 80–81, 84, 86, 88, 91, 511, 523, 971, 973, 977 Zuker, C. S., xxv, xxxvii, xl, 81, 91, 709–711, 714, 716, 717, 723, 724–728, 798, 798, 848, 858 Zumkley, H., 291, 294, 891, 903 Zumpe, D., 363, 379

Zuniga, J. R., xxxi, xlv, 789, 801, 804, 953, 957 Zupko, K., 57, 58, 61, 70, 73, 76, 88, 559, 569, 573 Zusho, H., 266, 270, 604, 613, 629–632, 638 Z˙ uwal⁄ a, K., 661, 677 Zwaardemaker, H.C., xxii, xliv, 204, 205, 218, 228, 984, 999 Zwart, A., 994, 996 Zweiman, B., 443, 451, 456, 459, 467, 476 Zwillenberg, D., 962, 963, 965 Zwozdziak, W., 963, 966 Zyzak, D. R., 170, 180, 414, 438

Subject Index

Absolute threshold, 203, 207, 389 classical conditioning, 390 operant procedures, 389–390 Acceptance-rejection reflexes, 682 Accessory olfactory bulb segregated pathways, 972 synaptic plasticity, 975–976 ACE inhibitors, 462, 951 Acetaminophen, 62 Acetylcholine (ACh), 150, 153–154, 484 piriform cortex, 191–192 Acetylcholinesterase (AChE), 154 Acetyl salicylic acid, 562 Acid multiple ion channels, 721 Acid-sensing ENaC-like channels, 721–722 Acid transduction proposed models, 722 Acid transduction mechanisms, 721–722 Acinar cells Bowman’s glands, 53 Acoustic rhinometry, 441–443 Acquired immunodeficiency syndrome, 463, 605–606 Across-fiber patterns, 747–748 Acrylates, 217 Activities of daily living, 963–964 Acute necrotizing ulcerative gingivitis, 941 Acute rhinitis, 467 Acute rhinosinusitis, 467 Acute viral-related rhinitis, 467 Adaptation, (see also Desensitization, Habituation), 222 sensory, 84, 217

Addiction, 540 Addison’s disease, 470 Adenocarcinoma paranasal sinuses, 598 Adenoid hypertrophy, 467 Adenylyl cyclase, 80 regulation, 80 types, 80 Adoption designs, 332 Adoption methods, 332–334 Adrenal steroids, 464 Adrenocortical insufficiency, 470 Adult learning, 357–358 Adult olfactory neuroepithelium neurogenesis, 93–107 Age globose basal cells, 119 neurogenesis, 98 odor perception, 216 receptor cell number, 36 Ageusia, 890, 948 Aggression-eliciting pheromone male mouse, 361–363 Aging, 952 olaction, 1, 36, 98, 216 taste, 790 Air-dilution olfactometry, 204 Airflow, 231–232 Airflow dynamics olfaction, 452–453 Airflow sensation odors, 452 Ala, 4 Alanine, 558 Alcohol breast milk, 835 1103

[Alcohol] dehydrogenase, 56 sniff test, 462 Alinamin, 204 Alkaline phosphatase, 56 Allergic rhinitis, 472, 599 Allesthesia, 907 Alum, xix, xxvi Aluminum, 555 Alzheimer’s disease, xxii, xxxi, 39 imaging, 600–601 neuropathology, 503–513 epithelium, 39, 504–511 odor identification, 238 olfactory bulb pathology, 512 olfactory cortex pathology, 512–513 olfactory function, 479–483 studies, 481–482 olfactory mucosa studies, 39, 506–508 olfactory tissue, 40 single staircase, 206–207 UPSIT scores, 480 Ambergris odorants, 277 structure, 279 Amblystoma, 115 Ambrettolide structure, 278 Ambrox, 277 Amiloride-insensitive pathways salt taste transduction, 720 Amino acids, 464, 558 transduction mechanisms, 722–723 Amitriptyline, 367 Ammonia, 584 Amniotic fluid flavor carrier, 837

1104 [Amniotic fluid] neonatal attraction, 316 odors influence on neonates, 313 postnatal attraction, 315 Amphetamine, 914 Amphibians taste buds, 660–661 Amplitude-matching neurons, 296 Amygdala, 182, 261, 411, 688 Amygdala system, 425 odor-taste odor-toxicosis, 420–421 Amygdaloid complex, 173 Amyloid plaques, 509 Amyotrophic lateral sclerosis olfactory function, 489–490 Amytriptyline, 951 Analgesics, 464, 562 Anchored 9-point category scale, 853 Androst-16-en-3-ol structure, 278 Androstenone, 61, 350 Anesthetics, 464 Angiotensin-converting enzyme (ACE) inhibitors, 951 Animal behavior, xxix–xxxi Animal spirits, xxi Animal studies kin recognition, 336–338 olfaction taste Annoyances, 586–587 Anorexia nervosa, 472 estrogen, 921–924 systemic infection and disease, 909 toxins, 910 Anosmia, xx–xxiii, 38, 461, 630–631, 635, 889 benzaldehyde, 329 clinical olfactometry, 237 isovaleric acid, 329–330 Anterior choanae, 2 Anterior cortical nucleus, 173 Anterior cranial fossa human intracranial view, 29 Anterior olfactory nucleus (AON), 146, 166–168, 171–172, 261 Antibiotics, 562 Anticancer agents, 464 Antifungals, 46 Antihistamines, 464 Antimicrobials, 464 Antimigraine medicines, 562 Antirheumatics, 464 Antithyroids, 464

Subject Index Antivirals, 464, 562 Apolipoprotein E, 483, 510 Aponeuroses, 4 Apoptosis, 62, 95 Apparent taste interactions, 809 Apparent within-substance interactions, 809 Appetite suppression, 563 Appetitive conditioning odor cues, 409–413 Approach paradigm, 404–405 Aquaporins, 31 Arachidonic acid epoxide, 61 Arochlor, 12, 54, 64 Aromascan A325, 301 Arousal maternal odors, 314 olfactory cues, 310 Array-based odor sensors, 297–304 Artificial neural networks (ANNs), 301–302 Artificial noses, 295–306 future directions, 306 Artificial odorant babies, 320 Artificial sensing system, 295 Ascending method of limits (AML), 206–207 Associational fibers, 169 Associative conditioning, 187 Astrocytes, 129 Auriculotemporal nerve (ATN), 947 Autoimmune syndromes, 941 Autonomic reflexes, 682 Autoradiography, xxviii Autoreceptors, 149 Aversive conditioning odor cues, 413–414 Avitaminosis, 950 Axonogenesis, 119 Axotomy, 36 Back-propagation-trained feed-forward networks electronic noses, 302–304 Bacteria, xv B2-adrenergic receptor, 84 B-adrenergic receptor kinase (BARK), 84 Bar-press stimulus presentation paradigm, 405–406 B-arrestin, 84 Basal cell division cell death, 98 molecular regulation, 119 Basal cell proliferation in vivo regulation, 99

Basal cells, 22, 33, 52, 118 ORNs, 76 Basal forebrain projections, 147–148 Basal ganglia, 688–689 Basic helix-loop-helix (bHLH), 121 Basic taste stimuli responses, 738–740 Bedwetting, 563 Behavior appetitive conditioning, 409–410 food preferences social transmission, 41 habituation/discrimination, 417 perception inferred, 867 recognition memory, 414, 415–416 Behavioral-genetic approach olfactory characteristics, 329 Behavioral-genetic research methods and designs, 329–331 Behavioral pheromones, 348 Behavioral responses influence of odors, 322, 385, 397 ontogeny, 825–832 preferences, 403–407 Behcet’s syndrome, 941 Bell’s palsy, 942–945, 953 Benzaldehyde anosmia, 329 structure, 279 Benzenoid musk, 277 Benzopyrene, 63 Biological IETS, 281 Biological spectroscope, 286 SOR, 281 Biological substrates, 315 Biology of twinning, 329–330 Biotransformation enzymes inhibitors, 64–65 Birds taste buds, 661 Bitter-almond odorants structure, 279 Bitter compounds detection, 708–709 diversity, 708–709 Bitterness intensity scaling solutions, 850–851 Bitter taste, 856–857 genetics, 848 PROP tasters, 857 role, 847–848 saliva, 644 transduction mechanisms, 708–712 proposed models, 712 Bitter transduction pathways, 711–712 Blanes’ cells, 142–143

Subject Index Blast injection device, 205 Blood flow neuroimaging history, 252–253 Blood group antigens, 123 Blood vessels, 54 BOLD functional magnetic resonance imaging (fMRI), 257 Bombyx mori, 345 Bone morphogenetic protein (BMP), 102–103, 116 Bony septum, 3 Book of the Dead, xvi Bosom Darwin’s hypothesis, 312 Bottle-fed infants maternal scents, 319 Bovine tongue, 652 Bowman’s glands, 35, 53, 54, 60 xenobiotic compound metabolism, 30 Bowman’s glands/duct complex, 118 Brain human drawing, 680 Brain contusion, 631, 960–961 Brain hemorrhage, 631, 960–961 Brainstem projections, 147–148 Brainstem tracts, 773–775 Brain tumors, 605 Breast influence on neonates, 313 maternal, 312 Breastfeeding, 312–314 learned recognition, 319 maternal scents, 319 novel foods, 837 Brief-access taste test, 865 Bruce effect, 365–368 Brush cells, 33 Bulbopetal cells, 147 Bulimia nervosa, 951 Bulk acoustic wave (BAW), 299 Bulla ethmoidalis, 7 Burning mouth syndrome (BMS), 952 magnitude-matched taste intensities, 788 Buserelin, 563 Buspirone, 367 Butanol threshold detection, 215 Cadmium, 555, 576, 579–580, 581 Calbindin, 34, 510–511 Calcitonin, 563 Calcium olfactory transduction, 83 Calcium/calmodulin-dependent PDE (PDE1C2), 83

1105 Calmodulin, 83 cAMP, xxvi desensitization, 84 elevation, 80 odorant detection, 79–81 production, 79 response element (CRE), 85 Camphor neutralization, 218 structure, 279 Camphoraceous odorants structure, 279 Cancer chemosensory abnormalities nutritional implications, 890–891 hypophagia, 909 treatment, 910 Caparrapi oxide strong-weak isomer pairs, 292 Capsaicin, 907 Captopril, 950–951 Carbohydrates, 883–884 Carbon black/polymer composite materials, 299 Carbonic anhydrase, 27 Carboxylesterase localization, 56 Carnosine, 123–124, 150–151 Cartilaginous septum, 3 Carvone enantiomers, 286 Catastrophe theory, 296 Category scales, 210 Caudolateral orbitofrontal secondary cortical taste area gustatory responses, 687–688 Cavernous plexuses, 11 Cavum, 2 Cell death neurogenesis, 98 OSN, 95, 96 Cell division, 118–119 Cellular differentiation, 119–122 biochemical and functional aspects, 123–124 Central gustatory nuclei functional development, 777–778 Central gustatory structures sensory ganglia, 760–764 Central nervous system disorders, 947–950 inhaled toxicant protection, 60 xenobiotic transport to, 549–564 Central olfactory pathways modulation, 186–187 nonolfactory responses, 186–187 sensory physiology, 181–194 Central olfactory structures, 165–176

Central taste anatomy and neurophysiology, 679–699 Centrifugal axons, 140, 145 terminal, 144 reconstruction, 145 Cephalexin, 562 Cephalic phase cardiovascular response, 888 Cephalic phase gastrointestinal response, 887 Cephalic phase pancreatic endocrine response, 888 Cephalic phase pancreatic exocrine response, 887–888 Cephalic phase renal response, 888–889 Cephalic phase salivary response, 887 Cephalic phase thermogenic response, 888 Cerberus, 116 Cerebellum odorant-induced activation, 266–267 CGAP1 olfactory transduction, 83 cGMP, xxvi, 80 olfactory transduction, 81–82 cGMP-dependent protein kinase (PKG), 84 Chemical field effect transistor (ChemFET), 299 Chemical mixtures olfactory perception, 217–223 trigeminal responses, 993–994 Chemical stimuli hedonic tone, 734–736 intensity, 734–736 quality, 734–736 Chemometric techniques, 301 Chemosensory abnormalities nutritional implications, 890–891 Chemosensory assessment, 210 Chemosensory disorders dietary management guidelines, 893–894 Chemosensory dysfunction, 2, 216 Chemosensory learning chemosensory genetics, 353–358 Chemosensory trigeminal stimulation human psychophysical and physiological studies, 983–995 Chemosensory trigeminal system anatomy and physiology, 982–983 China, xvii Chlorodinitrobenzene, 59 Choanal atresia, 467 Chocolate aroma, 221

1106 Cholecystokinin, 150, 154, 563 Choline acetyl transferase, 664 Cholinergic system discrimination learning, 413 Cholinesterase-inhibiting pesticide exposure, 538 Chorda tympani nerve, xxi, xxxi, 667, 938 fibers, 945 Chordin, 116 Christianity, xvi, xvii Chromatin negative gonadal dysgenesis, 470 Chromium, 576, 580 Chronic renal failure, 950 Chronic rhinosinusitis, 467 Chronogenetics, 332 Cigarette smoke, xxvii, 64, 216, 563 Cilia, xxv, 13, 17, 23–28, 463 beating frequency, 55 respiratory epithelium, 54 Ciliogenesis, 120 Cineole structure, 279 Cis-3-hexenol structure, 279 Classical conditioning absolute threshold, 390 Clinical assessment, 961–963 Clinical disorders evaluation and management, 935–953 Clomipramine, 951 Cluster analysis, 300–304 CNO (see Nervus terminalus) CNS transport specificity, 561 CNV, 243 CN V, 669–670, 947 nasal cavity, 11–12 CN VII, 942–943 taste fibers, 944 CN IX, 669, 946–947 CN X, 669, 947 Cobalt, 555 Cocaine, 558, 600, 914, 951 Codex atlanticus, xx Cognitive factors, 817–818 Cognitive processes, 358–359 rat, 358–359 Cognitive processing of odors OERP, 239–240 Collapsible nasal valve, 441 Cologne water neutralizing camphor, 218 Color-blindness, 330 Columella, 3 Combinatorial array model piriform cortex, 188

Subject Index Computed tomography, xxiii, 594 Computerized tomography (CT), xxiii Computer simulated flow, 448 Conchae development, 115–118 Conchal type sphenoid sinus, 9 Conditioned operant responses taste reinforcers, 866 Conditioned stimulus, 911 Conditioned taste aversion, xxxi, 683, 905–924 estrogen, 920–921 feeding systems, 905–909 illness systems, 909–910 learning, 910–913 neural connection, 912 neural mechanism, 913–918 Conducting polymer film, 301 illustration, 298 sensor array, 301 Congenital abnormalities, 599 Congenital anosmia, 469–470, 599 imaging, 603–604 Conspecific chemical signals influence neonatal behavior, 309–314 Contemporary taste testing, 786–793 Contingent negative variation (CNV), 243 Copper deficiency, 950 Copulin, 363 Cortex, 411–412, 425–426 odor-taste odor-toxicosis, 421–422 Coumarin, xviii, 59, 62 Counseling, 964 CPR, 57–58 Cranial nerve damage, 960 CREB activation, 84–85 Cribriform plate, xx, xxii, 5–6 Crista galli, 5, 29 Cross-modal matching procedures, 210 Crying, 314 Cushing’s syndrome, 470–471 Cyanide-metabolizing enzyme rhodanese, 56 Cyanide sensitivity families, 332 sensitivity, 58 Cybernetics defined, 295 Cyclic GMP desensitization, 84 Cyclooctane structure, 279

Cyclopentadecanolide, 276 Cytoarchitechtonic, 254 Cytoarchitecture olfactory epithelium, 55 Cytochrome P450, 57–58 Cytochrome P450 reductase (CPR), 57–58 Cytokines olfactory mucosa, 101–102 Cytosolic GST gene families, 58 Data processing electronic nose, 300–304 Death sensory neurons neurogenesis stimulation, 96 Deglution, xxxi, 463 De Humani Corporis Fabrica, xx Dendrites formation, 120 Dendritic knobs, 53, 120 Deglution, 463 Depression, xxxii, 951, 961 Depressor nasi septi labii, 4 Desensitization (see Adaptation), 84 Desipramine, 367, 951 Detection and recognition threshold tests, 206–207 Detection threshold, 867–873 comparison, 869–873 Developing mice nervus terminalis neural cell adhesion molecule immunoreactivity, 1014–1016 Developmental abnormalities, 599 Diabetes, 950 chemosensory abnormalities nutritional implications, 892–893 Diagnostic testing, 937–938 2,6-dicholorobenzonitrile, 62 Diet primary chemosensory disorders, 889–890 Difference threshold tests, 203, 208 Differential X chromosome inactivation, 330 Dimethylcyclohexane structures, 285 Dimethyl disulfide (DMDS), 363–364, 364 Dimethyl-sila-cyclohexane structures, 285 Direct scaling experiments, 806–907 Disability evaluation, 963–964 Discriminant analysis (DA), 301 Discriminant factorial analysis (DFA), 301

Subject Index Discrimination, 682, 732–734 Discrimination learning, 409–414 biochemical substrates, 412–413 Discrimination scaling late childhood/adolescence, 831–832 Discrimination tasks, 394 Dissimilar tasting substances mixtures, 813–829 qualitative changes, 815–816 DMDS, 363–364, 364 Dopadecarboxylase, 151 Dopamine, 150, 151 olfactory epithelium, 102–103 olfactory mucosa, 101–102 Down syndrome, 330 OERP, 239 olfactory function, 483–484 Doxepin, 951 Draw tube olfactometer of Zwaardemaker, 204, 205 Drosophila, 334–335 Duct cells Bowman’s glands, 52 Dyes, 557–558 Dynamic air-dilution olfactometers, 205 Dysgeusia, xxxi, 890, 948 Dysosmia, 889–890 Dystrophic neurites, 505–509 Early canine studies, 386–387 Early childhood taste, 830–831 Early olfactory experience influence adult social preferences, 320–322 Ear surgery, 945 Echo-planer imaging (EPI), 257 Ectohormones, 345 EEG, 229 description, 251 electrodes, 243–244 analogy, 230 EGF family olfactory mucosa, 101–102 EGF/TGF olfactory epithelium, 102–103 Egypt, xvi Electrical activation olfactory epithelium, 232 Electric Taste, xxi Electric-taste thresholds, 786 Electroantennogram, xxiv Electrochemical sensors electronic nose, 299 Electroencephalography (EEG), 229 description, 251 electrodes, 243–244 analogy, 230

1107 Electrogustometry, 784, 788–789 localized electric current, 785–786 Electron-lucent microvillous cell, 33 Electron microscopy, 19, 21, 93, 120 Electronic noses applications, 304–306 approaches, 304 hybrid technology, 300 technology, 295, 297 Electronic nose system elements, 297 Electro-olfactogram (EOG), xxiv, 80, 123, 229, 243 Electron-opaque microvillous cell, 33 Electroretinogram (ERG), 229 Embryo head human, 116, 117 Embryonic development structural aspects, 35–36, 117 Embryonic tongue human, 666 Enantiomers, 280, 286–287 Enchondroma, 596 Endocrine disorders, 470–471 Endogenous signaling molecules modulation, 61 Endohormones, 345 Endopiriform nucleus, 172–173 Endoscopic ethmoidectomy, 473 Endotracheal intubation, 945 End-stage liver disease, 950 Enkephalin, 150, 154 Ensheathing cells, xxvii, 129 photomicrograph, 122 types, 104–105 Entorhinal cortex, 173–174, 261, 409 Enuma elish, xvi Environmental applications electronic nose, 305–306 Environmental influences twins, 330–331 Environmental medical unit, 536 Environmental testing multiple chemical intolerance, 541–542 Environmental toxicants, 575–587 animal studies, 581–584 historical background, 575–577 therapy, 579 Enzyme induction, 63–64 EOGs, 80, 123, 229, 243 EPI, 257 Epilepsy odor memory impairment, 214 olfaction, 495–496 Epileptic seizures, 950

Epithelium interkinetic nuclear migration, 118 sodium channels functional development, 775–777 types, 52–53 Equimolar comparison, 810 Erection-eliciting pheromone rat, 364 Estrogen conditioned taste aversion, 918–924 Ethanol, 885–886 Ethmoidal sulcus, 6 Ethmoid bone schematic drawing, 5 Ethmoid bulla (bulla ethmoidalis), 7 Ethmoid complex, 5–7 Ethmoid labyrinth, 7 Ethyl citronellyl oxalate, 276 Ethyl-methoxypyrazine structure, 279 Eudesmol strong-weak isomer pairs, 292 Evans blue, 557 Event-related potentials vs. evoked potentials, 230 Event time, 816 Evoked potentials (EPs) description, 251 Experimental design functional neuroimaging experiments, 257–258 Expressed olfactory receptors response, 288 External carotid artery nasal cavity, 10 External nose lateral view, 2 External tufted cell, 141 Extinction, 918 Extracellular matrix, 125 Extraepithelial cell migration, 122 Extralingual taste buds, 658 Eye irritation homologous chemical series, 988–991 Eye irritation thresholds, 992 Facial nerve (CN VII), 942–943 taste fibers, 944 Facial reactions first year, 829 Factorial plot comparison, 809–810 Familial dysautomonia (Riley-Day syndrome), 951 Familial olfactory imprinting, 320 Family designs, 332 Family methods, 332–334 Family studies kin recognition, 338

1108 Fat, 884–885 PROP tasters, 857 Feature detection systems, 296 Fechner’s law, 806 Feed-forward networks approaches, 304 Feeding ontogeny, 833–838 young children, 837–838 Ferrocene, 62, 284 electron-density maps, 284 Fetal fluids odor mixtures, 314–315 Fetus feeding, 833 taste, 825 FGF family olfactory mucosa, 101–103 Fiber optic sensor, 299 Fila olfactoria olfactory nerve, 119 Filter paper method, 849–850 Fish taste buds, 660 Fixed intensity level comparison rules, 808 Fixed stimulus level comparison rules, 808–810 FLASH scan, 257 Flavonol ST (FST), 58 Flavor, xxxi Flavored milk, 835–836 Flavor perception human ontogeny, 823–839 Flavors, xxxi neural representation, 699 retronasal perception feeding, 834–835 Fluoxetine, 367 fMRI, 253–254, 257, 796 description, 252 odorant-induced activation, 258–259 Foliate papillae, 655–657 Follistatin, 116 Food acceptability primary chemosensory disorders, 889–890 Food avoidance cancer treatment, 910 Food choices genetic taste markers, 857–858 Food industry applications electronic noses, 304 Food intake estrogen, 919–920

Subject Index Food odor, 187 piriform cortex, 191 Food preferences social transmission, 419–420 Forced approach-avoidance paradigm, 405 Formaldehyde, 59, 585 sensitization, 538 Formal learning tasks, 418–419 Foster-Kennedy syndrome, 605 Fragile X syndrome, 330 Fragrance synthesis industry, 275–276 Frontal bone, 7–8 Frontal sinus, 7–8 Frontonasal prominence, 116 Fruit ripe vs. overripe, 219 Functional groups vibrational theory, 282–283 Functional magnetic resonance imaging (fMRI), 253–254, 796 description, 252 odorant-induced activation, 258–259 Functional neuroimaging description, 251 human temporal and spatial resolution, 252 human olfaction, 251–269 technical considerations, 255–258 properties, 254–255 Fungiform papillae, 657–658 induction and differentiation, 765–766 PROP tasting, 854–855 Fungiform taste bud human, 668 Fusiform somata, 142 Gadolinium, 598 Galaxolide, 277 Galectin-1, 126 Galen, 651 Galvanic currents saliva, 645 GAPs, 23, 130 Gas flow models, 447–448 Gasoline neutralizing camphor, 218 Gas sensors polypyrrole films electronic nose, 300 Gastroesophageal reflux disease (GERD), 942 GDNF family olfactory mucosa, 101–102 Gender odor perception, 216

Generalization, 732–734 Genetic manipulations recognition memory, 416–417 Genetics mice, 329–330, 335–336 olfactory perception, 329–340 Genetic taster status, 798 GFAP, 28, 105 Gingiva disorders, 941 Glands nasal respiratory epithelium, 13 Glass sniff bottles, 204 Glial acidic fibrillary protein (GFAP), 105 Glial cells, 130 Glial growth factor (GGF2), 104 Gliomas, 469 Globose basal cells, 22, 33, 118 Glomeruli, 141 formation, 129–131 Glossopharyngeal nerve (CN IX), 669, 946–947 Glutamate, 150, 151 release modulation, 149 Glutamatergic system discrimination learning, 412–413 Gold, 555 Golgi cells, 142 G-proteins, xxv, xxvi, 35, 79 Granule cells, 140, 142, 152 EPL synaptic connection, 144 mitral cells, 143 Greater petrosal nerve, 667 Greece, ancient, xvi, xx Green odorants structure, 279 Growth factors, 100–104 defined, 100 GSH, 58 GST, 58 GTPase-activating proteins (GAPs), 23 Guanylyl cyclase, 27 classes, 81–82 stimulation, 81–82 Guanylyl cyclase-activating protein (GCAP), 82 Guanylyl cyclase-D, 82 Gulf War veterans, 538 Gustatory anatomy, 679–680 Gustatory assessments evaluation, 788–793 Gustatory disorders classification, 936 Gustatory function assessment, 786–790

Subject Index [Gustatory function] human contemporary measurement, 783–798 regional testing, 784–786 whole-mouth testing, 784–786 Gustatory function testing, 963 Gustatory ganglia functional development, 777 Gustatory neural coding, 731–753, 747–753 Gustatory neurons, 699 basic response properties, 737–747 convergence and tuning breadth, 741–745 information coded, 734–737 Gustatory papillae, 654–655 blood supply, 658–659 maintenance, 766 Gustatory system functional development, 775–778 Gustatory transduction molecular physiology, 707–724 Gustducin-coupled taste receptors identification, 710–711 Gustin saliva, 645 Habituation, 190, 191 Habituation/discrimination paradigm, 417–418 biochemical substrates, 417 cellular correlates, 418 lesions, 417 Hallucinations, 472 Hamster vaginal attraction pheromone, 363–364 Head and neck examination, 962 Head injury, 2, 38, 604–605, 629–636, 959–964 activities of daily living, 634–635 clinical assessment, 631–634 counseling and information resources, 635–636 disability evaluation, 634–635 history of, xxiii, xxxi mechanisms, 2, 630–631, 960–961 medicolegal considerations, 635 patient history, 631–632 physical examination, 632–633 prognosis, 634 radiography, 633 treatment, 634 Head-orientation preference for odor, 315 Head trauma, (see Head injury), xxiii, xxxi Heat shock protein, 510

1109 Hedonic responses, 734 animals assessment, 864–866 odors by infants, 321 vs. signal processing, 866–867 Helional, 287 Heme oxygenase (HO), 82 Heroin intranasal use, 600 Herpes simplex virus 1 genome, 942–943 Hiatus semilunaris, 7 Higher-order gustatory relays, 775 Hindered functional groups, 283–284 Hippocampal system, 425 odor-taste odor-toxicosis, 421 Hippocampus, 262, 412 appetitive conditioning, 410–411 recognition memory, 414 History, 961–962 HIV chemosensory abnormalities nutritional implications, 891 Holoprosencephaly, 599 Homologous chemical series, 988–991 Horizontal basal cells (HBC), 22, 33, 118 cellular differentiation, 122 Horseradish peroxidase, 145, 558–559 HSP70, 28 Huddling olfactory cues, 310–311 Human brain drawing, 680 Human embryo head, 116, 117 Human embryonic tongue, 666 Human flavor perception ontogeny, 823–839 Human functional neuroimaging temporal and spatial resolution, 252 Human fungiform taste bud TEM, 668 Human gustatory function contemporary measurement, 783–798 regional testing, 784–786 whole-mouth testing, 784–786 Human nasal cavity anatomy, 1–14, 55 epithelia, 52 Human neonates, 311–314 chemoreceptive systems, 311–312 nipple localization/sucking, 312–314 Human nose schematic sagittal sections, 38 Human olfactory bulb embryonic development, 128 plastic section, 30 Human olfactory dendritic endings scanning electron micrograph, 18

Human olfactory epithelium SEM, 19 Human olfactory function psychophysical measurement, 203–223 Human olfactory memory, 424–425 Human olfactory neurons life span, 36 Human olfactory system functional mapping, 254 Human perception taste mixtures, 805–820 Human peripheral taste system development, 664–666 Human sense of smell history of electrophysiological research, 229–230 Human taste perception genetics, 847–858 Human tongue, 653–654, 665 innervation, 666–667 Human trigeminal chemosensory responses quantitative structure-activity relationships, 991–992 Hungary water, xviii Hunger sensory function, 882–883 taste processing, 690–692 Huntington’s disease imaging, 602 olfactory function, 488–489 Hydroxysteroid ST (HSST), 58 Hypersalivation, 948 Hypertension chemosensory abnormalities nutritional implications, 891 Hypertrophied adenoids, 467 Hypogeusia, 890 diagnosis, 790 Hypogonadotropic hypogonadism with anosmia, 1016 Hyposmia, 445, 461–473, 479–495, 889 Hypothalamus, 263, 688 Hypothyroidism, 471, 950 chemosensory abnormalities nutritional implications, 892 IDPN, 64 Identical twins, 329–330, 337 Identification confusion matrix tests, 213 Idiopathic dysgeusia, 953 IgA, 55 IGF family olfactory mucosa, 101–103

1110 Imaging parameters for functional neuroimaging studies, 256–257 Imipramine, 951 Imitrex, 562 Immediate response, 863 Immune dysregulation, 537 Immunoglobulin, 559 Indane musk structure, 278 Individual recognition olfactory cues, 317–320 Industrial chemicals and dusts, 217 Inelastic electron tunneling spectroscopy (IETS), 281 Infants (see also Neonates) amniotic environment odor, 316 bottle-fed maternal scents, 319 feeding, 833–837 hedonic responses, 321 milk, 834–837 mother’s olfactory signature, 319 newborn, 826–829 premature, 826 solid foods, 837 Inferior turbinate, 2, 9 Information resources, 964 Ingestion taste reactivity, 732 Inhaled toxicants detoxication, 59 protection, 60 Inherited taste blindness, 856 Innate aversions, 905–907 Innate preferences, 905–907 Inositol-1,4,5-triphosphate (IP3), xxvi, 76 odorant detection, 81 Insula, 266 Insular-opercular (primary) taste cortex, 685–687 Intake early childhood, 830–831 newborn infant, 828 Intensity-difference threshold, 393 Intensity discrimination, 211, 212, 793 Intensity scaling early childhood, 831 olfaction, 210–211 PROP taster status, 851–852 Internal tufted cells, 151 Intrabulbar axons, 145 Intraepithelial fascicles, 29 Intraepithelial glands nasal respiratory epithelium, 13

Subject Index Intranasal viral infections, 38 Intraorally delivered taste stimuli oromotor responses, 866 Inverted papilloma, 596–597 Iridanes strong-weak isomer pairs, 292 Irritant gases, 584–585 Isoamyl butyrate threshold detection, 215 Isobutylmethoxypyrazine (IBMP), 80 Isosteric molecules, 284–286 Isotope substitution, 285–286 Isovaleric acid anosmia, 329–330 Jacobson’s cartilage, 3 Jacobson’s organ (see Vomeronasal organ) Juniper, 355 Just noticable difference (JND), 208, 787 Juvenile angiofibroma, 597 Juvenile recognition recognition memory, 415–417 Juxtaglomerular cells (see Periglomerular cells) Kallmann’s syndrome, xxxii, 38, 127, 214, 462, 471–472 human fetus, 1016–1018 twins, 332 Karanal, 277 structure, 278 Keratinizing squamous epithelium nares, 4 Keros type III olfactory fossa, 6 Kiesselbach’s plexus, 10 Kin recognition family studies, 338 nonhuman animal studies, 336–338 olfaction, 336–339 twin studies, 338 Korsakoff psychosis, 214 imaging, 602–603 Labeled lines, 748 Labeled magnitude scales, 788, 853, 854 Lacrimal bone, 9 Laminae, 129 formation, 127–129 Lamina papyracea, 5, 7 Lamina propria, 35, 53 Laminin, 125–126 Laryngoscopy, 945 Late childhood/adolescence, 831–832 Lateral crura, 3 Lateral lamellae, 5, 6

Lateral nasal wall maps, 14 schematic sagittal view, 8, 10 Lateral olfactory tract (LOT), 167–169 Lead, 576, 581 Learned aversions, 905–907 Learned preferences, 905–907 Learned taste avoidance, 909 Lectins, 556 Left orbitofrontal cortex, 264 Lesion studies appetitive conditioning, 410–411 recognition memory, 414–415 Leucine, 558 LHRH, 332 LHRH-immunoreactive neurons nervus terminalis ganglia, 1012–1013, 1016–1018 LHRH immunoreactivity nervus terminalis, 1007 LHRH neurons migration, 1016 olfactory epithelium, 1013–1014 origin, 1008–1010 Ligustral structure, 279 Limbic lobe, xxiv Limonene, 220 Linear solvation model, 991–992 Line scales, 210 Lingual gustatory papillae development, 764–765 sensory ganglia, 760–764 Lingual nerve injury, 961 Lingual papillae, 654–658 Lingual taste bud primordia, 666 Lingual taste buds distribution, 655 Lingual tonsil, 654 Lipase, 642 Lipophilic odorants, 6t1 Lithium chloride, 914–917 hypophagia, 910 Little’s area, 10 Liver disease chemosensory abnormalities nutritional implications, 893 Longitudinal twin study, 332 Low salivary flow rates remedies, 646–647 Lucifer dyes, 557 Lung inhaled toxicant protection, 60 Luteinizing hormone releasing hormone, 150 Lymphoma, 599

Subject Index Machado-Joseph disease, 952 MAChRs, 154 Macrocyclic musk structure, 278 Macronutrients sensory influences, 883–885 Magnetic resonance imaging (MRI), xxiii, xxviii, xxxii, 253, 594 functional, xxiv Magnetic resonance spectroscopy (MRS) description, 252 Magnetic source imaging (MSI), 229 Magnetoencephalography (MEG), 796 description, 251 Magnitude estimation, 210, 787, 853 bias, 211 Magnitude matching, 210, 787 Main olfactory bulb, 182–187 glomerular layer, 182–184 Main olfactory epithelium (MOE), 351 Major histocompatibility complex (MHC), 337, 356 odors, 320, 321 Malaria, xix Male mice aggression-eliciting pheromone, 361–363 Malignant neoplasms, 597–599 Malingering detection, 216 Maltol, 290 MamFas II, 121 Mammalian nasal mucosa inhaled substance metabolism, 56–57 Mammalian olfactory epithelium early observations, 17–21 morphology, 17–40 Mammalian olfactory respiratory cilia diagram, 23 Mammalian pheromones, 345–372 case studies, 359–369 Mammalian taste buds, 661 Mammals cognitive processes, 358–359 nonhuman odor preference determination, 403–407 psychophysical evaluation, 385–397 methodological issues, 387–389 Manganese, 555–556, 581 Manometer, 439 MAPK (mitogen-activated protein kinase) odorant stimulation, 85 Mash1, 121 Mass spectrometry-based devices, 299

1111 Mate recognition pair-bonding odor learning, 422 Maternal behavior odor learning, 423–424 Maternal breast, 312 Maternal odors nipple attachment and sucking, 313 Maternal olfactory signature following birth, 320 Maternal olfactory signatures neonatal discriminative responses, 317–318 Maternal pheromone rat, 359–361 Maxillary bone, 7 Maxillary sinuses, 7 Mayol strong-weak isomer pairs, 292 MDS, 212 Medial crura, 3 Medial olfactory pit, 1010 Medial olfactory placode, 1008–1010 Medical diagnostic applications electronic nose, 305 Medical evaluation, 462–463, 535, 936–938 Medical history, 462–463, 936 Medical imaging, 593–608 Medications, 950, 961 Medicine early, xviii–xx history, xix Medicolegal considerations, 964 Mediodorsal thalamus, 409, 411 MEG, 796 description, 251 Melanoma, 599 Membranous septum, 3 Memory, 222–223 Memory tests olfactory, 213–214 Meningiomas, 468–469 Mercury, 556, 576, 580–581 Metallocenes, 284 Metallothionein, 511 Metal oxide semiconductor field effect transistors sensors, 299 Metal oxide sensor electronic nose, 298 illustration, 298 Metastatic disease, 599 Methamphetamine, 59, 914 Methimazole, 64 Methylanthranilate strong-weak isomer pairs, 292 Methyl bromide, 585

1-methyl-4-phenyl-1,2,3,6tetrahydropyridine-induced Parkinsonism olfactory function, 487 3-methylcholanthrene, 63 MHC, 337, 356 odors, 320, 321 Mice cognitive processes, 358–359 developing nervus terminalis, 1014–1016 genetics, 329–330, 335–336 LHRH neuron migration polysialic acid, 1016 male aggression-eliciting pheromone, 361–363 olfactory cilia distal parts, 26 olfactory epithelial surface sections, 25 Microencapsulated scratch and sniff odorized strips, 204, 205 Microsmia (see Hyposmia) Microvilli, xxv TEM, 34 Microvillar cell, 33 Microvillous cells, 33–34 Midazolam, 562 Middle ages, xvii Middle meatus, 5 Migraines, 950 Milk fresh vs. off, 219 Milkfeeding infants, 834–837 Mitogen-activated protein kinase odorant stimulation, 85 olfactory transduction, 85 Mitosis, 93, 118 Mitral cells, 140, 151, 183–187 Mitral/tufted cell projection piriform cortex, 188 Mixture interactions, 810–812 determination, 807–810, 813–815 Mixture perception complexity, 816–817 MLP, 303 illustrated, 304 Model olfactory system design, 296 Moffett’s position, 472 MOE, 351 Monkey nasal cavity anatomy, 55 Monoaminergic system discrimination learning, 413

1112 Monoclonal antibody immunolabeling, 145 Monozygotic (MZ) twins, 329–330 Morphine, 914 MOSFET’s (metal oxide semiconductor field effect transistors) sensors, 299 Motion sickness, 563 Motor neuron disease odor identification, 238 MRI, 593, 594 MPTP, xxxii, 487 MPTP-induced Parkinsonism, xxxii MSI (see Magnetic source imaging), 229 Mucociliary clearance patterns schematic diagram, 8 Mucosa, 2 composition comparative aspects, 55 Mucous membranes, 2 Mucus, 35 Multidimensional scaling (MDS), 212 Multifunctional synapse, 144 Multilayer perceptron (MLP), 303 illustrated, 304 Multiple chemical hypersensitivity (see Multiple chemical intolerance) Multiple chemical intolerance, 525–543 clinical recognition, 535–537 evaluation, 535–537 exposure history, 534 historical background, 526 vs. insensitivity, 528 intolerance, 534–535 laboratory evaluation, 535–537 management, 540–542 medical history, 535 phenomenology, 529–530 physical examination, 535–536 prevalence, 530–533 prevention, 542–543 problem definition, 526–527 prognosis, 542–543 proposed care definitions, 528–529 proposed mechanisms, 537–540 psychological mechanisms, 539 recent developments, 526 self-reported, 532 symptoms, 526, 534–535 trial of avoidance, 537 triggering influences, 526 Multiple chemical sensitivities, 586–587 cognitive component, 240 OERP, 239 Multiple-choice odor identification tests, 212 Multiple memory systems, 425–426

Subject Index Multiple peripheral neural pathways disorders, 942–947 Multiple sclerosis imaging, 606 odor identification, 238 olfactory function, 489 Multiple sweet receptors, 714–715 Multiple system atrophy olfactory function, 488 Muscarinic ACh receptors (mAChRs), 154 Muscone strong-weak isomer pairs, 292 Musk odors chemical classes, 278 MZA, 332 MZ co-twins, 330 MZ twins, 329–330 MZ twins reared apart (MZA), 332 NAChRs, 154 Nafarelin, 563 Naming tests, 212 Naphthol, 59 Nares, 4 Nasal abnormalities pharmaceutical treatment, 443–444 simulated, 442–445 Nasal airflow patterns, 446–449 Nasal airflow perception, 451–452 Nasal airway functional physiology, 439–440 measuring properties, 441–442 Nasal airway models, 446–448 Nasal anatomy, 2–5 olfactory ability, 445–446 Nasal biotransformation enzymes, 56–59 functions, 59–63 Nasal cancer, 63 Nasal cavity, 5–14 aerodynamics, 440–442 anatomy, 52, 442–446 arterial supply, 9–10 autonomic supply, 12 comparative anatomy, 55 cutaway view, 446 development, 115–118 drugs administered via, 553–554 general sensory supply, 11–12 human anatomy, 55 epithelia, 52 innervation, 11–12 lymphatic drainage, 10–11 microcirculation, 11 monkey anatomy, 55

[Nasal cavity] rat anatomy, 55 schematic drawing, 466 therapeutic drugs, 561–563 tumors, 595–596 vasculature, 9–11 venous drainage, 10 volume, 445–446 Nasal cycle olfactory function, 451 ultradian rhythm, 450 Nasal disease cause, 466–467 Nasal endoscopy, 463, 632–633 Nasal flow receptors, 452 Nasal fossa, 2 Nasal irritants suprathreshold responses, 987–988 Nasal irritation, 992 Nasal lining maps, 14 Nasal localization homologous chemical series, 988–991 Nasal metabolism activation, 62 detoxication, 59 mucosal damage, 65 Nasal model, 448 Nasal mucosa mammalian inhaled substance metabolism, 56–57 metabolic activation, 62–63 xenobiotic toxicity, 62–63 Nasal musculature, 4–5, 5 Nasal obstruction surgery, 473 Nasal olfactory function, 440 Nasal/olfactory placode, 116 Nasal passages anatomy, 2 epithelia, 12–14 human anatomy, 1–14 Nasal patency changes, 451–452 Nasal pit, 116 Nasal polyps, 473 Nasal prominence, 116 Nasal pungency homologous chemical series, 988–991 Nasal respiratory function, 439 Nasal secretion composition, 54 control, 54

Subject Index Nasal septum, 2, 3 schematic diagram, 3 Nasal sill, 4 Nasal strips, 444–445 Nasal surgery, 443, 467–468 Nasal toxicity chemically induced, 65 Nasal tumors, 63 Nasal valve, 4, 441 dilation, 444–445 external view, 4 Nasal vestibule, 4 external view, 4 Nasal xenobiotic enzymes, 60 Nasal xenobiotic metabolism inhibition, 64–65 Nausea, 910 N-CAM, 123 immunoreactivity, 125 Near-threshold interactions, 812, 818–819 Neonates chemoreceptors, 315 human, 311–314 learning, 355–357 mice screening tests, 396–397 skeletal and autonomic reactions, 826–829 Neophilia, 908 Neoplasma, 465 Nerve growth factor (NGF) defined, 100 Nerve of Pinkus, 1001 Nervus terminalis, 12, 388 anatomy, 1004–1005 development, 1008–1010 function, 1002–1003, 1005 histochemical and immunocytochemical procedures, 1004 historical background, 1001–1002 humans, 1005–1007 development, 1006–1007 immunocytochemical studies, 1014–1016 immunocytochemical techniques, 1003–1004 LHRH neuron migration, 1010–1012 mammals, 1004–1005 mice embryology, 1007–1014 structure and function, 1001–1021 transport route, 1018–1021 Neural network, 305 Neural network architecture illustrated, 304 Neurocalcin olfactory transduction, 83

1113 Neurodegenerative diseases assessment, 238–239 odor perception, 479–495 prevention, 563 treatment, 563 Neurofibrillary tangles, 505–509 Neurogenesis, xxvii aging, 98–99 ensheathing cell regulation, 104–105 regulation by immature neuron density, 97 regulation by thyroxine, 97 regulation by usage, 97 Neuroglia, 129 Neuronal plasticity structural aspects, 35–36 Neuronal-specific enolase (NSE), 124 Neuron precursors proliferation, 127–129 Neurons strong-weak isomer pairs, 292 Neuropeptides, 664 recognition memory, 416 Neuropeptide Y (NPY), 76, 154 Neuropil, 129, 141 Neurotransmitters recognition memory, 416 Neurotrophin molecules peripheral taste organ development, 771–773 Neurotrophins, 103, 619 olfactory mucosa, 101–102 Newborn infant (see also Neonates) intake, 828 pain, 828–829 taste, 826–829 skeletal and autonomic reactions, 826–829 NGF defined, 100 NHDB, 147 electrical stimulation, 154 Nickel, 556 Nickelocene, 284 electron-density maps, 284 Nicolene, 59 Nicotine nasal spray, 563 Nicotinic ACh receptors (nAChRs), 154 Nipple localization olfactory cues, 310 Nipple search releasing odour, 370 Nitric oxide, 82 Nitro musk structure, 278 Nitropyrene, 59 Noggin, 116

Noise detecting signals, 752–753 Nonadienal structure, 279 Nongustatory papillae, 654 Nonhuman animal studies kin recognition, 336–338 Nonhuman mammals odor preference determination, 403–407 psychophysical evaluation, 385–397 methodological issues, 387–389 Nonvibrational interpretations, 283 Norepinephrine, 150, 152–153 piriform cortex, 191 NOS, 82 Nose (see Nasal) NO synthase (NOS), 82 6-n-propylthiouracil (PROP), 788, 798, 856–857 supertasters, 852–853 tasters, 852–853 taster status age, 855–856 ethnicity, 855–856 sex, 855–856 tasting tongue anatomy, 854–855 NPY, 76, 154 NSE, 124 Nuclear medicine, 594–595 Nucleus of the horizontal limb of the diagonal band (NHDB), 147 electrical stimulation, 154 Nucleus of the solitary tract (NTS), 680–683, 734 neurons, 774–775 projections, 773–774 Nutrients chemosensory influences, 886–889 Nutritional status primary chemosensory disorders, 889–890 Obesity chemosensory abnormalities nutritional implications, 892 OBPs, 76–78 Obsessive compulsive disorder, 472 OCAM/mamFasII, 121, 123 Odor, 586–587 airflow sensation, 452 theories, 279–282 Odorant-binding proteins (OBPs), 76–78 Odorant detection long-term response, 84–85

1114 Odorant-induced activation age comparison, 265 amygdala, 263 anterior olfactory nucleus and olfactory tubercle, 262 cerebellum, 267 fMRI revelation, 260 gender comparison, 264 PET, 259 Odorant-induced activity time course, 260 Odorant-induced cAMP, 79 Odorant intensity, 289–292 Odorant mixtures component discrimination, 219 component identification, 219–220 perception, 217–223 perception mechanisms, 220–223 Odorant polarity, 290 Odorant receptors (ORs), 78–79 Odorants choice, 256 descriptors, 278 fast vs. slow, 221–222 general requirements, 276–277 intensity-related effects, 244 intravenous administration, 204 nasal metabolism function, 60–61 perceptual interaction between, 218 piriform cortex, 188 presenting, 205 weak and strong, 290 Odorant signal transduction model, 77 Odorant transduction general mechanism, 76 Odor categories, 278–279 Odor classification schemes, xviii Odor descriptors, xviii, 277–278 Odor discrimination posttraumatic disorders, 631 Odor encoding temporal properties, 255 Odor evaluation room, 206 Odor identity spatial component, 254–255 Odor intensity structural correlates, 291 Odorless molecules, 289–290 Odor memory test, 213–231 Odor naming tests, 212 Odor-odor associations, 418–419 Odor of sanctity, xvii Odor perception neurodegenerative diseases, 479–495 Odor-place associations, 418

Subject Index Odor preference determination nonhuman mammals, 403–407 approach paradigm, 404–405 bar-press stimulus presentation paradigm, 405–406 forced approach-avoidance paradigm, 405 odor trail paradigm, 406 sniff-rate analysis paradigm, 406 Odor profiles, 277–278 Odor signatures, 321 Odor-tactile associations, 418 Odor-toxicosis odor-taste, 420–421 Odor trail paradigm, 406 Odotopes, 280 functional groups, 282 vs. vibrations, 282–283 Odotope theory, 289 OERP (see Olfactory event-related potential) Oil of juniper neutralizing camphor, 218 Olfaction airflow dynamics, 452–453 applying imaging, 254–255 biopsy, xxxi genetically modified mice screening tests, 396–397 kin recognition, 336–339 ontogeny, 825 psychophysical evaluation, 389–396 routes, 824 social behavior development neonatal mammals, 309–322 Olfactometry, 204, 205, 231, 387, 390, 391 Olfactory biopsy, xxxi bulbs, xxvi, xxvii, xxxii, cilia, xxv ensheathing cells, xxvii epithelium, xxvii, xxxi receptor cells, xxvii receptors, xxvii threshold, xxii Olfactory activation temporal lobe and insula localization, 242 Olfactory axons, 28, 29, 139–140, 149–151 glomeruli, 130 growth regulation, 125–126 Olfactory biopsies ultrastructural correlates, 37–39 Olfactory bulb, xxvi, xxvii, xxxii, 258–259, 411–412, 425–426

[Olfactory bulb] anatomy, 126–127, 139–145 appetitive conditioning, 410 cell death, 143 cell generation, 143 centrifugal fibers, 152–154 centrifugal innervation, 146–149 convergence ratios, 143 development, 126–131 external plexiform layer, 56, 141 glomerular layer, 129, 140 granule cell layer, 142–143 graphic reconstruction, 1002 human embryonic development, 128 plastic section, 30 internal plexiform layer, 142 intrinsic neurons, 151–152 laminar organization, 56, 139–140 mitral cell layer, 130, 142 modulators, 149–151 neurochemistry, 149–154, 617–618 neurotransmitters, 149–151 odor coding, 145–146 odor-taste odor-toxicosis, 421–422 olfactory nerve layer, 140 olfactory neuron maturation, 124–125 output neurons, 184–186 plasticity, 617–618, 619–620 stem cells, 620–622 principal cells, 151 projections, 167–169 sensory neuron differentiation, 97 synaptic connection, 143–145 synaptic contact, 96–97 Olfactory bulbar glomerulus TEM, 30 Olfactory central nervous system plasticity, 617–619 Olfactory cilia lengths, 23 mice distal parts, 26 number, 23 rat cross section, 21 distal segments, 22 Olfactory cortex anatomy, 165–176 bulbectomy, 618–619 deprivation, 618 plasticity, 618–619 recognition memory, 414 Olfactory cybernetics concepts, 295–297 Olfactory cyclic nucleotide-gated channel (OCNC), 80

Subject Index Olfactory dendritic endings, 23 human scanning electron micrograph, 18 Olfactory discrimination behavior, 393 Olfactory disorders, 461–473 central causes, 600–606 classification, 461–462 clinical evaluation, 462–463 medical history, 462–463 treatment, 472–473 Olfactory disturbance dysfunction causes, 463–472 genetic component, 37–38 nomenclature, 579 peripheral causes, 595–600 Olfactory ensheathing cells characteristics, 28 Olfactory ensheathing glia, 105 regenerative properties, 105 Olfactory epithelium, xxvii, xxxi, 1, 21, 258–259, 619 cellular anatomy, 32, 75–76 differentiation, 118–123 growth factors, 100, 102 human SEM, 19 mammalian early observations, 17–21 morphology, 17–40 mucus layer, 21 neurogenesis, 93–107 pathology, 463 pathway, 550, 561 plasticity, 616 rat SEM, 19 surface mice, 25 rat, 19 TEM, 18 Olfactory ERMFs, 242 Olfactory event-related potential (OERP), 229, 230–240 age, 236 amplitudes insterstimulus interval, 235–236 odor quality, 235 presentation procedures, 234–236 relation to airflow rate, 234–235 stimulus characteristics, 234–236 stimulus duration, 234 stimulus intensity, 234 applications, 237–238 first positive peak, 232 gender, 236 measures reliability, 236

1115 [Olfactory event-related potential] presented at different flow rates, 235 psychophysical and neuropsychological measures, 236–237 recording, 232–233 stimulation and recording parameters, 233 stimulation requirements, 230–232 subject variables, 236 testing conditions, 233–234 ultradian rhythm, 236 Olfactory fossa, 5 Olfactory function electrophysiological measurement, 229–244 human psychophysical measurement, 203–223 Olfactory function testing, 633–634 Olfactory glomeruli, 140 Olfactory impairment questionnaire, 635 Olfactory magnetic source imaging chemosensation applications, 241–243 description, 241 Olfactory maker protein (OMP), 27, 75, 123, 150–151 Olfactory memory, 409–426 human, 424–425 Olfactory mucosa, 1, 21–39, 51–66, 579 anatomy, 21–35 cell dynamics growth factor regulation, 104 cellular anatomy, 53 composition, 52–56 damage rodents, 552–553 enzymatic localization, 56–63 growth factors and receptors, 101 histological section, 118 metabolism, 63–65 P450s, 57–58 secretions, 54 transient aspects, 35–39 vertebrate composition, 18 Olfactory nerve pathway, 549 Olfactory neuroblastoma, 596 Olfactory neuroepithelium, 1, 14 mapping, 13 Olfactory neurogenesis, 93–94 molecular regulation, 100–106 in vivo regulation, 95–100 Olfactory neurons, 17, 595 cellular differentiation, 119–120

[Olfactory neurons] characteristics, 36 human life span, 36 maturation extrinsic influence, 124–126 Olfactory pathways anatomy, 139–154, 165–176 development, 118–131 plasticity growth factors, 619–620 thyroid hormones, 620 Olfactory perception genetics, 329–340 Olfactory phosphodiesterases, 82–83 Olfactory placode, 115 cells, 120 development, 116 Olfactory plasticity origin, 622 Olfactory preference questionnaire, 332 Olfactory receptor neurons, 1, 18, 22–29, 52–53, 62, 75–76, 78, 183, 396, 595 dendrite, 28 freeze-fracture platinum carbon replica TEM, 19 primary cultures, 81 regenerative capacity, 61 Olfactory receptors, xxv, xxvii response pattern, 287 Olfactory recognition studies, 318–319 Olfactory reference syndrome, 466, 472 Olfactory respiratory cilia mammalian, 18 diagram, 23 Olfactory responses functional dissociations, 985–987 Olfactory sensitivity, 206–209, 218, 331, 332 Olfactory sensory neurons disposable neuron model, 94, 95 life span, 94 replaceable neuron model, 96 Olfactory signal modulation, 26 onset, 26 termination, 26 transduction, 76 Olfactory signatures, 318–319, 336 maternal following birth, 320 underlying basis, 318–319 Olfactory slice, 256 Olfactory supporting cell microvilli, 30 Olfactory systems, 577 anatomy, 595

1116 [Olfactory systems] cybernetics, 295–306 future directions, 306 developmental anatomy, 115–131 human functional mapping, 254 physiology, 595 potential confounding, 864 trigeminal system psychophysical interaction, 994 Olfactory tests discrimination, 211 identification, 212 memory, 213 recognition, 212 signal detection threshold 206–208 supro-threshold, 209–214 Olfactory toxicants bloodstream, 559 Olfactory transduction molecular neurobiology, 75–85 Olfactory tubercle, 167, 172, 261 Olfactory xenobiotic metabolism modification, 63–65 OMP, 27, 75, 123, 150–151 OMP-positive cells OSN, 97 One-bottle intake tests, 861–863 Ontogenetic mechanisms neonates, 314–317 olfactory recognition, 319–320 Operant procedures absolute threshold, 389–390 Ophthalmic division trigeminal nerve, 11–12 Optical sensors, 299 Oral cavity disorders, 939–941 excessive dryness, 950 Oral mucosa disorders, 939–941 Orbitofrontal cortex, 192–193, 263–266, 409, 411, 412, 680 recognition memory, 414 taste, 693–695 Orbitofrontal cortex system, 425 odor-taste odor-toxicosis, 421 Orbitofrontal gyri, 254 Organ of Masera (see Septal organ) Organic materials electronic nose, 300 OR gene-labeled olfactory axons, 146 Orotopic representation, 746–747 ORs, 78–79 Oscilloscope, xxiv Osirus, xvi Osmetech plc, 301

Subject Index Osteomas, 596 Ostiomeatal complex, 7 Oxymetazoline, 443–444 Oxytocin, 154 Pain early childhood, 831 first year, 830 late childhood/adolescence, 832–833 newborn infant, 828–829 Palatability early childhood, 831 first year, 830 late childhood/adolescence, 832 newborn infant, 828–829 Palatine bone, 9 Panic disorder, 539 Papillae sensory ganglia, 764–773 Parabrachial nuclei in the pons (PBN), 775 Parabrachial nucleus, 683–684 Paracellular transport, 550, 561 Parallel evolution, 35 Paranasal sinuses, 2, 5–9 adenocarcinoma, 598 coronal CT, 6 tumors, 595–596 Paranasal sinusitis, 595 Paraseptal cartilages (Jacobson’s cartilage), 3 Parasympathetic pathway, 12 Parkinsonism-dementia complex of Guam olfactory function, 487–488 Parkinson’s disease, xxxii, 39 imaging, 601–602 neuropathology, 513–515 odor identification, 238 olfactory bulb pathology, 514–515 olfactory cortex pathology, 515 olfactory epithelial biopsy, 39 olfactory epithelium pathology, 514 olfactory function, 484–486 Parosmia (see also Dysosmia), xxiii, 630 Particle deposition and uptake, 448–449 Particulate guanylyl cyclase, 81 Patient habits changing, 646–647 Pattern code advantages, 752–753 PCA, 301 replicate samples, 303 PCD, 329 PDEs, 82–83

PDGF, 100 olfactory epithelium, 102–103 olfactory mucosa, 101–102 PEMEC threshold detection, 215 Pemphigus vulgaris, 941 Penicillamine, 950–951 Peptides, 563 Perceived intensity assessment, 389, 867–874 Perceived quality assessment, 391–396, 874–875 Perception human taste mixtures, 805–820 vs. visceral function, 731–732 Perceptron, 303 Perceptual constancy, 395–396 Perfume, xv–xvii Perfumer’s strip, 205 Periamygdaloid cortex, 173, 261 Perichondrial aponeurosis, 4 Periglomerular cells, 128, 140, 151–152, 183, 184 Periodontal structures disorders, 941 Peripheral nerves disorders, 943 functional development, 775 Peripheral taste fibers multiple sensitivity, 738–741 Peripheral taste organ development neurotrophin molecules, 771–773 Peripheral taste system, 651–670 human development, 664–666 Persia, xvi PET, 253, 594, 796 description, 252 Phantosmia, 461 Phencyclidine, 914 Phenobarbitol, 63 Phenol ST (PST), 58 Phenylacetic acid, 277–278 Phenyl ethyl methyl ethyl carbinol (PEMEC) threshold detection, 215 Pheromones, xxx definition, 346–352 dictionary, 346–347 scientists, 348–352 textbook, 347–348 history, 345–346 mammalian, 345–372 case studies, 359–369 early concerns, 352–353 Phorbol esters, 80

Subject Index Phosphodiesterases (PDEs), 82–83 Phospholipase C (PLC), 81 Physical attraction/orientation olfactory cues, 310 Physical examination (see Medical evaluation) Piezoelectric quartz crystal oscillator, 298 Pin cells mammalian taste buds, 661 Piperonal, 287 Piriform cortex, 147, 172–173, 187–192, 254, 259–261, 409 anterior vs. posterior regions, 190 modulation and nonolfactory responses, 191–192 Plain radiographs, 594 Plastic squeeze bottles, 204 Platelet-derived growth factor (PDGF), 100 olfactory epithelium, 102–103 olfactory mucosa, 101–102 P-menthane derivatives strong-weak isomer pairs, 292 Pocket smell test, 462 Poiseville equation, polyposis, 473 Poliomyelitis, xxvi Pollution control, 219 Polysialic acid, 1016 Positive predictive value taste tests, 792–793 Positron emission tomography (PET), xxiv, xxviii, xxix, 253, 594, 796 description, 252 Posterior choanae, 2, 117 Postsellar type sphenoid sinus, 9 Posttraumatic ageusia history, 961 Posttraumatic anosmia, 605, 635–636 Posttraumatic anosmia-ageusia syndrome, 960 Posttraumatic disorders odor discrimination, 631 Posttraumatic epilepsy, 961 Postviral anosmia, 38 Prechordal plate, 116 Preference early childhood, 830–831 first year, 829–830 late childhood/adolescence, 832 Pregnancy, 952 Premature infant taste, 826 Prenatal learning, 354–355 Presellar type sphenoid sinus, 9

1117 Presumptive bulb, 129 Primary olfactory cortex, 254 (see also Secondary olfactory structures) functional definition, 268 projections, 146–147 Primary olfactory regions, 259–261 Primates nucleus of the solitary tract, 680–681 taste processing hunger and satiety, 691–692 thalamic taste area, 684 Primer pheromones, 348 Priming pheromones, 364–369 Principal components analysis (PCA), 301 replicate samples, 303 Procion, 557 Progenitor cells, 143 Prognosis, 963 Progressive supranuclear palsy (PSP), xxxii olfactory function, 488 PROP (see 6-n-propylthiouracil) Protease inhibitors, 951 Protein, 884 taste, 689–690 Protein kinase A (PKA) desensitization, 84 Protein kinase C (PKC), 80 desensitization, 84 Protoglomeruli, 129 Protozoa, xv Provosts of Nancy, xvii Pseudohypoparathyroidism, 471 Pseudo-twins, 332 Psychiatric disorders, 472 Psychogalvanic skin response (PSR), 243 Psychophysical measurements environmental applications, 994–995 Psychophysical tests animals procedures, 386 humans procedures, 204–214 subject variables, 216–217 Psychophysics, xxii, xxxi Psychosocial test reliability, 214–215 unilateral vs. bilateral, 215–216 Puberty-accelerating pheromone male mice, 368 Public health, xix Pungency thresholds, 992 Purkinje cell degeneration (PCD), 329 Putative odorant receptors antibodies, 26–27 Pyridine threshold detection, 215 Pyriform aperture (anterior choanae), 2

Quality coding neuron types, 748–753 theories, 747–748 Quantitative olfactory testing, 203–223, 462 Quartz crystal microbalance illustration, 298 Quartz resonator electronic nose, 298 Rabbit nasal CYP2E1, 64 Radiation-induced xerostomia, 953 Radiography, 962–963 Raphe nuclei, 148 Ras-MAPK (mitogen-activated protein kinase) olfactory transduction, 85 Rat cognitive processes, 358–359 Ratings, 790 Rating scales, 210 Ratio scale, 210 Rat maternal pheromone, 359–361 Rat nasal cavity anatomy, 55 Rat olfactory cilia cross section, 21 distal segments, 22 Rat olfactory epithelial surface, 19 Rat olfactory epithelium SEM, 19 Receptor activation patterns, 287 Receptor expression studies, 287–288 Receptor-mediated bitter transduction, 709–710 Receptor sequences number, 288–289 Recognition memory, 414–418 biochemical substrates, 416–417 lesions, 417 Recognition thresholds, 793–794 Recombinant odorant-binding proteins (OBPs), 78 Recovery, 963 Reentrant loops, 426 Regional chemogustometry, 789 Regional taste deficits, 788–789 Regional taste function, 788–789 Regulators of G-protein-signaling (RGS) proteins, 23 Rejection taste reactivity, 732 Releasing pheromones, 359–364 Releasing responses, 345 Reliability olfactory tests, 214, 215, 236 taste tests, 792

1118 Renaissance, xx–xxi Renal disease chemosensory abnormalities nutritional implications, 893 Reynold’s number, 440 Reptile taste buds, 661 Respiration cerebellar involvement, 267 Respiratory epithelium, 12–13 SEM, 37 Respiratory function, 439 Response bias measure, 209 Retinal, 61 Retinoic acid, 61 RGS proteins, 23, 36 Rhesus monkey vaginal pheromone, 363 Rhinnitis pharmaceutical treatment, 443–444 Rhinomanometry, 443–444 Rhinoplasty, 468 Rhinosinusitis, xxxi, 466, 467 Rhodanese, 56, 59, 60 Right orbitofrontal cortex lesions, 264 Riley-Day syndrome, 951 Rod cells mammalian taste buds, 661 Rodent nucleus of the solitary tract, 681–683 Rodent olfactory neuroepithelium light microscopy, 18 Rodents, 387 (see also Rat) Rodent taste processing hunger and satiety, 690–691 Rodent thalamic taste area, 684–685 Roman Empire, xvi–xvii Rostromedial olfactory cortices, 167, 172 S-100 protein, 28, 32 Saliva buffering capacity, 643 chemical composition, 640–641 dentition, 643 digestion, 642 hyperviscosity, 950 ions, 640 oral flora, 643 proteins, 640–641 taste, 639–647 temperature control, 643 tissue permeability and lubrication, 641–642 wound healing, 642–643 Salivary glands disorders, 941–942 tongue, 658

Subject Index Salivary secretion drug effects, 646 reflex control, 640 Salivary stimulation, 647 Saliva substitutes, 647 Salt taste functional development, 775–777 Salt taste transduction amiloride-insensitive pathways, 720 proposed models, 720–721 Same-sex fraternal twins, 337 San Diego Smell Identification Test, 462 Sarcomas sinonasal cavities, 598 Satiety, 682–683, 683 olfactory, visual, taste sensoryspecific, 695–696 taste processing, 690–692 SAW, 299 devices electronic nose, 298 Scaling methodologies, 853–854 Scandinavian Odor-Identification Test, 462 test-retest reliability, 215 Schizophrenia, xxxii, 214, 951 imaging, 603 neuropathology, 515–518 olfactory bulb, 516–517 olfactory cortex, 517–518 olfactory epithelium, 516–517 olfactory function, 472, 490–494 Schneiderian membranes, 2 Schwann cells, 105 Schwannomas, 597 Scratch and sniff odorants, (see University of Pennsylvania Smell Identification Test) Seasonal affective disorder, 472 Secondary detoxication, 59–60 Secondary olfactory cortex, 254 Secondary olfactory regions, 262–266 Secondary olfactory structures, 165–167 associational connections, 169 chemoarchitecture, 170–171 connectivity, 171–174 feedback projections to olfactory bulb, 169–170 functional aspects, 174–175 neuromodulatory inputs, 170 Second messengers odorant detection, 79–84 Sedatives, 562 Self-organizing maps (SOMs), 302–304 Semilunar ganglion nasal cavity, 11–12

Sense of smell human history of electrophysiological research, 229–230 psychophysics, 203–223 Sensitivity olfactory tests, 206–209 taste tests, 792 Sensor output electronic nose, 300–304 Sensor technology electronic nose, 297–300, 298 Sensory arrays raw data response, 302 Sensory epithelium SEM, 37 Sensory function hunger, 882–883 Sensory ganglia, 760–764 central targets, 773–775 development, 760–762 neurites, 762–764 peripheral receptive field, 769–771 peripheral targets, 764–773 Sensory neuron differentiation, 97 Sensory neuron survival synaptic contact, 96–97 Septal organ, 389 Septoplasty, 468 Septum maps, 14 Seromucous glands nasal respiratory epithelium, 13 Serotonin (5-HT), 150, 152 Sewage odor changes, 219 Sex attractant pheromone, 364 Sexual behavior, xxx odor learning, 422–423 Sex differences, 216, 236 Shape-based theories SOR, 280 Shear injuries, 630–631 Short axon cells, 140, 152 Short-range pheromone, 369 Signal detection tests, 208 Signal detection theory (SDT), 208–209 Signal-modulating molecules, 27 Signal plus noise (SN), 208 Signals olfactory neurogenesis regulation, 100 Sila compounds, 284–285 Silicon, 284 Silver, 576 Similar tasting substances, 807–813 Single-photon emission computed tomography (SPECT), xxiv, 594 description, 252

Subject Index Single staircase (SS), 206–207, 207 Sinonasal cavities sarcomas, 598 Sinonasal infectious disease, 595 Sinonasal tract alterations, 630 Sinus disease cause, 466–467 Sjogren’s syndrome, 941 salivary function, 645–646 Small stimulus volumes, 863 Smell loss, 634 nutritional implications, 881–894 Smell Identification Test (see UPSIT), 213 Smell threshold test, 462 Smelling chemical group, 282 Smoking cessation, 216, 563 Sniff somatosensory or motor components, 256 Sniff bottle, 204, 205 Sniffing, 266–267 Sniffing and nonsniffing paradigms, 256 Sniffin Sticks test test-retest reliability, 215 Sniff-rate analysis paradigm, 406 Social behavior odor learning, 422–424 Social cues, 908 Social recognition, 317 Sodium appetite, 683–684 Sodium chloride, 886 Sodium salt transduction ion channels, 718 mechanisms, 718–721 Soft tissue anatomy, 9–14 Solid foods infants, 837 Solitary chemosensory cells, 659 Soluble guanylyl cyclase, 81 Solvation equations applicability, 992 Solvents, 559, 585–586 Somatostatin, 150, 154 SOMs, 302–304 Sonic hedgehog (sHH), 116 SOR, 275–293 current state, 275–276 theories, 279–282 Sour taste saliva, 644 Spatial processing, 220–221 Specific hungers, xxxi

1119 Specificity taste tests, 792 SPECT, 594 description, 252 Sphenoid bone, 8–9 Sphenoid sinus, 8–9 types, 9 Sphenopalatine artery nasal cavity, 10 Spices, xv–xix Spina bifida and symmelia, 330 Spoiled T1-weighted gradient echo (FLASH) scan, 257 Spot-35, 34 Spot-35 protein, 510–511 Squamous cell carcinoma, 598, 939–940 Squeeze bottle, 205 Stapedectomy, 945 Staphylococcus, xviii Statistical analysis functional neuroimaging experiments, 257–258 Stem cells (see Basal cells), xxviii Steroid musk structure, 278 Steroids metabolic modulation, 61 Stimulus control and presentation, 204, 863–864, 867 generalization, 393–394, 394 generation and control, 255, 256, 387–388 identification, 794, 795 magnitude, 211 quality, 794 Strange male pregnancy blocking pheromone, 365–368 Strene oxide, 59 Stress, 359 Strongest Component Model, 218 Structure-odor relationship (SOR), 275–293 current state, 275–276 theories, 279–282 Subepithelial capillaries, 11 Subepithelial structure, 53–54 Substance P, 150, 154 Substitute parents fostering newborn young, 320 Sucking first year, 829 newborn infant, 828 olfactory cues, 310 Sulcus, 6 Sulcus terminalis, 654 Sulfotransferases (ST), 58

Sulfuraceous character molecular vibration, 283 Sumatriptan, 562 Summated response comparison, 809 Superficial muscular aponeurotic system (SMAS), 4 Superoxide dismutase, 510 Supporting cells, 29–33, 52 cellular differentiation, 121–122 ion and water regulation, 30 ultrastructural observations, 30 Suprathreshold intensity, 873–874 Suprathreshold interactions, 812–813, 819–820 Suprathreshold scaling procedures, 209–211 Suprathreshold stimuli assessment, 390–391 Surface acoustic wave (SAW), 299 devices electronic nose, 298 Sustentacular cells, 52, 76, 118 Sweet compounds diversity, 712–713 Sweet modifiers diversity, 712–713 Sweet receptors physical characterization, 713 SAR-inferred models, 713–714 search, 717 Sweet taste innate preferences, 906–907 loci genetic mapping, 717 PROP tasters, 857 transduction mechanisms, 712–718 biochemical and molecular biological identification, 715–717 proposed models, 717–718 Switching device schematic diagram, 231 Symptoms implications, 935–936 Synthetic processing vs. analytic processing, 751–752 Synuclein, 509–510 Systemic lupus erythematosus, 941 Targeted odors, 27 Taste, 635 aging, 790 astringency, 698 aversive and illness response, 917–918 chemotopic correlates, 746–747 conditioned reactions life span, 907–908 constant stimulation, 746

1120 [Taste] fat, 697–698 human imaging studies, 698–699 illness response, 913–915 ingestive response, 913 insular-opercular taste cortex, 685 intravascular, xx multimodal representations, 692–698 neural representation, 699 nutritional implications, 881–894 ontogeny, 824–825, 825–832 orbitofrontal cortex, 693–695 processing information, 683 qualities, xx, xxi quality coding, 685 quality processing, 747–753 saliva, 639–647 sensitivities random distribution, 737–738 texture, 696–697 visceromotor integration, 736–737 Taste bud cells molecular markers, 663–664 Taste buds, xx, xxi, xxxi, 658 cycle and life span, 769 distribution, 654–658 formation, 767–768 functional development, 775 induction, 766–767 innervation, 666–667, 784 intravascular, xx mammalian, 661 numbers, 767–768 qualities, xx, xxi receptors, xxv reptile, 661 sensory ganglia, 760–764, 764–773 size, 767–768 vertebrate cell types, 659–664 Taste cells ENaC-like channels, 718–720 Taste complainers, 791 Taste compounds discrimination, 874–875 generalization, 875–876 Taste confusion matrix (TCM), 795 Taste detection thresholds, 786–787, 848–849 Taste dysfunction causes, 938–952 Taste function behavioral assessment animals, 863–864 biological correlates, 796–798 psychophysical evaluation nonhuman mammals, 861–876

Subject Index Taste-guided behavior, 731–737 ingestive decisions, 731–734 Taste hypophagia, 913 Taste inputs neural discrimination, 749–750 Taste intensity rating scales, 787–788 Taste perception human genetics, 847–858 Taste processing, 680–690 hunger, 690–692 satiety, 690–692 temporal and spatial aspects, 745–747 Taste quality, 816 Taste-quality frequency profiles, 795 Taste-quality processing assessment, 790 Taste receptor cells, xxv gustatory sensitivities, 737–738 Taste responses time course, 745–746 Taste signal detection and discrimination animals assessment, 866–867 Taste stimulus discrimination, 793–795 Taste system development, 759–778 Taste tests appropriateness, 790–791 clinical setting suitability, 790–793 future, 793–798 interpretation, 792 management, 791 Taste transduction future prospects, 724 Taurine, 149–150, 150, 558 T&T olfactometer, 462 TBI, 960 history, 961 TCM, 795 Temperature, 744–745 Template matching, 296 Temporal lobe epilepsy OERP, 239 Temporal processing, 221–222 Terbinafine, 951 Terminating molecules, 27 Terrestrial mammals olfactory phenotypes, 317 Tertiary olfactory structures projections, 170 Testosterone, 367 Tetrachlorodibenzo-p-dioxin, 63 Tetralin musk structure, 278

TFGB family olfactory mucosa, 101–103 Thalamic taste area, 684–685 Thalamus, 263 3 [H]-tymidine, 93 Thiamine propyldisulfide (Alinamin), 204 Thiocyanate, 59 Three-drop technique, 784–785 Threshold detection, xxxi early childhood, 831 first year, 830 late childhood/adolescence, 831 preference, xxxi Thresholds, xxii, xxxi, 790 Threshold testing classical conditioning, 392 operant conditioning, 392 Thyroid hormones, 97 olfactory pathways, 620 Timberol structure, 279 Tobacco smoking, 216–217, 473 Toluene, 586 Tonalid structure, 278 Tongue bovine, 652 disorders, 941 formation, 764–765 human, 653–654, 665 innervation, 666–667 injury, 960 salivary glands, 658 sensory ganglia, 764–773 Total taste intensity, 817 Touch, 744–745 Toxic agents rodent olfactory mucosa, 582–583 Toxicant-induced loss of tolerance, 530 phenomenology, 531 Toxic insult, 577–579 Toxic metals, 579–584 Transcellular transport, 549 Transcytosis, 549 Transduction mechanism schematic, 281 Trans-2-hexenal, 277 structure, 278 Transmitter glutamate, 149 Transneuronal transport, 560–561 Transport consequences, 563–564 mechanisms, 559–561 Traseolide structure, 278

Subject Index Traumatic brain injury (TBI), 960 history, 961 Treatment, 952–953, 963 Trench mouth, 941 Triad threshold procedure, 794 Triangle test, 203 Trifluoperazine, 951 3-trifluoromethylpyridine, 62 Trigeminal chemosensation, 981–995 Trigeminal chemosensory responses human quantitative structure-activity relationships, 991–992 Trigeminal nerve (CN V), xxii, 669–670, 947 nasal cavity, 11–12 Trigeminal receptor systems potential confounding, 864 Trigeminal responses functional dissociations, 985–987 Trigeminal stimulants pure odorants, 984–985 Trisomy 21, 330 Trypan blue, 557 Tufted cells, 128, 140, 183–187 Tumor necrosis factor (TNF) cell death, 98 Tumors, 63, 468–469 Tunica mucosa nasi, 2 Tuning breadth, 747–748 Turbinates, 2, 5, 440, 442–453 Turbulent flow, 440–441 Turner’s syndrome, 330, 470, 952 Twin-family design, 332 Twin research olfactory sensitivity, 331–332 Twins reared apart, 332 types, 329–330 determination, 330 Twin studies kin recognition, 338 Twin study logic and methods, 330–331 Two-alternative forced-choice procedure, 786 Two-bottle intake tests, 861–863 Tympanoplasty, 945 Type I cells mammalian taste buds, 661–662 Type II cells mammalian taste buds, 661–662 Type III adenylyl cyclase (AC3), 80

1121 Type III cells mammalian taste buds, 662 Type IV cells mammalian taste buds, 662–663 Type V cells mammalian taste buds, 663 Tyrosine hydroxylase (TH), 151 Ubiquitin, 31 UDP glucuronosyltransferases (UDPGTs), 58 UDP glycosyltransferases (UGTs), 58 UDPGTs, 58 UGTs, 58 Ultradian rhythm nasal cycle, 450 Unassigned candidate transduction elements, 723–724 Unconditioned response, 911 Unconditioned stimulus, 911 Undecanal, 290 University of Pennsylvania Smell Identification Test (UPSIT), 204, 212–213, 216, 331, 332, 462, 467, 480–495, 584, 585, 629, 633 malingering, 216 UPL2 model, 218 Upper respiratory infection, 463–466 UPSIT (see University of Pennsylvania Smell Identification Test) Uptake, 559–560 UST-SAs, 332 Vaccines, 563 Vagus nerve (CN X), 669, 947 Validity taste tests, 792–793 Vallate papillae, 655, 665 Valproic acid, 558 Vasoactive intestinal peptide (VIP), 563 Vasopressin, 154, 563 VEG protein, 77 Vertebrate olfactory mucosa composition, 18 Vertebrate taste buds cell types, 659–664 Vibration theory, 289 SOR, 280 Virtual chemical sensors, 299 Viruses, 2, 38, 464–465, 556–557 Visinin-like protein (VILIP) olfactory transduction, 83

Visual event-related potentials, 229 Vitamin B12, 563 VNO (see Vomeronasal organ) Vomeronasal epithelium, 30 Vomeronasal nerve development, 1008–1010 Vomeronasal organ (VNO), 3, 9, 17, 34, 351, 388, 967–976 chemosensory role, 969–970 microvilli, 27 signaling, 19 signal transduction, 27 species distribution, 969 structure and function, 968–969 Vomeronasal receptor cells, 27 Vomeronasal receptor genes, 972 Vomeronasal receptor neurons (VRNs) transduction mechanisms, 971 Vomeronasal stimuli, 970–971 Vomeronasal system information coding, 972–973 neural projections, 973–975 Vomiting, 910 Von Ebner salivary glands, 642 proteins, 644–645 Warlocks, xvii Water flow models, 446–447 Whole-mouth taste intensity, 786–788 Whole-mouth taste quality, 788 Wine, xvi cork taint, 219 Witches, xvii Withdrawal symptoms, 529 Wright’s confusion matrix, 213 Xenobiotic metabolic activity, 40, 56 Xenobiotic-metabolizing capacity, 53 Xenobiotic-metabolizing P450, 57–58 Xerostomia, 941 Yes/no identification tests, 212 Zidovudine, 562 Zinc, 150–151 Zinc binding odorant intensity, 291–292 Zinc deficiency, 950 Zinc sulfate, xxvi, 96 Zinc supplementation, 579

About the Editor

RICHARD L. DOTY is Director of the Smell and Taste Center and Professor in the Department of Otorhinolaryngology: Head and Neck Surgery, University of Pennsylvania Medical Center, Philadelphia. The author or coauthor of more than 300 professional publications, he serves as an editorial consultant to over 50 scientific journals. He is the recipient of many awards, including the James A. Shannon Award (1996) from the National Institutes of Health, the Scientific Sense of Smell Award (2000) from the Olfactory Research Fund, and the Outstanding Scientists of the 20th Century Award (2000) from the International Biographical Centre, Cambridge, England. Dr. Doty received the B.S. degree (1966) in psychology from Colorado State University, Fort Collins, the M.A. degree (1968) from California State University, San Jose, in conjunction with the National Aeronautics and Space Administration, Moffett Field, and the Ph.D. degree (1971) from Michigan State University, East Lansing. He received post-doctoral fellowship training at the University of California, Berkeley (1971–1973), and the University of Pennsylvania, Philadelphia (1973–1975).