Progress in Brain Research Volume 93 The Human Hypothalamus in Health and Disease: Proceedings of the 17th International Summer School of Brain Research, Held at the Auditorium of the University of Amsterdam

PROGRESS IN BRAIN RESEARCH VOLUME 93 THE HUMAN HYPOTHALAMUS IN HEALTH AND DISEASE

Recent volumes in PROGRESS IN BRAIN RESEARCH Volume 71: Neural Regeneration, by F.J. Seil, E. Herbert and B.M. Carlson (Eds.) - 1987. Volume 72: Neuropeptides and Brain Function, by E.R. de Kloet, V.M. Wiegant and D. de Wied (Eds.) - 1987. Volume 73: Biochemical Basis of Functional Neuroteratology, by G. J. Boer, M.G.P. Feenstra, M. Mirmiran, D.F. Swaab and F. van Haaren (Eds.) - 1988. Volume 74: Transduction and Cellular Mechanisms in Sensory Receptors, by W. Hamann and A. IggO (Eds.) - 1988. Volume 75: Vision within Extrageniculo-striate Systems, by T.B. Hicks and G. Benedek (Eds.) - 1988. Volume 76: Vestibulospinal Control of Posture and Locomotion, by 0. Pompeiano and J.H.J. Allum (Eds.) - 1988. Volume 77: Pain Modulation, by H.L. Fields and J.-M. Besson (Eds.) - 1988. Volume 78: Transplantation into the Mammalian CNS, by D.M. Gash and J.R. Sladek, Jr. (Eds.) - 1988. Volume 79: Nicotinic Receptors in the CNS, by A. Nordberg, K. Fuxe, B. Holmstedt and A. Sundwall (Eds.) - 1989. Volume 80: Afferent Control of Posture and Locomotion, by J.H.J. Allum and M. Hulliger (Eds.) - 1989. Volume 81: The Central Neural Organization of Cardiovascular Control, by J. Ciriello, M.M. Caverson and C. Polosa (Eds.) - 1989. Volume 82: Neural Transplantation: From Molecular Basis to Clinical Applications, by S. Dunnett and S.-J. Richards (Eds.) - 1990. Volume 83: Understanding the Brain through the Hippocampus, by J. Storm-Mathison, J. Zimmer and O.P. Ottersen (Eds.) - 1990. Volume 84: Cholinergic Neurotransmission: Functional and Clinical Aspects, by S.-M. Aquilonius and P.-G. Gillberg (Eds.) - 1990. Volume 85: The Prefrontal Cortex: Its Structure, Function and Pathology, by H.B.M. Uylings, C.G. van Eden, J.P.C. de Bruin, M.A. Corner and M.G.P. Feenstra (Eds.) 1991. Volume 86: Molecular and Cellular Mechanisms of Neuronal Plasticity in Normal Aging and Alzheimer’s Disease, by P.D. Coleman, G.A. Higgins and C.H. Phelps (Eds.) 1990. Volume 87: Role of the Forebrain in Sensation and Behavior, by G. Holstege (Ed.) - 1991. Volume 88: Neurology of the Locus Coeruleus, by C.D. Barnes and 0. Pompeiano (Eds.) 1991. Volume 89: Protein Kinase C and its Substrates: Role in Neuronal Growth and Plasticity, by W.H. Gispen and A. Routtenberg (Eds.) - 1991. Volume 90: GABA in the Retina and Central Visual System, by R.R. Mize, R.E. Marc and A.M. Sillito (Eds.) - 1992. Volume 91: Circumventricular Organs and Brain Fluid Environment, by A. Ermisch, R. Landgraf an H.-J. Ruhle (Eds.) - 1992. Volume 92: The Peptidergic Neuron, by J. Joosse, R.M. Buijs and F.J.H. Tilders (Eds.) 1992.

PROGRESS IN BRAIN RESEARCH VOLUME 93

THE HUMAN HYPOTHALAMUS IN HEALTH AND DISEASE Proceedings of the 17th International Summer School of Brain Research, held at the Auditorium of the University of Amsterdam (The Netherlands), 26- 30 August, 1991

EDITED BY

D.F. SWAAB, M.A. HOFMAN, M. MIRMIRAN, R. RAVID and F.W. VAN LEEUWEN Netherlands Institute for Brain Research, Meibergdreef 33, I105 AZ Amsterdam (The Netherlands)

AMSTERDAM

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ELSEVIER LONDON - NEW YORK 1992

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TOKYO

0 1992 Elsevier Science Publishers B.V. All rights reserved. No part of this publication may be reproduced, stored in a retrieval system or transmitted in any form or by any means, electronic, mechanical, photocopying, recording or otherwise without the prior written permission of the Publisher, Elsevier Science Publishers B.V., Copyright and Permissions Department, P.O. Box 521, 1000 AM Amsterdam, The Netherlands. No responsibility is assumed by the Publisher for any injury and/or damage to persons or property as a matter of products liability, negligence or otherwise, or from any use or operation of any methods, products, instructions or ideas contained in the material herein. Because of the rapid advances in the medical sciences, the publisher recommends that independent verification of diagnoses and drug dosages should be made. Special regulations for readers in the U.S.A.: This publication has been registered with the Copyright Clearance Center Inc. (CCC), Salem, Massachusetts. Information can be obtained from the CCC about conditions under which the photocopying of parts of this publication may be made in the U.S.A. All other copyright questions, including photocopying outside of the U.S.A., should be referred to the Publisher. ISBN 0-444-89538-8 (volume) ISBN 0-444-80104-9 (series) Elsevier Science Publishers B.V. P.O. Box 211 1000 AE Amsterdam The Netherlands

Printed on acid-free paper Printed in The Netherlands

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List of Contributors Ansseau, M., Department of Psychiatry, Academic Hospital of Liege, Domaine Universitaire du Sart Tilman, B.35, 4000 Liege 1 , Belgium. Bergen, H., Rockefeller University, York Avenue and 66th Street, New York, NY 10021, U.S.A. Braak, E., Department of Anatomy, Johann Wolfgang Goethe University, Theodor Stern Kai 7, 6000 Frankfurt am Main 70, Germany. Braak, H., Department of Anatomy, Johann Wolfgang Goethe University, Theodor Stern Kai 7, 6000 Frankfurt am Main 70, Germany. Bray, G.A., Pennington Biomedical Research Center, Louisiana State University, 6400 Perkins Road, Baton Rouge, LA 70808-4124, U.S.A. Breder, C.D., Department of Neurology, University of Chicago, 947 East 58th Street, Box 271, Chicago, IL 60637, U S A . Champier, J., Laboratory of Anatomy and Pathology, Faculty of Medicine Alexis Carrel, Rue Guillaume Paradin, 69372 Lyon CCdex 08, France. Chan-Palay, V.L., Neurological Clinic, University Hospital, Frauenklinikstrasse 36, Zurich 8091, Switzerland. Charlton, H.M., Department of Human Anatomy, University of Oxford, South Parks Road, Oxford OX1 3QX, U.K. Charnay, Y., Laboratory of Neuropathology, Be1 Air Clinic, Geneva, Switzerland. Chigr, F., Laboratory of Anatomy and Pathology, Faculty of Medicine Alexis Carrel, Rue Guillaume Paradin, 69372 Lyon CCdex 08, France. De Wied, D., Rudolf Magnus Institute, Vondellaan 6, 3521 GD Utrecht, The Netherlands. Epelbaum, J., INSERM U 159, Paul Broca Center, 75014 Paris, France. Gooren, L.J.G., Department of Endocrinology, Free University, P.O. Box 7057, 1007 MB Amsterdam, The Netherlands. Goudsmit, E., Netherlands Institute for Brain Research, Meibergdreef 33, 1105 AZ Amsterdam, The Netherlands. Heuser, I., Max-Planck Institute of Psychiatry, Clinical Institute, Kraepelinstrasse 10,8000 Munich 40, Germany. Hofman, M.A., Netherlands Institute for Brain Research, Meibergdreef 33, 1105 AZ Amsterdam, The Netherlands. Holsboer, F., Max-Planck Institute of Psychiatry, Clinical Institute, Kraepelinstrasse 10, 8000 Munich 40, Germany. Jentsch, B., Neurological Clinic, University Hospital, Frauenklinikstrasse 36, Zurich 8091, Switzerland. Jordan, D.;Laboratory of Anatomy and Pathology, Faculty of Medicine Alexis Carrel, Rue Guillaume Paradin, 69372 Lyon CCdex 08, France. Kok, J.H., Department of Neonatology, Academic Medical Center, Meibergdreef 15, 1105 AZ Amsterdam, The Netherlands. Kopp, N., Laboratory of Anatomy and Pathology, Faculty of Medicine Alexis Carrel, Rue Guillaume Paradin, 69372 Lyon CCdex 08, France. Kremer, H.P.H., Department of Neurology, Academic Hospital Leiden, P.O. Box 9600, 2300 RC Leiden, The Netherlands. Legros, J.J Department of Endocrinology, Academic Hospital of Likge, Domaine Universitaire du Sart Tilman, B.35, 4000 Likge 1, Belgium. McEwen, B.S., Laboratory of Neuroendocrinology, The Rockefeller University, 1230 York Avenue, New York, NY 10021-6399, U.S.A.

..

VI Martin, J.B., Department of Neurology, UCSF School of Medicine, Room S-224, 513 Parnassus Avenue, San Francisco, CA 94143-0410, U.S.A. Mengod, G., Department of Neurochemistry, CID-CSIC, Jordi Girona 18- 26, Barcelona 08034, Spain. Mirmiran, M., Netherlands Institute for Brain Research, Meibergdreef 33, 1105 AZ Amsterdam, The Netherlands. Moore, R.Y ., Center for Neuroscience, University of Pittsburgh, CNUP Office, Biomedical Science Tower W 1657, Pittsburg, PA 15261, U.S.A. Najimi, M., Laboratory of Anatomy and Pathology, Faculty of Medicine Alexis Carrel, Rue Guillaume Paradin, 69372 Lyon CCdex 08, France. Neijmeijer-Leloux, A., Netherlands Institute for Brain Research, Meibergdreef 33, 1105 AZ Amsterdam, The Netherlands. Palacios, J.M., Department of Neurochemistry, CID-CSIC, Jordi Girona 18 - 26, Barcelona 08034, Spain. Pfaff, D.W., Rockefeller University, York Avenue and 66th Street, New York, NY 10021, U.S.A. Phillips 111, J.A., Division of Genetics, Department of Pediatrics, Vanderbilt University, Nashville, TN 37232, U.S.A. Probst, A., Institute of Pathology, Department of Neuropathology, University of Basel, CH-4003, Basel, Switzerland. Rance, N.E., Department of Pathology, University of Arizona College of Medicine, 1501 N. Campbell Avenue, Tucson, AZ 85724, U.S.A. Rasmussen, D.D., Department of Reproductive Medicine 0802, University of California at San Diego, 225 Dickinson Street, San Diego, CA 92103-1990, U.S.A. Ravid, R., Netherlands Institute for Brain Research, Meibergdreef 33, 1105 AZ Amsterdam, The Netherlands. Repaske, D.R., Department of Pediatrics, Division of Endocrinology, CB 7220, 509 Burnett Womack, University of North Carolina, Chapel Hill, NC 27599-7220, U.S.A. Reppert, S.M., Children’s Service, Massachusetts General Hospital, 32 Fruit Street Boston, MA 021 14, U.S.A. Riskind, P.N., Neurology Service, Massachusetts General Hospital, Boston, MA 021 14, U.S.A. Ronken, E., Rudolf Magnus Institute, Vondellaan 6, 3521 GD Utrecht, The Netherlands. Saper, C.B., Department of Pharmacological and Physiological Sciences, University of Chicago, 947 East 58th Street, Box 271, Chicago, IL 60637, U.S.A. Scanlon, M.F., Department of Medicine, University of Wales College of Medicine, Heath Park, Cardiff CF4 4XW, U.K. Schwanzel-Fukuda, M., Rockefeller University, York Avenue and 66th Street, New York, NY 10021, U.S.A. Scherbaum, W.A., Medical and Outpatient Clinic, University of Ulm, Robert Kochstrasse 8, D-7900 Ulm, Germany. Spengler, D., Max-Planck Institute of Psychiatry, Clinical Institute, Kraepelinstrasse 10, 8000 Munich 40, Germany. Summar, M.L., Division of Genetics, Department of Pediatrics, Vanderbilt University Medical Center, T2404 Medical Center North, Nashville, TN 37232, U.S.A. Swaab, D.F., Netherlands Institute for Brain Research, Meibergdreef 33, 1105 AZ Amsterdam, The Netherlands. Van de Poll, N.E., Netherlands Institute for Brain Research, Meibergdreef 33, 1105 AZ Amsterdam, The Netherlands. Van Cool, W.A., Department of Neurology, Academic Medical Center, Meibergdreef 15, 1105 AZ Amsterdam, The Netherlands. Van Goozen, S.H.M., Psychonomics, Faculty of Psychology, University of Amsterdam, Roeterstraat 15, 1018 WB Amsterdam, The Netherlands. Van Leeuwen, F.W., Netherlands Institute for Brain Research, Meibergdreef 33, 1105 AZ Amsterdam, The Netherlands. Van Zwieten, E.J., Netherlands Institute for Brain Research, Meibergdreef 33, 1105 AZ Amsterdam, The Netherlands. Weesner, G., Rockefeller University, York Avenue and 66th Street, New York, NY 10021, U.S.A. Wiegant, V.M., Rudolf Magnus Institute, Vondellaan 6, 3521 GD Utrecht, The Netherlands.

VII

Witting, W., Netherlands Institute for Brain Research, Meibergdreef 33, 1105 AZ Amsterdam, The Netherlands. Wood, M.J., Department of Human Anatomy, University of Oxford, South Parks Road, Oxford OX1 3QX,U.K. Zheng, L.M., Rockefeller University, York Avenue and 66th Street, New York, NY 10021, U.S.A.

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Preface The 17th International Summer School of Brain Research held in Amsterdam from August 26 to August 30, 1991, was the first international multidisciplinary conference on the human hypothalamus in health and disease. The summer school was organized by the Netherlands Institute for Brain Research (NIBR), an institute of the Royal Netherlands Academy of Sciences. The history of the NIBR dates back to the beginning of this century. It was in 1901, at a meeting of the International Association of Academies held in Paris, that the anatomist Wilhelm His proposed to place research into the nervous system on an international footing. This proposal resulted in the formation of the International Academies Committee, which set itself the task of “organizing a network of institutions throughout the civilized world, dedicated to the study of the structure and functions of the central organ . . .”. This resulted in the founding of several institutes for brain research, among them the NIBR, which was founded in 1909. The committee pointed out that “the time is not far distant when the study of the millions of brain cells will have to be divided amongst researchers in the way the astronomers have been obliged to divide the millions of stars into various groups”. The purpose of the summer school was, however, not to divide the millions of hypothalamic neurons among the participants, but rather to share and assemble the current knowledge on the human hypothalamus in health and disease. The human hypothalamus is involved in growth, development, birth, rhythmicity, water balance, thermoregulation, feeding, sexual behavior and many other functions. Alterations in hypothalamic structures and functions are thought to be involved in diseases such as anorexia nervosa, bulimia, depression, Cushing’s disease, diabetes insipidus, Prader-Willi syndrome, polycystic ovaries syndrome and the malignant neuroleptic syndrome as well as disturbances in sleep, circadian rhythms and temperature regulation. In addition, the hypothalamus is affected in neurodegenerative diseases and might be responsible for particular symptoms in, e.g., Alzheimer’s, Parkinson’s and Huntington’s diseases, Down’s syndrome and possibly also in multiple sclerosis. Moreover, the development of this brain region is presumed to be affected by hormones given during pregnancy (e.g., diethylstibestrol (DES)), in adrenogenital syndrome and in Turner’s, Klinefelter’s and Kallman’s syndromes. In spite of this impressive list, only recently a start was made with collecting data on the normal development, sexual differentiation and aging of the human hypothalamus, and the first information on how the pathophysiology of each of these conditions is reflected in the morphology of the human hypothalamus is now beginning to appear in the literature. Such data should provide unique information on possible functions of the different hypothalamic nuclei. In the past few years, with the advent of powerful molecular, physiological, neuroanatomical and morphometric techniques, such questions have been attacked

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with new vigor. Different groups from various disciplines in many countries work on the human hypothalamus and provide new and exciting data. This volume intends to integrate this new information in the form of review chapters for students, researchers and clinicians. In addition, we hope this book will initiate further multidisciplinary international collaborative research efforts in this field. We were very grateful to see the spontaneity with which a great number of scientists agreed to come to Amsterdam and contribute to this volume. We would like to acknowledge the generosity of the Royal Netherlands Academy of Sciences, the University of Amsterdam and Neurosciences Amsterdam, under whose auspices this summer school was held, as well as many other generous financial supporters without whose contributions this summer school would not have been possible. We would also like to thank Tini Eikelboom, Olga Pach and Aad Janssen of the NIBR secretariat for their invaluable organizational and editotial help. Dick F. Swaab Michel A. Hofman Majid Mirmiran Rivka Ravid Fred W. Van Leeuwen

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Acknowledgements The 17th International Summer School of Brain Research was organized under the auspices of: The Royal Netherlands Academy of Sciences The University of Amsterdam Neurosciences Amsterdam Financial support was also obtained from: CIBA-Geigy Dr. Saal van Zwanenberg Foundation EURAGE Merck Sharp & Dohme Merrell Dow Research Institute Netherlands Society for the Advancement of Natural Sciences, Medicine and Surgery Organon International B.V. Remmert Adriaan Laanfonds Sandoz Serv ier SmithKline Beecham Stichting Steunfonds VOGG (support foundation of parents of the mentally handicapped) Van den Houten Foundation

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Contents List of Contributors Preface

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Acknowledgements

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V IX XI

Section I - Structure of the Human Hypothalamus 1. Anatomy of the human hypothalamus (chiasmatic and tuberal region) H. Braak and E. Braak (Frankfurt am Main, Germany) . . . .

3

Section I1 - Clinical Manifestations of Hypothalamic Diseases 2. Endocrine functions of the hypothalamus and alterations in neuroendocrine function - focus on thyrotropin and growth hormone M.F. Scanlon (Cardiff, U.K.) ............................

19

3. Neurologic manifestations of hypothalamic disease J.B. Martin and P.N. Riskind (San Francisco, CA, U.S.A. and Boston, MA, U.S.A.) ...................................

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Section I11 - Technical Potentialities and Pitfalls in the Use of Human Material

4. In situ hybridization histochemistry in the human hypothalamus G . Mengod, E. Goudsmit, A. Probst and J.M. Palacios (Barcelona, Spain, Amsterdam, The Netherlands and Basel, Switzerland) ...........................................

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5 . Receptor localization in the human hypothalamus

J.M. Palacios, A. Probst and G. Mengod (Barcelona, Spain and Basel, Switzerland) .....................................

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6. Human hypothalamic and pituitary neuroendocrine function during in vitro perifusion D.D. Rasmussen (San Diego, CA, U.S.A.) . . . . . . . . . . . . . . . .

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XIV

7. Brain banking and the human hypothalamus - factors to match for, pitfalls and potentials R. Ravid, E.J. Van Zwieten and D.F. Swaab (Amsterdam, The Netherlands) ...........................................

Section IV

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Biological Rhythms

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8. The organization of the human circadian timing system R.Y. Moore (Pittsburgh, PA, U.S.A.) ....................

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9. Prenatal development of a hypothalamic biological clock S.M. Reppert (Boston, MA, U.S.A.) .....................

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The fourth C.U. Ariens Kappers lecture

10. The human hypothalamus: comparative morphometry and photoperiodic influences M.A. Hofman and D.F. Swaab (Amsterdam, The Netherlands)

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11. Circadian rhythms and the suprachiasmatic nucleus in perinatal development, aging and Alzheimer’s disease M. Mirmiran, D.F. Swaab, J.H. Kok, M.A. Hofman, W. Witting and W.A. Van Goo1 (Amsterdam, The Netherlands) . . . .

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Section V - Development, Aging and Dementia 12. Ontogeny of peptides in the human hypothalamus in relation to sudden infant death syndrome (SIDS) N. Kopp, M. Najimi, J. Champier, F. Chigr, Y. Charnay, J. Epelbaum and D. Jordan (Lyon and Paris, France and Geneva, Switzerland) ...........................................

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13. LHRH neurons: functions and development M. Schwanzel-Fukuda, L.M. Zheng, H. Bergen, G. Weesner and D.W. Pfaff (New York, NY, U.S.A.) ................

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14. The human hypothalamus in relation to gender and sexual orientation D.F. Swaab, L.J.G. Gooren and M.A. Hofman (Amsterdam, The Netherlands) .......................................

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15. Hormonal influences on morphology and neuropeptide gene expression in the infundibular nucleus of post-menopausal women N.E. Rance (Tucson, AZ, U.S.A.) .......................

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xv 16. The human hypothalamo-neurohypophyseal system in relation to development, aging and Alzheimer’s disease E. Goudsmit, A. Neijmeijer-Leloux and D.F. Swaab (Amsterdam, The Netherlands) ..................................

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17. The hypothalamic lateral tuberal nucleus: normal anatomy and changes in neurological diseases H.P.H. Kremer (Leiden, The Netherlands) . . . . . . . . . . . . . . . .

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18. Galanin tuberomammillary neurons in the hypothalamus in Alzheimer’s and Parkinson’s diseases V.L. Chan-Palay and B. Jentsch (Zurich, Switzerland) . . . . . .

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Section VI - Osmoregulation 19. Animal models for osmoregulatory disturbances F.W. Van Leeuwen (Amsterdam, The Netherlands) .........

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20. Autoimmune hypothalamic diabetes insipidus (“autoimmune hypothalamitis”) W.A. Scherbaum (Ulm, Germany) .......................

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21. The molecular biology of human hereditary central diabetes insipidus D.R. Repaske and J.A. Phillips I11 (Chapel Hill, NC, U.S.A. and Nashville, TN, U.S.A.) .............................

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22. The use of linkage analysis and the Centre d’Etude Polymorphisme Humain (CEPH) panel of DNA in the study of the arginine vasopressin, oxytocin and prodynorphin gene loci M.L. Summar (Nashville, TN, U.S.A.) . . . . . . . . . . . . . . . . . . .

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Section VII - Hypothalamus and Reproduction

23. Animal models for brain and pituitary gonadal disturbances H.M. Charlton and M.J. Wood (Oxford, U.K.)

. . . . . . . . . . . 321

24. Genetic, hypothalamic and endocrine features of clinical and experimental obesity G.A. Bray (Baton Rouge, LA, U.S.A.) . . . . . . . . . . . . . . . . . . .

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25. Hypothalamic involvement in sexuality and hostility: comparative psychological aspects N.E. Van de Poll and S.H.M. Van Goozen (Amsterdam, The Netherlands) ...........................................

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Section VIII - Hypothalamus and Stress

26. Re-examination of the glucocorticoid hypothesis of stress and aging B.S. McEwen (New York, NY, U.S.A.) ...................

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27. The role of corticotropin-releasing hormone in the pathogenesis of Cushing’s disease, anorexia nervosa, alcoholism, affective disorders and dementia F. Holsboer, D. Spengler and I. Heuser (Munich, Germany)

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28. Endogenous pyrogens in the CNS: role in the febrile response C.B. Saper and C.D. Breder (Chicago, IL, U.S.A.)

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Section IX - Psychiatric Diseases 29. Endorphins and schizophrenia V.M. Wiegant, E. Ronken and D. De Wied (Utrecht, The 433 Net herlands) ........................................... 30. Neurohypophyseal peptides and psychopathology J.J. Legros and M. Ansseau (Liege, Belgium)

Subject Index

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455

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SECTION I

Structure of the Human Hypothalamus

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D.F. Swaab, M.A. Hofman, M. Mirmiran, R. Ravid and F.W. van Leeuwen (Eds.) Progress in Brain Research, Vo1. 93

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0 1992 Elsevier Science Publishers B.V. All rights reserved.

CHAPTER 1

Anatomy of the human hypothalamus (chiasmatic and tuberal region) Heiko Braak and Eva Braak Department of Anatomy, J. W. Goethe University, 0-6000 Frankfurt/M 70, Germany

Introduction

The hypothalamus has pivotal significance in the regulation of both the endocrine and the autonomic nervous system. It, furthermore, influences consciousness, sleep and behavior. Increasing age and a variety of diseases may impair many of these functions and may have deleterious effects on the morphology of hypothalamic structures (Daniel and Prichard 1975; Treip, 1984; Saper, 1990). The present architectonic analysis is largely based on examinations of relatively thick (100 - 400 pm) Nissl preparations counterstained for intraneuronal lipofuscin deposits. In such preparations, individual neurons recede into the background, but the large number of cells superimposing each other facilitates recognition of the borders and internal organization of individual nuclei. Moreover, pigment-Nissl preparations can be used for differentiating nerve cell types in the brain of the human adult. This has been shown by means of a Golgi deimpregnation technique which allows perception of the distinguishing features of lipofuscin deposits through transparent cell bodies and processes of impregnated neurons (Braak, 1983). Lipofuscin granules are particularly stable and remain more or less unchanged by delayed fixation. Using the pigment deposits as a natural marker for classification of neuronal types, therefore, permits - as a prerequisite for studies of disease-related changes - the use of human autopsy material (Braak, 1980, 1984; Braak and Braak, 1987).

Antero-posteriorly the hypothalamus sensu lato extends from the pallidum to the zona incerta and subthalamic nucleus (Kuhlenbeck and Haymaker, 1949). The hypothalamus sensu stricto, in contrast, comprises only the walls and the base of the third ventricle below the hypothalamic sulcus. Unfortunately, considerable discrepancies exist regarding subdivisions of the hypothalamus and the nomenclature of its nuclear grays (Malone, 1910; Griinthal, 1930; LeGros Clark, 1936; Brockhaus, 1942; Kuhlenbeck and Haymaker, 1949; Wahren, 1959; Diepen, 1962;Braak andBraak, 1987; Saper, 1990). Most authors agree in distinguishing three regions, namely the chiasmatic (preoptic) region, followed by the tuberal region and the mamillary complex. The chiasmatic region lies over and anterior to the optic chiasm and includes the walls of the preoptic recess. The magnocellular neurosecretory nuclei define its posterior border (Braak and Braak, 1987). The cone-shaped tuberal region surrounds the infundibular recess and extends to the neurohypophysis, while the mamillary bodies dominate the mamillary region abutting upon the midbrain tegmentum. This short review is limited to the chiasmatic and the tuberal region. A feature common to both territories is the scantness of myelinated fibers separating off the mamillary region which is rich in myelin (Wahren, 1959; Diepen, 1962). The hypothalamic gray

Both the chiasmatic and the tuberal region harbor

4

n

O

D

Fig. 1. Chiasmatic and tuberal region of the proper hypothalamus in the human adult. The diagram shows the main landmarks and nuclear grays that are encountered as coronal sections are traced aiitero-posteriorly (u - h ) , sagittal sections mediolaterally ( i - m ) and horizontal sections infero-superiorly ( n -s). Each of the sections is spaced apart by 800 pm. uc, Anterior commissure; an, accessory neurosecretory nucleus; em, corpus mamillare; cu, cuneate nucleus; db, nucleus of the diagnonal band; dm. dorsomedial nucleus;fm, fasciculus mamillo-thalamicus;f, fornix; hg, hypothalamic gray; infundibular nucleus; in, intermediate nucleus; oc, optic chiasm; o f ,optic tract;pe, periventricular nucleus;ph, posterior hypothalamic nucleus;pm, posteromedial nucleus;pv, paraventricular nucleus; re, retrochiasmatic nucleus; sc, suprachiasmatic nucleus; so, supraoptic nucleus; st, nucleus of the stria terminalis; su, subthalamic nucleus; fh,thalamus; tl, lateral tuberal nucleus; f m , tuberomamillary nucleus; un, uncinate nucleus; vm, ventromedial nucleus.

v,

many nuclei with relatively clear-cut boundaries. The nuclear grays are embedded in an ill-defined assembly of small nerve cells referred to as the hypothalamic gray (Fig. 1-hg; Braak and Braak, 1987). Combined pigment-Nissl preparations

render distinction of four neuronal types within the reaches of the hypothalamic gray possible. The type I neurons occur quite frequently and are characterized by unusually large and intensely stained lipofuscin granules. Type I1 cells are marked by

Fig. 2. Neuronal types occurring within the chiasmatic and tuberal region. Pigment-Nissl preparation (aldehydefuchsin, Darrow red, 12 pn). I - IV, Nerve cell types of the hypothalamic gray. I , Type 1 neuron with coarse pigment granules; I I , type I1 neuron with dense accumulations of small lipofuscin granules; I l l , sparsely pigmented type 111 neuron; IV, type IV neuron devoid of pigment. SI, Main cell type of the supraoptic nucleus; SII, medium-sized SII neuron dusted with pigment. PI, Main cell type of the paraventricular nucleus; PII, medium-sized PI1 neuron filled with finely granulated pigment; PIII, third cell type of the paraventricular nucleus with a few intensely stained pigment granules. in, Non-pigmented and strongly basophilic neurons of the intermediate nucleus; sc, non-pigmented small cell of the suprachiasmatic nucleus; me, melanin-containing neuron located within the retrochiasmatic nucleus; vm, sparsely pigmented nerve cell of the ventromedial nucleus; dm, pigment-laden neuron of the dorsomedial nucleus; if, sparsely pigmented nerve cell of the infundibular (arcuate) nucleus; f l , pigment-laden medium-sized cell of the lateral tuberal nucleus; fin, large neuron of the tuberomamillary nucleus.

5

6

medium-sized to small pigment granules which are tightly packed together and fill one pole of the cell body. A third type contains only a fine scattering of dust-like granules while type IV neurons, in general, exhibit large bipolar or multipolar cell bodies devoid of lipofuscin accumulations. Type IV cells are rich in basophilic material (Fig. 2; Braak and Braak, 1987). The magnocellular neurosecretory complex

Most prominent among the well-defined nuclei of the chiasmatic region is the magnocellular neurosecretory complex consisting of the supraoptic nucleus, the paraventricular nucleus and satellites of both. A feature of these nuclei is a conspicuously dense capillary network. The supraoptic and the paraventricular nuclei are linked together by clusters of cells that tend to be arranged around and along blood vessels. These ectopic cells are referred to as the accessory neurosecretory nucleus (Figs. 1an, 3). The magnocellular neurosecretory nuclei synthesize the hormones vasopressin and oxytocin which are transported via the hypothalamo-hypophyseal tract and released into blood vessels of both the infundibulum and the neurohypophysis (hypothalamo-hypophyseal system: Bargmann, 1949, 1954). Neurosecretory material (carrier proteins, neurophysins) can be visualized by special staining techniques. In the adult very little of it is seen in the magnocellular neurosecretory complex; however, in the infant much material is present not only in the cell bodies but in the cellular processes as well (Fig. 3). In experimental animals, the magnocellular neurosecretory nuclei generate - in addition to axons heading towards the neurohypophysis - descending projections to a number of lower brain-stem nuclei and the intermediolateral column of the spinal cord (hypothalamo-autonomic projection: Saper et al., 1976; Swanson and McKellar, 1979). The supraoptic nucleus is associated with posterior portions of the optic chiasm and proximal portions of the optic tract. It consists of a superolateral, superomedial and inferomedial subunit which are linked together by loosely arranged strands of cells (Fig. 1-so).

Combined pigment-Nissl preparations reveal two specific neuronal types in the supraoptic nucleus. The predominating SI cells exhibit a large rounded multipolar cell body with peripherally arranged Nissl substance. Close to the eccentric nucleus there is a clear area with a few faintly stained lipofuscin granules (Fig. 2 3 4 . The SII cells generate only a few thick dendrites. Their cell bodies are smaller than those of the SI cells. The cytoplasm appears dusted with pigment which frequently penetrates into the proximal dendrites (Fig. 2-SZZ). Small cells with characteristics of those found in the surrounding hypothalamic gray can be encountered only occasionally within the boundaries of the supraoptic nucleus. The paraventricular nucleus is an elongated plate of nerve cells close to the ependymal lining of the third ventricle (Fig. 1-pv). Sagittal sections running parallel to the walls of the ventricle reveal the boomerang-like shape of the nucleus (Fig. li). The boundaries of the nucleus are less well defined than those of the supraoptic nucleus and many small cells which, according to their cytological characteristics belong to the hypothalamic gray, can be found within the limits of the paraventricular nucleus. Ectopic neurosecretory paraventricular cells can be recognized similarly in locations far away from the parent nucleus, such as within the pallidum or the bed nucleus of the stria terminalis. The dominating cell type (PI) closely resembles the large multipolar neurons found in the supraoptic nucleus (Fig. 2-PZ).The second neuronal type (PII) also reminds one of the medium-sized cells of the supraoptic nucleus with dust-like pigmentation (Fig. 2-PZZ). The paraventricular nucleus harbors a third specific cell type (PHI) that has no counterpart in the supraoptic nucleus. PI11 cells are relatively small and contain a few coarse pigment granules that can readily be distinguished from the dust-like particles seen in PI1 neurons (Fig. 2-PIZZ). With the use of immunocytochemical techniques it has been shown that vasopressin and oxytocin are located in separate neuronal types, and it is generally accepted that the soma-size of vasopressinergic neurons is larger than that of oxytocinergic ones (Dierickx and Vandesande, 1979). It appears temp-

routinely fixed autopsy material simply stained for lipofuscin pigment and Nissl substance. Speculations explaining the existence of the third cell type in the paraventricular nucleus cannot presently be made. The fact that a similar cell type is missing in the supraoptic nucleus may be of help in identifying further characteristics of this strange cell type. The sexually dimorphic intermediate nucleus

The small but particularly interesting intermediate nucleus is a well-defined compact structure located at the level of the optic chiasm halfway between the paraventricular and supraoptic nucleus (Figs. 1-in, 4). Many descriptions of the human hypothalamus do not mention this nuclear gray which first has been delineated by Brockhaus (1942). The chiasmatic region in the brain of the rhesus monkey shows the presence of this nucleus (Fig. 5 ; H. Braak, un-

Fig. 3. Coronal section showing the neurosecretory system of an infant. The supraoptic nucleus, paraventricular nucleus and the cell masses of the accessory neurosecretory nucleus are well displayed and even the hypothalamo-hypophyseal tract can be recognized (1-month-old infant, aldehydefuchsin, 400 pm). (For abbreviations see legend to Fig. 1.)

ting, therefore, to consider the large specific cells as purportive vasopressinergic neurons and the medium-sized to small cells as the oxytocinergic ones. Provided that this assumption is correct, this would offer unique possibilities to the neuropathologist who would then be able to distinguish vasepressinergic neurons from oxytocinergic ones in

Fig. 4. Coronal section through anterior portions of the chiasmatic region of the human hypothalamus (aldehydefuchsin; Darrow red, 400 pm), Note the well delineated intermediate nucleus halfway between the supraoptic nucleus and the paraventricular nucleus. (For abbreviations see legend to Fig. 1.)

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published observation). Its existence in other primate and subprimate mammalian species has as yet not been confirmed. The fact that the nucleus has escaped recognition by most of the more recent researchers can hardly be understood. Swaab and Fliers (1985) have described an area in the human brain that in all probability corresponds to the intermediate nucleus of Brockhaus. The most remarkable feature of this nucleus is, that its volume is considerably larger in men than in age-matched women. The nucleus, therefore, exhibits a clear sexual dimorphism (Swaab and Fliers, 1985; Swaab and Hofman, 1988; Hofman and Swaab, 1989). The data presently available on sexual dimorphism in various hypothalamic nuclei of subhuman mammalian species (Bleier et al., 1982) are not sufficient to determine whether the human intermediate nucleus corresponds to any of these architectonic units. A single neuronal type forms the intermediate nucleus. The medium-sized bipolar nerve cells are marked by many large and sharply defined Nissl bodies. Moreover, they are devoid of lipofuscin deposits and can, on account of this, be easily distinguished from ectopic neurosecretory nerve cells which may also occur close to the intermediate nucleus (Fig. 2-in;Braak and Braak, 1987).

Fig. 5 . Coronal section through the chiasmatic region of the rhesus monkey (aldehydefuchsin, Darrow red, 400 pm). The intermediate nucleus is seen as a basophilic structure at about the same position as in the human brain. (For abbreviations see legend to Fig. 1.)

The suprachiasmatic nucleus The suprachiasmatic nucleus is a small nuclear gray located below the paraventricular nucleus and close to the ependymal lining of the third ventricle (Fig. 1sc; Stopa et al., 1984; .Moore, 1989). The nucleus shows relatively clear-cut boundaries in a wide variety of mammals with the exception of higher primates and man (Cassone et al., 1988). Considerable ambiguity is met if one attempts to trace the borderlines of this nucleus in the human brain using classical histological techniques. This fact has even cast doubt on its existence in man (Griinthal, 1930; LeGros Clark, 1936; Defendini and Zimmerman, 1978). The nucleus is formed by particularly small fusiform neurons that are almost devoid of

basophilic material and pigment (Fig. 2-sc). These small cells synthetize a number of different neuropeptides and, using immunocytochemical techniques, the nucleus can be apportioned into subdivisions each dominated by a chemically distinct group of nerve cells (Mai et al., 1991). A direct retinal projection reaches the nucleus (Moore, 1973; Sadun et al., 1984). Its efferents influence the production of melatonin in the pineal gland. The nucleus, accordingly, is considered to represent the endogenous clock of the brain playing a significant role in the control of biological rhythms (Moore, 1979,1982; Sadunet al., 1984; Stopaet al., 1984; Swaab et al., 1985). A remarkable loss of suprachiasmatic nerve cells is found t o occur during the course of Alzheimer’s disease and is considered

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to cause disturbances in circadian rhythms frequently observed in individuals suffering from this disorder (Swaab et al., 1985; Mirmiranet al., 1989). The retrochiasmatic nucleus and the melanincontaining hypothalamic nerve cells The extended area of the retrochiasmatic nucleus encompasses the fibers of the supraoptic commissures and harbors a large number of different cell types, many of which resemble those of the hypothalamic gray (Fig. 1-rc).Interspersed between these cells are numerous ectopic neurosecretory supraoptic neurons and small islands of tightly packed nerve cells that, according to their cytological features, belong to the lateral tuberal nucleus. In addition, there are large nerve cells displaying the characteristics of tuberomamillary nerve cells. Some of the small retrochiasmatic nerve cells are conspicuous because they contain a brownish pigment. These cells belong to the melanin-containing hypothalamic nerve cells which are spaced widely apart from each other but generally tend to be located close to the ependymal lining of the third ventricle (Fig. 2-me). In the human brain neuromelanin is formed as an oxidative byproduct of catecholamine synthesis and can be used as a natural post-mortem marker of catecholaminergic neurons (Bazelon et al., 1967; Bogerts, 1981). The small cells, in fact, represent the hypothalamic dopaminergic system (Spencer et al., 1985). It appears noteworthy that this system remains unchanged even in severe cases of Parkinson’s disease while all the other melanin-containing nuclei are severely involved such as the substantia nigra, the locus coeruleus and the dorsal vagal area (Matzuk and Saper, 1985). Until now, neither cell loss nor development of Lewy bodies or neurofibrillary tangles have been shown to occur in the small hypothalamic dopaminergic nerve cells with increasing age or during the course of degenerative diseases. The ventromedial, posteromedial and dorsomedial nuclei The voluminous ventromedial nucleus is a con-

spicuous structure of the tuberal region. The long axis of the pear-shaped nuclear gray runs in a superomedial-inferolateral direction (Fig. 1-vm). The cell density is higher in the peripheral portions than in the center of the nucleus. Moreover, a narrow, cell-sparse zone surrounds the nucleus, facilitating its delineation from adjoining nuclear grays (Fig. 6). The closely related posteromedial nucleus is smaller and partly fills the space between the ventromedial nucleus and the mamillary body (Fig. 1-pm). Both nuclei are formed by small uni- or bipolar nerve cells which are sparsely pigmented or devoid of lipofuscin deposits (Fig. 2-vm). The ventromedial nucleus is interconnected with many neighboring areas but also generates major projections to the magnocellular nuclei of the basal forebrain (Jones et al., 1976). These nuclei in turn send axons to virtually all portions of the cerebral cortex. It can be assumed, therefore, that the ventromedial nucleus influences via these pathways higher cortical functions and behavior. The dorsomedial nucleus is poorly differentiated in the human brain and covers the anterior and superior poles of the ventromedial nucleus. Large numbers of cells which, according to their cytological features, belong to the hypothalamic gray, invade peripheral portions of the nucleus. The medium-sized nerve cells of the dorsomedial nucleus are markedly richer in lipofuscin deposits than those of the ventromedial nucleus (Fig. 2-dm). The periventricular and infundibular nuclei The closest topographical relation to the neurohypophysis is borne by the infundibular nucleus (Spatz et al., 1948)filling profound portions of the tuber cinereum. The horseshoe-shaped nucleus incompletely surrounds the lateral and posterior entrance of the infundibulum and is anteriorly supplemented by the retrochiasmatic nucleus. Superiorly the nucleus is in continuation with the narrow periventricular nucleus (Figs. 1-if, pe, 6). The uni- or bipolar cells of the infundibular nucleus are tightly packed together and have their long axis preferentially arranged parallel to the infundibulum (Fig. 2-ij’). The cells generate an abun-

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dance of fine fibers to both the infundibulum and the neurohypophysis. Numerous cells located within the infundibular nucleus, the periventricular nucleus, the hypothalamic gray and other parvocellular areas synthesize substances which either stimulate or inhibit endocrine cells of the pituitary (parvocellular neurosecretory system: Spatz et al., 1948). These substances are transported via the tuberoinfundibular tract and released into the portal hypophyseal vessels.

Fig. 6 . Coronal section through the tuberal region of the human hypothalamus. Well defined nuclei (ventromedial nucleus, infundibular nucleus with periventricular nucleus, lateral tuberal nucleus) are surrounded by the less sharply delineated tuberomamillary nucleus and the hypothalamic gray (aldehydefuchsin. Darrow red, 400 pm). (For abbreviations see legend to Fig. 1 .)

The lateral tuberal nucleus Basolateral portions of the tuber cinereum contain the lateral tuberal nucleus and the tuberomamillary nucleus which considerably increase in size with phylogenetic advance. Both nuclei are formed by highly characteristic neuronal types that can easily be distinguished from any other hypothalamic neuronal type in pigment-Nissl preparations. The two nuclei should, therefore, not be included in illdefined terms such as “lateral hypothalamic area” (LeGros Clark, 1936). The lateral tuberal nucleus most probably occurs only in primates and is particularly large in the human brain (Strenge, 1975; Fujii, 1982). Major portions are located in basolateral portions of the tuberal region with extensions into both the chiasmatic and the mamillary regions. Many globose or columnar subunits condense to form the nuclear gray, while smaller subunits in the vicinity may lack continuation to the main portion (Fig. 1 4 ) . The basolateral surface of the tuber cinereum frequently reveals slight elevations formed by the underlying cell aggregations of the lateral tuberal nucleus (lateral eminence). The nucleus is composed of only a single neuronal type of medium cell size. These cells can be recognized immediately by the eccentrically placed nucleus and the large deposit of coarse and densely packed lipofuscin granules which fills the other pole of the cell body (Fig. 2 4 . Cells located in the center of the nuclear gray have larger distances between each other than those in the periphery. So far, little is known concerning the connections and functional significance of the lateral tuberal nucleus. Nevertheless, it appears important to note that it exhibits a pronounced cell loss in cases of Huntington’s disease. The adjoining tuberomamillary nucleus, in contrast, remains more or less unchanged (Vogt and Vogt, 1951; Wahren, 1964; Kremer et al., 1990). Another enigmatic feature is its heavy involvement in cases of dementia with argyrophilic grains, a degenerative disorder of unknown etiology (Braak and Braak, 1989; Itagaki et al., 1989; Masliah et al., 1991). Clinically, the disorder

11

resembles Alzheimer’s disease. The pathological alterations, however, differ from Alzheimer-related changes. The key feature is the occurrence of spindle-shaped grains within the neuropil of a few cortical areas and subcortical nuclei. The entorhinal region is the most severely affected cortical territory while the lateral tuberal nucleus bears the brunt of the subcortical changes. The neighboring tuberomamillary nucleus remains virtually without change (Braak and Braak, 1989). In Alzheimer’s disease, the lateral tuberal nucleus shows presence of numerous profiles immunoreactive with Alz-50. This affection can be considered as an early stage of Alzheimer-related cytoskeletal pathology. Neurofibrillary tangles and neuropil threads, in contrast, occur only rarely and can mainly be observed in very severely affected brains (Fig. 7; Kremer et al., 1991). The lateral hypothalamus is considered to be significant in the regulation of food intake. Severe involvement of the lateral tuberal nucleus in the absence of changes in the tuberomamillary nucleus is a feature common to dementia with argyrophilic grains and Huntington’s disease. Both disorders are frequently associated with severe cachexia. It appears tempting, therefore, to assume a probable significance of the lateral tuberal nucleus for the regulation of feeding behaviors (Kremer et al., 1991); nevertheless this speculation certainly has to await confirmation.

The tuberomamillary nucleus The tuberomamillary nucleus extends mainly through the posterior tuberal and anterior mamillary territories. It almost totally surrounds the lateral tuberal nucleus (Fig. 1-tm). Part of the nucleus accompanies the fornix (perifornical portion); another one extends into the mamillary region where it incompletely surrounds the mamillary nuclei (perimamillary portion). Tuberomamillary neurons show a pronounced tendency to spread out into adjoining areas, a fact that frequently renders definition of nuclear borders difficult. Typically, the density of the tuberomamillary neurons diminishes towards peripheral portions, and these ill-

Fig. 7. Coronal section through the tuberal region of the human hypothalamus specifically silver-stained for neurofibrillary changes of the Alzheimer type. Note the severe affection of the tuberomamillary nucleus and posterior hypothalamic nucleus. In this case, there is also an involvement of the lateral tuberal nucleus (Gallyas technique for neurofibrillary changes, 100 am). (For abbreviations see legend to Fig. 1.)

defined areas become gradually mixed up with nerve cells of the hypothalamic gray (Fig. 6 ) . The large nerve cells forming the nucleus display a ragged outline with lipofuscin granules clustered into flocks (Fig. 2-tm). The densely stained Nissl material is preferentially concentrated at peripheral portions of the soma and at infoldings of the nuclear membrane. Many of the tuberomamillary cells contain acidophilic granules within the soma (Issidori-

des et al., 1978), a characteristic feature which can also be found within the morphologically related large neurons of the magnocellular basal forebrain nuclei (Ulfig, 1991). The tuberomamillary neurons synthesize gammaaminobutyric acid as neurotransmitter and contain a variety of additional substances including histamine, adenosin and galanin (Panula et al., 1990). These neurons have widespread projections and significantly contribute to the system of nonthalamic projections into the cerebral cortex (Saper and German, 1987). Quite a number of subcortical non-thalamic nuclei project to the cortex (cholinergic magnocellular nuclei of the basal forebrain, serotonergic oral raphe nuclei, dopaminergic paranigral nucleus and nucleus parabrachialis pigmentosus, noradrenergic locus coeruleus). The cortical projection of the tuberomamillary nucleus is of about the same magnitude as that of the magnocellular forebrain nuclei (Saper, 1990). All of the above-mentioned non-thalamic nuclei projecting into the cortex have been found to be severely damaged in Alzheimer’s disease (German et al., 1987). It, therefore, is not surprising to find the tuberomamillary nucleus heavily affected as well. A key feature of Alzheimer’s disease is the deposition of insoluble fibrous material that normally does not occur in the human brain. Bundles of abnormal filaments appear in the form of neurofibrillary tangles within the ceIl bodies and neuropil threads within the processes of affected nerve cells (Braak et al., 1986). During thecourseof Alzheimer’s disease, almost all tuberomamillary nerve cells develop a neurofibrillary tangle and numerous neuropil threads (Fig. 7; Saper and German, 1987; Simpson et al., 1988; Braak and Braak, 1991). The lateral tuberal nucleus, in comparison, is involved to a much smaller degree and shows neurofibrillary tangles only in the end-stage of the disease (Fig. 6; Braak and Braak, 1991). Oddly enough, most review articles on Alzheimer-related changes d o not mention the tuberomamillary affection. A similar pattern of pathology is also observed in cases of Parkinson’s disease with numerous Lewy bodies within both the cell bodies and the dendrites

of the tuberomamillary nerve cells (Langston and Forno, 1978). Again, Lewy bodies are virtually absent within the lateral tuberal nucleus. Tanglebearing and Lewy body-bearing nerve cells will ultimately die. Thus, a considerable loss of tuberomamillary nerve cells can generally be observed in cases of Alzheimer’s disease and Parkinson’s disease. Summary and conclusions The hypothalamus sensu stricto consists of the chiasmatic, the tuberal and the mamillary region. The present study is confined to the poorly myelinated chiasmatic and tuberal region. Both regions harbor many nuclear grays with relatively clear-cut boundaries embedded in an ill-defined nerve cell assembly referred to as the hypothalamic gray. Prominent components of the chiasmatic region are the magnocellular neurosecretory complex (supraoptic nucleus, paraventricular nucleus, accessory neurosecretory nucleus), the sexually dimorphic intermediate nucleus, the suprachiasmatic and retrochiasmatic nuclei. The dominating structure of the tuberal region is the complex of the ventromedial, posteromedial and dorsomedial nuclei supplemented by the periventricular and infundibular nuclei. Lateral portions of the tuber cinereum harbor the lateral tuberal nucleus and the tuberomamillary nucleus. The lateral tuberal nucleus exhibits pronounced cell loss in Huntington’s chorea and is also severely involved in cases of dementia with argyrophilic grains. The large nerve cells of the tuberomamillary nucleus show particularly severe affection in both Alzheimer’s (intraneuronal neurofibrillary changes) and Parkinson’s disease (Lewy bodies). Acknowledgements This work was supported by grants from the Deutsche Forschungsgemeinschaft. The skillful assistance of Mrs. Babl, Fertig, Schneider (preparations), Szasz (drawings) and Mr. Muller (language) is gratefully acknowledged.

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14 Masliah, E., Hansen, L.A., Quijada, S., DeTeresa, R., Alford, M., Kauss, J. and Terry, R. (1991) Late onset dementia with argyrophilic grains and subcortical tangles or atypical progressive supranuclear palsy? Ann. Neurol., 29: 389 - 396. Matzuk, M.M. and Saper, C.B. (1985) Preservation of hypothalamic dopaminergic neurons in Parkinson’s disease. Ann. Neurol., 18: 552- 5 5 5 . Mirmiran, M.D., Swaab, D.F., Witting, W., Honnebier, M.B.O.M., van Cool, W.A. and Eikelenboom, P. (1989) Biological clocks in development, aging and Alzheimer’s disease. Brain Dysfunct., 2: 57-66. Moore, R.Y. (1973) Retinohypothalamic projection in mammals: a comparative study. Brain Rex, 49: 403 - 409. Moore, R.Y. (1979) The anatomy of central neural mechanism regulating endocrine rhythms. In: D.T. Krieger (Ed.), Endocrine Rhythms, Raven Press, New York, pp. 63 - 87. Moore, R.Y. (1982) The suprachiasmatic nucleus and the organization of a circadian system. Trends Neurosci., 5 : 404 - 407. Moore, R.Y. (1989) The geniculo-hypothalamic tract in monkey and man. Brain Rex, 486: 190- 194. Panula, P., Airaksinen, M.S., Pirvola, U. and Kotilainen, E. (1990) A histamine-containing neuronal system in human brain. Neuroscience, 34: 127- 132. Sadun, A.A., Schaechter, J.D. and Smith, L.E.H. (1984) A retinohypothalamic pathway in man: light mediation of circadian rhythms. Brain Rex, 302: 371 - 377. Saper, C.B. (1990) Hypothalamus. In: G. Paxinos (Ed.), The Human Nervous System, Academic Press, New York, pp. 389 - 413. Saper, C.B. and German, D.C. (1987) Hypothalamic pathology in Alzheimer’s disease. Neurosci. Lett., 74: 364 - 370. Saper, C.B., Loewy, A.D., Swanson, L.W. and Cowan, W.M. (1 976) Direct hypothalamo-autonomic connections. Brain Rex, 117: 305-312. Simpson, J., Yates, C.M., Watts, 0.and Fink, G . (1988) Congo red birefringent structures in the hypothalamus in senile dementia of the Alzheimer type. Neuropathol. Appl. Neurobiol., 14: 381 - 393. Spatz, H., Diepen, R. and Gaupp, V. (1948) Zur Anatomie des Infundibulum und des Tuber cinereum beim Kaninchen. Zur Frage der Verkniipfung von Hypophyse und Hypothalamus. Dtsch. 2. Nervenheilk., 159: 230-268. Spencer, S . , Saper, C.B., Joh, T., Reis, D.J., Goldstein, M. and Raese, J.D. (1985) Distribution of catecholamine-containing neurons in the normal human hypothalamus. Brain Res., 328: 73 - 80. Stopa, E.G., King, J.C., Lydic, R. and Schoene, W.C. (1984) Human brain contains vasopressin and vasoactive intestinal polypeptide neuronal subpopulations in the suprachiasmatic region. Brain Rex, 297: 159- 163. Strenge, H. (1975) Uber den Nucleus tuberis lateralis im Gehirn des Menschen. Eine pigmentarchitektonische Studie. Z . Mikrosk. Anat. Forsch., 89: 1043- 1067.

Swaab, D.F. and Fliers, E. (1985) A sexually dimorphic nucleus in the human brain. Science, 228: 1112 - 1115. Swaab, D.F. and Hofman, M.A. (1988) Sexual differentiation of the human hypothalamus: ontogeny of the sexually dimprphic nucleus of the preoptic area. Dev. Brain Res., 44: 314-318. Swaab, D.F., Fliers, E. and Partiman, T.S. (1985) The suprachiasmatic nucleus of the human brain in relation to sex, age and senile dementia. Brain Res., 342: 37 - 44. Swanson, L.W. and McKellar, S. (1979) The distribution of oxytocin- and neurophysin-stained fibers in the spinal card of the rat and monkey. J. Comp. Neurol., 188: 87 - 106. Treip, C.S. (1984) The hypothalamus and pituitary gland. In: J. Adams, J.A.N. Corsellis and L.W. Duchen (Eds.), Greenfield’s Neuropathology, 4th edn., Arnold, London, pp. 748 - 778. Ulfig, N. (1991) Distribution of intracytoplasmatic acidophilic granules in the magnocellular nuclei of the basal forebrain in normal individuals and patients suffering from Parkihson’s disease. Clin. Neuropathol., 10: 11 - 15. Vogt, C. and Vogt, 0. (1951) Precipitating andmodifying agents in chorea. J. Nerv. Ment. Dis., 116: 601 -607. Wahren, W. (1959) Anatomie des Hypothalamus. In: G. Schaltenbrand and P. Bailey (Eds.), Einfuhrung in die Stereotaktischen Operationen mit einem Atlas des Menschlichen Gehirns, Vol. 1, Thieme, Stuttgart, pp. 119- 151. Wahren, W. (1964) Zur Pathoklise des Nucleus tuberis lateralis. In: W. Bargmann and J.P. Schadk (Eds.), Progress in Brain Research, Vol. 5, Elsevier, Amsterdam, pp. 161 - 170.

Discussion D.F. Swaab: Is also in your experience the intermediate nucleus as you call it sexually dimorphic? There is no doubt in my mind that the intermediate nucleus is the same nucleus which we call “the sexually dimorphic nucleus” (SDN) of the preoptic area (Swaab and Fliers , 1985). The name “intermediate nucleus” might also give rise to confusion since, in the first place, every nucleus in the hypothalamus is intermediate between some other nuclei, and, in the second place, Feremutsch (1948) has described the “intermediate nucleus”. But this is clearly not the same one as the SDN. By this term he means the scattered cells and islands of vasopressin- and oxytocin-containing cells between the SON and PVN. H. Braak: Yes, the intermediate nucleus is the same as the SDN and it is sexually dimorphic. Allen and coworkers (1989) recently published a paper on nuclei of the chiasmatic region; they described a nuclear gray obviously corresponding to the intermediate nucleus of Brockhaus - they referred to it as the interstitial nucleus of the anterior hypothalamus no. 1 - but found no sexual dimorphism of this nucleus. This is in contradiction to our own observations confirming the results of Swaab and Fliers (1985). I agree with the remark on Feremutsch (1948). However, the original description of Brockhaus (1942) is quite clear.

15

C.B. Saper: Comment from chair to question by D.F. Swaab: I agree with Dr. Braak. Reading Brockhaus’s original paper (1942) it is quite clear that he distinguished the intermediate nucleus from the accessory supraoptic cells that are nearby. Our immunocytochemical studies with oxytocin and vasopressin antisera confirm this distinction. J. Joosse: Lipofuscin occurs in neurons of numerous invertebrates. It has been studied in detail in snail and molusc brains, in relation to aging and whether or not it can be mobilized. They gave negative results. Is there any indication of metabolic changes in lipofuscin content of human brain neurons and what might be the functional meaning of its presence? H. Braak: There are no clear indications for metabolic changes of lipofuscin deposits in the human brain. D.F. Swaab: 1 should like to make a brief comment to Prof. Joosse’s question on the possible effects of lipofuscin of the neurons. Neurons containing lipofuscin, such as the supraoptic nucleus (SON) and paraventricular nucleus (PVN) cells, remain perfectly intact. There is no loss of cells in the SON or PVN in aging or Alzheimer’s disease (Goudsmit et al., 1990). On the other hand, as Dr. Braak has shown (Braak and Braak, 1987), there is virtually no lipofuscin in the SCN. However, the SCN is clearly affected in aging and Alzheimer’s disease (Swaab et al., 1985). So there seems to be no relationship between lipofuscin accumulation and neuronal death in aging. H. Braak: I agree, another example is the inferior olive which is laden with pigment but without any notable loss of nerve cells as age advances. C.B. Saper: Drs. Heiko and Eva Braak are to be congratulated on their elegant pigmento-architectonic studies. I have two questions on the correlation of these observations with recent immunocytochemical observations made in humans and other species. Firstly, you have equated the large type I cells in the supraoptic and paraventricular nuclei with vasopressin neurons and the smaller type I1 cells with oxytocin cells. Our own observations in humans indicate that vasopressin cells in the paraventricular nucleus tend to cluster in the ventrolateral quadrant, while oxytocin cells tend to be more dorsally and diffusely located. Are type I and type I1 cells differentially distributed in a way that can be correlated with the immunocytochemical data? H. Braak: As yet we have not studied the distribution pattern in detail. There is obviously a specific pattern for each cell type and we will have to examine this in the near future. C.B. Saper: Secondly, you have equated the tuberomamillary nucleus with the cell group of the same name in the rat, whose neurons contain GABA and histamine, and project to the cerebral cortex. This cell group, as defined in your pigmentoarchitectonic studies, and originally by Malone (1910), is enormous. I wonder whether you might not have included in the tuberomamillary nucleus other large, similar-appearing neurons in the lateral hypothalamus, that project to the cerebral cortex in the rat, but do not contain histamine or GABA?

H. Braak: Much work of comparative neuroanatomists has to be done to clearly show the homology of the structures that are now referred to as the tuberomamillary nucleus in the various mammalian species. H.P.H. Kremer: Is there neuronal loss in the lateral tuberal nucleus in cases of dementia with argyrophilic grains in cats, described by you? H. Braak: We have not quantitatively counted nerve cells of the lateral tuberal nucleus in this disorder. However, we did not observe gliosis or obvious loss of nerve cells. The characteristic of the disorder is simply a dense accumulation of argyrophilic grains in the neuropil of the lateral tuberal nucleus. N. Kopp: Is there the same phylogenetic increase in volume of the medial part of the tuberal region as for the lateral tuberal nucleus? H. Braak: The medial part of the tuberal region is much more conservative than the lateral part. Both the lateral tuberal nucleus and the tuberomamillary nucleus increase considerably in size with phylogenetic advance. M.A. Hofman: Do you mean that these nuclei increase in size as one climbs the scale naturae from mouse to man, even if differences in hypothalamic volume between mammals are taken into account. H. Braak: I guess there is no scale from mouse to man, but there is an indication for an increase in relative volume of the lateral tuberal nucleus and the tuberomamillary nucleus as the primate scale is ascended. J.M.B.V. De Jong: Is lipofuscin different from ceroid lipopigment? H. Braak: The material stored in neuronal ceroid lipofuscinoses differs from normal lipofuscin (it is less inert, more soluble, stains less distinctly with aldehyde-fuchsin, etc., than lipofuscin). By the way, only a few types of nerve cells in the human brain accumulate the storage material; so this storage disease does not affect all nerve cells in the same manner. W.A. Scherbaum: I was interested in your data on lipofuscin distribution in the hypothalamus. When I looked at unfixed sections of human hypothalamus using the indirect immunofluorescence test, it appeared evident to me that lipofuscin is not present in fetal tissue, and its quantity in the cytoplasm of neurosecretory cells of adults increases with advancing age. So my questions are: do the results obtained with autofluorescence methods correspond to the results you showed by applying aldehyde-fuchsin staining; and secondly, do you have data on the appearance of lipofuscin during ontogeny? H. Braak: Lipofuscin is virtually absent in the fetal brain. It starts to developin early childhood in certain nuclei (e.g., inferior olive), other nuclei follow later. The adult pattern of lipofuscin distribution is seen from the second decade onwards. The pattern seen by fluorescence differs from that seen in aldehyde-fuchsinstained preparations. D.D. Rasmussen: In your paper you mentioned various hypothalamic cell types based in part on size and shape. My ques-

16 tions are: how plastic are these cell types? Do monopolar cells ever become bipolar and vice versa? Do neurons extend or retract processes under different physiological conditions, changing their shape? H. Braak: As far as can be judged by looking at pigment-Nissl preparations (showing only the proximal stems of the dendrites) we have not encountered profound changes in the appearance of these cells during ageing or during the course of degenerative diseases. This does not exclude that alterations may occur in distal portions of the dendrites. Shrinkage of the cell soma can - in certain nuclei - be observed in Alzheimer’s and Parkinson’s disease. R.Y. Moore: I shall first offer an anecdotal comment. Some years ago we had the opportunity to study the thalamus of Albert Einstein. There we found someof the largest neuronal accumulations of lipofuscin I have encountered. There is every indication that Einstein functioned quite well in the period before his death, providing further indication that lipofuscin accumulation is not inimical to function. Secondly, I would like to ask Professor Braak for his views of the human homologue of the lower mammalian preoptic area. H. Braak: It is very difficult to make homologies between animal brains and the human brain. One example that 1 have described is the nucleus intermedius. I believe it will require a great deal of very careful comparative neuronatomical study to establish these homologies.

References Allen, L.S., Hines, M., Shryne, J.E. and Gorski, R.A. (1989) Two sexually dimorphic cell groups in the human brain. J. Neurosci., 9: 497 - 506. Braak, H . and Braak, E. (1987)The hypothalamus of the human adult: chiasmatic region. Anat. Embryo/., 176: 315 - 330. Brockhaus, H. (1942) Beitrag zur normalen Anatomie des Hypothalamus und der Zona incerta bei Menschen. J. Psychol. Neurol., 51: 96- 196. Feremutsch, K . (1948) Die Variabilitat der cytoarchitektonischen Struktur des menschlichen Hypothalamus. Monatschr. Psychiatr. Neurol., 116: 257-283. Goudsmit, E., Hofman, M.A., Fliers, E. and Swaab, D.F. (1990) The supraoptic and paraventricular nuclei of the human hypothalamus in relation to sex, age and Alzheimer’s disease. Neurobiol. Aging, 11: 529 - 536. Malone, E. (1910) Uber die Kerne des menschlichen Diencephalon. Abhandl. Konigl. Preuss. Akad. Wiss. (Physik.-Math. Klmse), 1 : 1 - 32. Swaab, D.F. and Fliers, E. (1985) A sexually dimorphic nucleus in the human brain. Science, 228: 1112- 1115. Swaab, D.F., Fliers, E. and Partiman, T. (1985) The suprachiasmatic nucleus of the human brain in relation to sex, age and dementia. Brain Res., 342: 37 - 44.

SECTION I1

Clinical Manifestations of Hypothalamic Diseases

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D.F. Swaab, M.A. Hofman, M. Mirmiran, R. Ravid and F.W. van Leeuwen (Eds.) Progress in Brain Research, Vol. 93 0 1992 Elsevier Science Publishers B.V. All rights reserved.

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CHAPTER 2

Endocrine functions of the hypothalamus and alterations in neuroendocrine function - focus on thyrotropin and growth hormone M.F. Scanlon Section of Endocrinology, Metabolism and Diabetes, Department of Medicine, University of Wales College of Medicine, Heath Park, CardgJ U.K.

Introduction

Over the last three decades major advances in the understanding of hypothalamic-pituitary control mechanisms have led to important diagnostic and therapeutic developments. The early work of Geoffrey Harris which established the concept of hypothalamic-pituitary control via the microvascular portal system was confirmed by the isolation of Guillemin and Schally in the late 1960’s and early 1970’s, of a variety of chemical mediators of these brain actions: hypothalamic regulatory peptides. In more recent times the two most elusive hypothalamic regulatory peptides, corticotrophin releasing hormone and growth hormone releasing hormone have been isolated and characterised through the efforts of Vale and Guillemin. It is now generally felt that all of the most important hypothalamic regulators of anterior pituitary function have been characterised. The hypothalamic peptides have been widely used in clinical research and as diagnostic tools in the investigation of normal and abnormal anterior pituitary function. There have also been major and occasionally unexpected therapeutic developments such as the development and application of new long acting dopamine agonists for treatment of hyperprolactinaemic states (and Parkinson’s disease), long acting somatostatin analogues for treatment of growth hormone (GH) hypersecretion and gastro-

enteropancreatic tumours, and long acting gonadotropin-releasing hormone (GnRH) analogues for inducing therapeutic hypogonadism. In this review I will briefly discuss some recent advances in the understanding of the hypothalamic control of anterior pituitary function focussing on thyrotropin (TSH) and growth hormone, two proteins with widely differing patterns of release and peripheral actions. This study is the synthesis of more extensive recent reviews on these subjects (Dieguez et al., 1988; Page et al., 1989b; Scanlon, 1991). Thyroid stimulating hormone (TSH)

(a) Circadian and ultradian changes There is a clear circadian variation in basal TSH levels in both animals and man (Vanhaelst et al., 1972; Fukuda et al., 1977; Rose and Nisula, 1989). Circulating levels increase before the onset of sleep reaching their peak between 23:OO and 04:OO hours and falling to a nadir around 11:OO hours. There does not appear to be any sex-related difference in amplitude or frequency of circadian TSH changes (Brabant et al., 1989), unlike the secretory pulses of GH. TSH release is pulsatile and both amplitude and frequency of pulse are greater at night (Greenspan et al., 1986; Brabant et al., 1987, 1990; Rossmanith et al., 1988). Sleep reduces the amplitude but not the frequency (Parker et al., 1987)of TSH pulses

20

but the underlying mechanisms are unknown. Patients with severe primary hypothyroidism show increased pulse amplitude by day but a reduction of the nocturnal increase in pulse amplitude (Weeke and Laurberg, 1976; Samuels et al., 1990). An interesting recent observation is that the secretory pulses of TSH, a-subunit and the gonadotrophins (which together constitute the glycoprotein family of hormones) show a high degree of concordance consistent with the operation of a common hypothalamic pulse generator (Samuels et al., 1990). The central mechanisms underlying TSH pulsatility and rhythmicity are unknown. They are probably mediated in part by signals from the suprachiasmatic nuclei of the hypothalamus, paired structures situated just above the optic chiasm that initiate intrinsic circadian rhythmicity, the timing of which can be influenced by non-visual nerve impulses arising in the retina (Moore, 1983). It is clear that TSH rhythms are not secondary to peripheral factors such as thyroid hormones, haemo-concentration or glucocorticoid changes. Furthermore, circadian changes in circulating TSH levels can be detected using ultrasensitive assays in hyperthyroid patients (Evans et al., 1986), suggesting that central mechanisms can override to an extent the powerful negative feedback effects of thyroid hormones at the pituitary level. Basal TSH levels show a small acute rise following the lowering of glucocorticoid levels by means of 1 I-@-hydroxylaseinhibition with metyrapone (Re et al., 1976), suggesting that endogenous glucocorticoids exert a small inhibitory influence on TSH secretion. Furthermore, pharmacological doses of glucocorticoids inhibit basal TSH secretion and abolish the circadian variation in basal TSH (Sowers et al., 1977)and this mechanism may well underlie the reduction in basal and TRHstimulated TSH levels and in circadian TSH changes which occur in depression (Souetre et al., 1988; Bartalena et al., 1990a) and following major surgery (Bartalena et al., 1990b). However, total abolition of t&e circadian rhythm of cortisol with metyrapone did not cause disruption of overall circadian TSH changes although there was a small but significant

decrease in the acrophase and amplitude of the TSH profile (Salvador et al., 1985). Although both dopamine and dopamine agonists, administered acutely, abolish the circadian change in TSH secretion (Sowers et al., 1982; Brabant et al., 1989) it seems unlikely that endogenous dopaminergic pathways play a primary role in determining circadian TSH changes. The nocturnal elevation in TSH is not due to a decline in dopaminergic inhibition since the dopaminergic inhibition of TSH release is greater at night than during daytime hours (Scanlon et al., 1980; Perez Lopez et al., 1982; Rossmanith et al., 1988). Dopamine is a determinant of TSH pulse amplitude but not frequency (Rossmanith et al., 1988). It appears that dopaminergic pathways mediate a “fine tuning” control system which serves to dampen down TSH pulsatility presumably in order to maintain basal TSH levels, and hence thyroid function, in as steady a state as possible. The mechanisms by which the TSH response to dopamine blockade is greater at night than during the day are unclear. It cannot be explained by increased central dopaminergic activity since the PRL response to dopamine blockade is the same at night as during daytime hours (Salvador et al., 1988). Similarly, a-adrenergic pathways do not play a primary role in determining TSH circadian rhythmicity since a-adrenergic blockade with thymoxamine, which penetrates the blood-brain barrier, did not affect the circadian pattern of TSH change although plasma TSH levels were slightly decreased throughout the entire period of study (Valcavi et al., 1987). Serotonin, although present in high concentrations in the suprachiasmatic nucleus, does not play any major role in circadian TSH changes (Golstein et al., 1979). The pineal hormone melatonin which in animals exerts an inhibitory effect on hypothalamic-pituitary-thyroid function (Gordon et al., 1980), does not exert any effect on circadian TSH changes in man (Strassman et al., 1988). Recent evidence suggests that TSH can regulate its own secretion through an increase in dopamine receptors at thyrotroph level (Foord et al., 1985), a finding which could explain the higher TSH and unaltered PRL responses to dopamine

21

blockade at the time of greatest TSH secretion (Salvador et al., 1988). A further possible contributor to circadian TSH changes is the diurnal variation in the activity of anterior pituitary 5 ’ monodeiodinase which promotes intrapituitary T, to T, conversion in the rat. Circadian changes in the activity of this enzyme have recently been described (Murakami et al., 1988). (b) Effects of temperature, age and calorie restriction Acute exposure to cold causes an acute rise in circulating TSH levels in both animals and human neonates but this phenomenon is much less marked in adults (Jackson, 1982). This TSH response to cold can be attenuated by either passive immunisation with anti-TRH antibodies or a-adrenergic blockade indicating that adrenergic release of hypothalamic TRH may mediate the phenomenon (Jackson, 1982). Lesions affecting the temperature regulating the centre of the hypothalamic preoptic nucleus abolish the TSH response to cold stress but do not cause hypothyroidism (Morley, 1981). Ageing itself probably causes a slight decrease in TSH secretion but this is without clinical effects. In one study of a large healthy elderly population, basal TSH levels were low in 5% and accompanied by a reduced TSH response to TRH (Finucane et al., 1989). In addition the TSH pulse amplitude is reduced with preservation of the frequency of pulsatility and overall pattern of circadian change. The underlying mechanism is unclear but it has been suggested that the TSH changes may reflect an adaptive mechanism to the reduced need for thyroid hormones in the elderly (Van Coeverden et al., 1989). Calorie restriction leads to reduced basal and TRH-stimulated TSH levels in both animals and man despite reduced T, levels (Vinik et al., 1975; Hugues et al., 1984). The components of the TSH suppression in man are a reduction in the overall basal TSH level and the nocturnal elevation in TSH levels due to a decrease in TSH pulse amplitude but not frequency (Romijn et al., 1990). In the more extreme clinical setting of anorexia nervosa, the reduced basal and stimulated TSH levels may be a con-

sequence of increased circulating cortisol levels but there is no doubt that the mechanisms underlying this phenomenon are complex. Passive immunisation with somatostatin antibodies abolishes the starvation-induced decline in TSH secretion in rats (Hugues et al., 1986) indicating a mediating role of hypothalamic somatostatinergic pathways secondary to hitherto unknown metabolic signals (Fig. 1). There is no evidence of increased dopaminergic inhibition of TSH during calorie restriction (Mora et al., 1980; Rojdmark, 1983) and TRH administration does not reverse the acute decline in TSH levels during fasting (Spencer et al., 1983). In addition to alterations in hypothalamic function, pituitary 5 ’ deiodinase activity is altered by calorie restriction since blockade of T, to T, conversion with iopanoic acid derivatives can enhance the TSH response to TRH (Burman et al., 1983). It is likely that all these factors interact to reduce thyroid function in response to acute food deprivation hence leading to energy conservation.

(c) Effects of stress, non-thyroidal illness and neuropsychiatric disorders Acute stress in animals leads to a fall in circulating TSH levels. In man, surgical stress is associated with an acute reduction in basal TSH (Kehlet et al., 1979;

Fig. 1 . Schematic representation of the interaction of signals which have been implicated in mediating TSH responses to cold and calorie restriction. (From Scanlon, 1991, with permission.)

22

Zalaga et al., 1985) and a longer term abolition of the nocturnal elevation in basal TSH levels (Bartalena et al., 1990b). This occurs despite a fall in free T, levels, whilst free T, levels are unaltered (Wartofsky and Burman, 1982; Bartalena et al., 1990b). In animals both opioidergic and dopaminergic pathways have been implicated in this phenomenon (Judd and Hedge, 1982) whilst in man, circulating glucocorticoids and dopaminergic pathways may play a mediating role (Zalaga et al., 1985; Bartalena et al., 1990b). The effects of both calorie restriction and these stress phenomena bear some resemblance to the altered neuroregulation of TSH which occurs in non-thyroidal illness (the “euthyroid sick” syndrome) and certain neuropsychiatric disorders. Although in non-thyroidal illness basal TSH levels are usually normal ,they may be low or sometimes even slightly raised (Wehman et al., 1985; Hamblin et al., 1986). Despite the frequent therapeutic use of pharmacological agents such as glucocorticoids and dopamine which suppress TSH acutely (Vierhapper et al., 1982; Wehman et al., 1985) endogenous central inhibition of thyrotroph function is common as illustrated by the abolition of the nocturnal elevation in basal TSH levels in up to 60% of acutely ill patients in the presence of low free T,, and elevated reverse T, levels (Romijn and Wiersinga, 1990). However, true central hypothyroidism is rare in such patients who usually but not always show normal free T, levels (Faber et al., 1987; Romijn and Wiersinga, 1990). It seems clear that, in addition to peripheral alterations in thyroid hormone economy usually manifest as lowered free T,, elevated reverse T, and normal free T, levels, there is central suppression of thyrotroph function in patients with severe nonthyroidal illness such as heart failure, infection, diabetes mellitus (Kabadi et al., 1984; Small et al., 1986) or chronic renal failure (Pokroy et al., 1974). The precise initiating signals and underlying mechanisms are unknown although alterations in opioidergic, dopaminergic and somatostatinergic activity may each contribute. In addition peripheral, glucocorticoid-mediated inhibitory feedback probably plays an important role particularly in the

‘I

$ i i - l ~ ~ l

Fig. 2. Schematic representation of the interaction of some central and peripheral signals which have been implicated in mediating TSH responses to stress and illness. (From Scanlon, 1991, with permission.)

acute setting (Delitala et al., 1987). Finally recent evidence indicates that activation of the cytokine pathways involving tumour necrosis factor-a! and interleukin-10, which inhibit TSH and stimulate ACTH release in animals (Dubuis et al., 1988; Ozawa et al., 1988) may be crucial mediating events in the coordination of the thyroidal and adrenal responses to stress and non-thyroidal illness (Fig. 2). Abnormalities in TSH secretion also occur in anorexia nervosa and endogenous depression with a reduced TSH response to TRH being found quite commonly (Mora et al., 1980; Loosen, 1988). A much more frequent phenomenon is loss of the nocturnal elevation in basal TSH levels which together with the low free thyroid hormone, ferritin and SHBG levels may indicate central hypothyroidism (Bartelena et al., 1990a). Once again the mechanisms are unclear: dopamine is not involved in central TSH suppression in anorexia nervosa (Mora et al., 1980) but both cortisol and body temperature changes have been implicated in depression (Souetre et al., 1988; Bartelena et al., 1990a). Without doubt the debate about whether central hypothyroidism exists in patients with stress, severe non-thyroidal illness, depression or anorexia nervosa will continue. It is of interest therefore that thyroid hormone treatment of patients with nonthyroidal illness is of no benefit or may even cause deterioration (Brent and Hershman, 1986) whilst thyroid hormones may enhance the therapeutic benefits of tricyclic antidepressant therapy in depression (Stein and Avni, 1988).

23

Growth hormone (GH) Our understanding of GH control has increased considerably over the last decade (Dieguez et al., 1988). GH itself has two separate pathways of action (Fig. 3). It stimulates general body growth through the mediation largely of IGF-1 which is produced mainly by the liver under the direct influence of GH. However, IGF-1, and probably several other growth factors, also play important paracrine roles in the control of the growth and function of many tissues. These locally produced growth mediators may also be to some extent under the control of GH. GH also plays an important role in metabolism, simulating lipolysis, increased production of non-esterified and free fatty acids and the production of ketones. In excess, GH causes carbohydrate intolerance which may lead on to frank diabetes mellitus. Various products of each of these separate pathways of GH action feed back on both the hypothalamus and the anterior pituitary to control the function of the somatotroph cells. In consequence, the secretion of GH is determined, at any given point in time, by the interplay of a variety of different factors. The basic secretory pattern of GH is pulsatile in both animals and man with a characteristic sleepentrained circadian rhythm. Marked GH release occurs with the onset of slow wave sleep (SWS). It is now clearly established that hypothalamic control of somatotroph function is mediated by two peptides, somatostatin which is inhibitory and a Hypothalamus.

r

\

GH-releasing hormone (GHRH) which is stimulatory. It has been demonstrated by passive immunisation techniques in free-living rats and by direct measurement, albeit in anaesthetised animals, that these two peptides probably work in concert such that pulses of GH release are a consequence of a pulse of GHRH release accompanied by a reduction in somatostatin release into hypophyseal portal blood (Plotsky and Vale, 1985). When GHRH is administered to normal human subjects or GH-deficient children, the majority of whom have hypothalamic GHRH deficiency, GH responses can be very variable. When repeated doses of GHRH are administered to normal subjects at 2 h intervals, the GH response to the second bolus of GHRH is abolished (Shibasaki et al., 1985). Furthermore, continuous infusion of GHRH causes GH release initially but GH secretion falls to baseline levels within 6 h (Vance et al., 1985). Is this reduction in responsiveness of the somatotroph due to desensitisation and depletion of intracellular stores of GH, or to the operation of various inhibitory feedback pathways? A variety of studies have indicated that although desensitisation of the somatotroph does occur if GHRH is administered at a high enough dose over a long enough period of time in vitro (Bilezikjian and Vale, 1984; Ceda and Hoffman, 1985; Edwards et al., 1988), this is in no way comparable in terms of degree to the marked desensitization of the gonadotroph which follows repeated GnRH administration (Dieguez et al., 1984). Indeed, disruption of the normal 90 min pulsatile secretory pattern of GnRH (and hence gonadotrophin) release by continuous GnRH administration causes the development of hypogonadism in both males and females. This is rapidly reversible and is now used therapeutically in a wide array of conditions such as hormone-dependent carcinoma o€ breast or prostate , precocious puberty and endometriosis. Loss of the pulsatile pattern of hypothalamic GnRH release probably underlies the “functional” hypogonadotropic hypogonadism and is a frequent accompaniment of chronic disease, stress, depression and body weight disturbances (Crowley et al., 1985). Further studies have shown that loss of re-

’ 1

Growth

Metabolism

LipoIysis tI NEFA/FFA Ketones

Somatomedin-C IGF- 1

CHO Intolerance Diabetes Mellitus

Tissue growth

Fig. 3. Pathways of GH action and feedback control.

24

sponsiveness of the somatotroph to GHRH in vitro has at least two components: firstly there is a reduction in the Bmaxresponse to GHRH which is probably due to depletion of intracellular stores since this can be reversed by treatment with somatostatin along with GHRH. Secondly, however, the cyclic AMP response to repeated administration of GHRH is also reduced and this cannot be reversed by co-incubation with somatostatin (Edwards et al., 1988). These data suggest that there is uncoupling of the GHRH receptor from adenylate cyclase, a process which is known to occur in other systems following repeated agonist administration. Although it has been demonstrated that repeated administration of GHRH leads to about a 50% reduction in the numbers of GHRH receptors, this does not, in itself, reduce GH responsiveness to GHRH and probably only 30 - 40% occupancy of such receptors is necessary for maximal responsiveness (Bilezikjian et al., 1986).Despite these in vitro observations, however, there are no data which demonstrate that desensitisation of the somatotroph is important in vivo. There is accumulating evidence that depletion of intracellular stores of GH does not explain the loss of GH responsiveness to GHRH in vivo. The GH response to a-adrenergic agonism is preserved 2 h after pretreatment with GHRH (Valcaviet al., 1988) and the GH response to hypoglycaemia and arginine infusion are not only preserved but enhanced (Shibasaki et al., 1985; Page et al., 1988). Furthermore the simultaneous administration of maximal doses of GHRH and insulin leads to increased GH release compared with either agent alone (Page et al., 1987b) suggesting. the operation of separate pathways. The GH response to L-dopa administration is abolished by prior GHRH administration (Page et al., 1988). These data indicate that the somatotroph is not depleted of GH and does not become refractory to all stimuli following GHRH pretreatment. Insulin-induced hypoglycaemia, arginine, clonidine and alpha-adrenergic activation do not require the release of endogenous hypothalamic GHRH in order to cause GH secretion and could be influencing somatostatinergic tone. In contrast, L-dopa may well act via the release of en-

dogenous GHRH, though an effect on SS remains possible since dopamine agonism with bromocriptine potentiates the GH response to GHRH (Vance et al., 1987). The results of further studies have suggested that the reduction in the GH response to GHRH after prior GHRH treatment is probably mediated by acute inhibitory feedback pathways operating through hypothalamic somatostatinergic pathways. Melmed and colleagues (1988) in the United States have shown in a variety of studies that IGF-1 itself has an inhibitory feedback role directly at the level of the somatotroph, to limit GH responsiveness to GHRH, forskolin, cyclic AMP, TPA, glucocorticoids and T,. This is in addition to the known effects of IGF-1 to stimulate somatostatin release from the hypothalamus in vitro (Berelowitz et al., 1981). An important point about such effects of IGF-1 is that they are slow, occurring over several hours, and may therefore constitute a system for “setting” the background level of responsiveness of the somatotroph to other signals. In the more acute setting there are convincing data suggesting that GH itself can act as a negative feedback inhibitor of somatotroph responsiveness. It has been known for some years that GH causes stimulation of somatostatin release from the rat hypothalamus in vitro (Sheppard et al., 1978). More recently it has been demonstrated in human in vivo studies that GH administration leads to abolition of subsequent GH responsiveness to GHRH over a short time period before there is any detectable rise in circulating IGF-1 levels in response to the administered GH (Ross et al., 1987a). This may constitute an acute inhibitory feedback pathway for the control of somatotroph function. Recent evidence has highlighted the importance of cholinergic pathways in the control of GH secretion and has indicated probable interaction of a “cholinergic-somatostatinergic hypothalamic unit” with peripheral feedback pathways mediated by glucose and GH. Cholinergic activation with pyridostigmine leads to enhanced GH responsiveness to GHRH whereas cholinergic muscarinic blockade with drugs such as atropine or pirenzepine leads to abolition of

25

GH(mU/I) 30 -

GH(mU/I) 9-

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10 -

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.

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responses to all known GH secretagogues with the exception of insulin-induced hypoglycaemia (Dieguez et al., 1988). Although such evidence does not exclude a direct pituitary action of acetylcholine to stimulate GH release, such a pathway cannot account for the abolition of the GH response to exogenously administered GHRH by cholinergic blockade. In the in vivo setting it is possible that cholinergic mechanisms are operating at the hypothalamic level through modulation of the somatostatinergic neurone such that there is a tonic inhibitory cholinergic control of somatostatin release (Fig. 5 ) . Furthermore, such cholinergic modulation of somatostatin may underlie the sleep-induced release of GH. Since pirenzepine does not penetrate the blood-brain barrier it seems likely that the cholinergic modulation of GH pulses and sleep-related GH release is exerted at median eminence level. However, the site of origin of such cholinergic input is unclear at present. The relative sparing of the GH response to insulin-induced hypoglycaemia by cholinergic blockade is consistent with the hypothesis that cholinergic pathways and glucose modulate somatostatin-producing neurones in a hierarchical fashion in that hypoglycaemia can override the cholinergic influence when appropriate physiologi:a1 demands are made. Investigation of this choli-

,

2

3

Time(hours) Fig. 4. Abolition of slow wave sleep (SWS) related GH release in six normal males by cholinergic muscarinic blockade with pirenzepine (PIR) 100mg p.0. at 12:OO midnight. Blocks indicate periods of SWS on control (N) and experimental (M) nights. (From Peters et al., 1986, with permission.)

this response (Casanuevaet al., 1983; Massara et al., 1984, 1986a,b). In addition cholinergic muscarinic blockade completely abolishes physiological nocturnal slow wave sleep-related GH release (Fig. 4) and also GH responses to exercise and food (Leveston and Cryer, 1980; Casanuevaet al., 1984; Peters et al., 1986; Page et al., 1987a, 1989a). Finally cholinergic muscarinic blockade abolishes the GH

Catecholamines Opiates GABA

Acetyl Choline

Glucose

Fig. 5. Major pathways in neuroregulation of GH.

26

nergic system in relation to inhibitory feedback control by GH itself indicates that the GH response to GHRH which is abolished by prior GH or GHRH administration, can be restored by activation of cholinergic pathways with pyridostigmine (Massara et al., 1986a; Ross et al., 1987b). Thesedata suggest that the inhibitory GH-mediated pathway may well act via modulation of the release of endogenous somatostatin. Pharmacological utilisation of this pathway may lead to a more predictable response to repeated administration of GHRH in the in vivo situation. Despite the potent action of muscarinic receptor blocking drugs to abolish GH release, these agents are totally ineffective in the medical management of acromegaly, a condition in which the normal pulsatile and sleep-related patterns of GH release are lost. We have found that pirenzepine does not affect either basal, GHRH- or TRH-induced GH release in alargenumberof suchpatients(J0rdanet al., 1986). A possible explanation for this is depicted as follows: the current hypothesis is that cholinergic hypothalamic neurones exert a tonic inhibitory control over hypothalamic somatostatinergic neurones. If this effect is blocked using muscarinic cholinergic blocking drugs such as pirenzepine or atropine, one will obtain a surge of somatostatin release into hypophyseal portal blood with appropriate GH suppression. The acromegalic somatotroph cells are not receptive to hypothalamic somatostatinergic signals (whereas they are receptive to peripheral somatostatin administration) which implies a local intrapituitary block perhaps due to disturbance of hypophyseal portal microvascular arrangements within the pituitary gland itself as a consequence of tumour development. This is entirely in accord with current views that pituitary tumours are autonomous and not under hypothalamic control. Perspectives

How then might this increased understanding of cholinergic control mechanisms be of therapeutic benefit? Two possible areas are being explored at present. We have demonstrated that cholinergic

muscarinic receptor blockade with pirenzepine can abolish the nocturnal GH surge in both normal subjects and in patients with insulin-dependent diabetes mellitus (Peters et al., 1986; Page et al., 1987a). GH hypersecretion can lead to altered acute metabolic control in diabetes and has also been implicated in the development of a variety of microvascular complications in this condition, particularly retinopathy. In general a safe and well-tolerated means of abolishing nocturnal GH secretion (most GH is secreted at night during slow wave sleep) would be helpful in improving acute control and perhaps treatment of long-term microvascular disease. Our preliminary studies indicate that cholinergic muscarinic blockade at night over a 1 week period does indeed lead to reduction of the dawn phenomenon in diabetes, and associated improvements in acute metabolic control which are presumed to be consequent upon the quite marked GH inhibition which occurs (Atiea et al., 1989). References Atiea, J.A., Creagh, F., Page, M.D., Owens, D.R., Scanlon, M.F. and Peters, J.R. (1989)Early morning hyperglycaemiain insulin-dependent diabetes mellitus: acute and sustained effects of cholinergic blockade. J. Clin. Endocrinol. Metab., 69(2): 390-395. Bartalena, L., Placidi, G.F., Martino, E., et al. (1990a) Nocturnal serum thyrotropin (TSH) surge and the TSH response to TSH-releasing hormone: dissociated behaviour in untreated depressives. J. Clin. Endocrinol. Metab., 71: 650. Bartalena, L., Martino, E., Brandi, L.S., et al. (1990b) Lack of nocturnal serum thyrotropin surge after surgery. J. Clin. Endocrinol. Metab., 70: 293. Berelowitz, M., Szabo, M. and Frohman, L.A. (1981) Somatomedin-C mediates growth hormone negative feedback by effects on both hypothalamus and the pituitary. Science, 212: 1279- 1281. Bilezikjian, L.M. and Vale, W.W. (1984) Chronic exposure of cultured rat anterior pituitary cells of GRF causes partial loss of responsiveness to GRF. Endocrinology, 115: 2032 - 2034. Bilezikjian, L.M., Seifert, H. and Vale, W. (1986) Desensitization to growth hormone-releasing factor (GRF) is associated with down-regulation of GRF-binding sites. Endocrinology, 118: 2045 - 2052. Brabant, G., Brabant, A., Ranft, U., et al. (1987) Circadian and pulsatile thyrotropin secretion in euthyroid man under the influence of thyroid hormone and glucocorticoid administration. J. Clin. Endocrinol. Metab., 65: 83.

27 Brabant, G., Ocran, K., Ranft, U., von zur Muhlen, A. and Hesch, R.D. (1989) Physiological regulation of thyrotropin. Biochimie, I 1 : 293. Brabant, G., Frank, K., Ranft, U., et al. (1990) Physiological regulation of circadian and pulsatile thyrotropin secretion in normal men and women. J. Clin. Endocrinol. Metab., 70: 403. Brent, G.A. and Hershman, J.M. (1986) Thyroxine therapy in patients with severe non-thyroidal illness and low serum thyroxin concentration. 1. Clin. Endocrinol. Metab., 63: 1. Burman, K.D., Smallridge, R.C., Burge, J.R., Carlson, D. and Wartofski, L. (1983) Ipodate restores the fasting-induced decrement in thyrotropin secretion. J. Clin. Endocrinol. Metab., 57: 597. Casanueva, F.F., Betti, R., Cella, S.G., Muller, E.E. and Mantegassa, P. (1983) Effects of agonists of cholinergic neurotransmission on growth hormone release in the dog. Acf a Endocrinologica, 103: 15- 20. Casanueva, F.F., Villanueva, L., Cabranes, J.A., CabezasCervato, J. and Fernandez-Cruz, A. (1984) Cholinergic mediation of growth hormone secretion elicited by arginine, clonidine and physical exercise in man. J. Clin. Endocrinol. Metab., 59: 526- 530. Ceda, G.P. and Hoffman, A.R. (1985) Growth hormonereleasing factor desensitisation in rat anterior pituitary cells in vitro. Endocrinology, 116: 1334- 1340. Crowley, W.F., Filicori, M., Spratt, D.I. and Santoro, N.F. (1985) The physiology of gonadotrophin-releasing hormone (GnRH) secretion in men and women. In: R.O. Creep (Ed.), Recent Progress in Hormone Research, Academic Press, Orlando, FL. Delitala, G . , Tomasi, P. and Virdis, R. (1987)Prolactin, growth hormone and thyrotropin-thyroid hormone secretion during stress states in man. Bailliere’s Clin. Endocrinol. Metab., 1: 391. Dieguez, C., Foord, S.M., Shewring, A.G., et al. (1984) The effects of long-term growth hormone releasing factor (GRF 140)administration on growth hormone secretion and synthesis in vitro. Biochem. Biophys. Res. Comm., 121: 111 - 117. Dieguez, C., Page, M.D. and Scanlon, M.F. (1988) Growth hormone neuroregulation and its alteration in disease states. Clin. Endocrinol., 28: 109- 143. Dubuis, J.M., Dayer, J.M., Siegrist-Kaiser, C.A. and Burger, A.G. (1988) Human recombinant interleukin-10 decreases plasma thyroid hormone and thyroid stimulating hormone levels in rats. Endocrinology, 123: 2175. Edwards, C.A., Dieguez, C., Ham, J., Peters, J.R. and Scanlon, M.F. (1988) Evidence that growth hormone depletion and uncoupling of the regulatory protein of adenylate cyclase (N,) both contribute to the desensitisation of growth hormone responses to growth hormone releasing factor. J. Endocrinol., 116: 185- 190. Evans, P.J., Weeks, I., Jones, M.K., Woodhead, J.S. and Scanlon, M.F. (1986) The circadian variation of thyrotropin in

patients with primary thyroidal disease. Clin. Endocrinoi., 24: 343 - 348. Faber, J., Kirkegaard, C., Rasmussen, B., Westh, H., BuschSorensen, M. and Jensen, I.W. (1987)Pituitary-thyroidaxis in critical illness. J. Clin. Endocrinol. Metab., 65: 315. Finucane, P., Rudra, T., Church, H., et al. (1989) Thyroid function tests in elderly patients with and without an acute illness. Age Ageing, 18: 398 - 402. Foord , S.M., Peters, J.R., Dieguez, C., Shewring, A.G., Hall, R. and Scanlon, M.F. (1985) TSH regulates thyrotroph responsiveness t o dopamine in vitro. Endocrinology, 1 18: 1319- 1326. Fukuda, H., Greer, M.A., Roberts, L., Greer, S.A. and Panton, P. (1977) The effect of constant illumination on the circadian rhythms of plasma thyrotropin and corticosterone and on the estrous cycle in the rat. Endocrinology, 101: 1304. Golstein, J., Vanhaelst, L., Bruno, O.D. and L’Hermite, M. (1 979) Effect of cyproheptadine on thyrotrophin and prolactin secretion in normal man. Acta Endocrinol. (Copenh.), 92: 205. Gordon, J., Morley, J.E. and Hershman, J.M. (1980) Melatonin and the thyroid. Horm. Metab. Res., 12: 71. Greenspan, S.L., Klibanski, A., Schoenfeld, D. and Ridgway, E.C. (1986) Pulsatile secretion of thyrotropin in man. J. Clin. Endocrinol. Metab., 63: 661. Hamblin, P.S., Dyer, S.A., Mohr, V.S., et al. (1986) Relationship between thyrotropin and thyroxine changes during recovery from severe hypothyroxinemia of critical illness. J. Clin. Endocrinol. Metab., 62: 717. Hugues, J., Burger, A.G., Pekary, A.E. and Hershman, J.M. (1984) Rapid adaptations of serum thyrotropin, triiodothyronine and reverse triiodothyronine levels to shortterm starvation and refeeding. Acta Endocrinol. (Copenh.), 105: 194. Hugues, J.N., Enjalbert, A., Moyse, E.,etal. (1986)Differential effects of passive immunisation with somatostatin antiserum on adenohypophyseal hormone secretions in starved rats. 1. Endocrinol., 109: 169. Jackson, I.M.D. (1982) Thyrotropin releasing hormone. N. Engl. J. Med., 306: 145. Jordan, V., Dieguez, C., Rodriguez-Arnao, M.D., Gomez-Pan, A., Hall, R. and Scanlon, M.F. (1986) Influence of dopaminergic, adrenergic and cholinergic blockade and TRH administration on GH responses to GRF 1-29. Clin. Endocrinol., 24: 291 - 298. Judd, A.M. and Hedge, G.A. (1982) The roleof opioid peptides in controlling thyroid stimulating hormone release. Lsfe Sci., 31: 2529. Kabadi, U.M., et al. (1984) Impaired pituitary thyrotroph function in uncontrolled type I1 diabetes mellitus: normalization on recovery. J. Clin. Endocrinol. Metab., 59: 521. Kehlet, H., Klauber, P.V. and Weeke, J. (1979) Thyrotropin, free and total triiodothyronine, and thyroxine in serum during surgery. Clin. Endocrinol., 10: 131.

28 Leveston, S.A. and Cryer, P.E. (1980) Endogenous cholinergic modulation of growth-hormone secretion in normal and acromegalic humans. Metabolism, 29: 703 - 706. Loosen, P.T. (1988) Thyroid function in affective disorders and alcoholism. Endocrinol. Metab. Clin. North A m , , 17: 55. Massara, F., Ghigo,E., Goffi,S., Molinatti, G.M., Muller, E.E. and Camanni, F. (1984) Blockade of hp-GRF-40-induced GH release in normal men by a cholinergic muscarinic antagonist. J. Clin. Endocrinol. Metab., 59: 1025 - 1026. Massara, F., Ghigo, E., Molinatti, P., et al. (1986a) Potentiation of cholinergic tone by pyridostigmine bromide re-instates and potentiates the growth hormone responsiveness to intermittent administration of growth hormone-releasing factor in man. Acta Endocrinol., 113: 12- 16. Massara, F., Ghigo, E., Demislis, K., et al. (1986b) Cholinergic involvement in the growth hormone releasing hormoneinduced growth hormone release: studies in normal and acromegalic subjects. Neuroendocrinology, 43: 670 - 675. Melmed, S. (1988) Pituitary growth factors. In: Neuroendocrine Perspective, Vol. 6, Springer, New York, pp. 27 - 46. Moore, R.Y. (1983) Organization and function of a nervous system circadian oscillator. Fed. Proc., 42: 2783. Mora, B., Hassanyeh, F., Schapira, K., et al. (1980) Calorie restriction, thyroid status and inhibitory dopaminergic control of thyrotrophin secretion in man. In: J.R. Stockigt and S. Nagataki (Eds.), Proceedings of the Australian Academy of Science - Thyroid Research VIII, Proceedings of the VIIIth International Thyroid Congress, pp. 59 - 61. Morley, J.E. (1981) Neuroendocrine control of thyrotropin secretion. Endocrine Rev., 2: 396. Murakami, M., Tanaka, K. and Greer, M.A. (1988) There is a nyctohemeral rhythm of type I1 iodothyronine 5 '-deiodinase activity in rat anterior pituitary. Endocrinology, 123: 1631. Ozawa, M., Sato, K., Han, D.C., Kawakami, M., Tsushima, T. and Shizume, K. (1988) Effects of tumor necrosis factorcdcachectin on thyroid hormone metabolism in mice. Endocrinology, 123: 1461. Page, M.D., Koppeschaar, H.P.F., Dieguez, C., et al. (1987a) Cholinergic rnuscarinic receptor blockade with pirenzepine abolishes slow wave sleep-related growth hormone release in young patients with insulin-dependent diabetes mellitus. Clin. Endocrinol., 26: 355 - 359. Page, M.D., Koppeschaar, H.P.F., Edwards, C.A., et al. (1987b) Additive effects of growth hormone releasing factor and insulin hypoglycaemia on growth hormone release in man. Clin. Endocrinol., 26: 589 - 595. Page, M.D., Dieguez, C., Valcavi, R., Edwards, C., Hall, R . and Scanlon, M.F. (1988) Growth hormone (GH) responses to arginine and L-dopa alone and after GHRH pretreatment. Clin. Endocrinol., 28: 551 -558. Page, M.D., Bevan, J.S., Dieguez, C., Peters, J.R. and Scanlon, M.F. (1989a) Cholinergic blockade with pirenzepine improves carbohydrate tolerance and abolishes the G H response to meals in normal subjects. Clin. Endocrinol., 30: 519 - 524.

Page, M.D., Dieguez, C. and Scanlon, M.F. (1989b) Neuroregulation of growth hormone secretion. In: R.B. Heap (Ed.), Biotechnology in Growth Regulation, Butterworth Scientific, London, pp. 47 - 55. Parker, D.C., Rossman, L.G., Pekary, A.E. and Hershman, J.M. (1987) Effect of 64-h sleep deprivation on the circadian wave form of thyrotropin (TSH): further evidence of sleeprelated inhibition of TSH release. J. Clin. Endocrinol. Metab., 64: 157. Perez Lopez, F., Gonzalez Moreno, C.M., Abos, M.D., Andonegui, J.A. and Corvo, R.H. (1982) Pituitary responses to a dopamine antagonist at different times of the day in normal women. Acta Endocrinol. (Copenh.), 100: 481. Peters, J.R., Evans, P. J., Page, M.D., et al. (1986) Cholinergic muscarinic receptor blockade with pirenzepine abolishes slow wave sleep-related growth hormone release in normal adult males. Clin. Endocrinol., 25: 213 - 217. Plotsky, P.M. and Vale, W.W. (1985) Patterns of growth hormone-releasing factor and somatostatin secretion into the hypophyseal-portal circulation of the rat. Science, 230: 461 -463. Pokroy, N., Epstein, S., Hendricks, S. and Pimstone, B. (1974) Thyrotropin response to intravenous thyrotropin releasing hormone in patients with hepatic and renal disease. Horm. Metab. Res., 6 : 132. Re, R.N., Kourides, LA., Ridgway, E.C., Weintraub, B.D. and Maloof, F. (1976) The effect of glucocorticoid administration on human pituitary secretion of thyrotropin and prolactin. J. Clin. Endocrinol. Metab., 43: 338. Rojdmark, S. (1983) Are fasting-induced effects on thyrotropin and prolactin secretion mediated by dopamine? J. Clin. Endocrinol. Metab., 56: 1262. Romijn, J.A. and Wiersinga, W.M. (1990) Decreased nocturnal surge of thyrotropin in non-thyroidal illness. J. Clin. Endocrinol. Metab., 7 0 35. Romijn, J.A., Adriaanse, R., Brabant, G . , Prank, K., Endert, E. and Wiersinga, W.M. (1990) Pulsatile secretion of thyrotropin during fasting: a decrease of thyrotropin pulse amplitude. J. Clin. Endocrinol. Metab., 70: 1631. Rose, S.R. and Nisula, B.C. (1989) Circadian variation of thyrotropin in childhood. J. Clin. Endocrinol. Metab., 68: 1086. Ross, R.J.M., Borges, F., Grossman, A., et al. (1987a) Growth hormone pretreatment in man blocks the response to growth hormone-releasing hormone: evidence for a direct effect of growth hormone. Clin. Endocrinol., 26: 117 - 123. Ross, R.J.M., Tsagarakis, S., Grossman, A., et al. (198713) GH feedback occurs through modulation of hypothalamic somatostatin under cholinergic control: studies with pyridostigmine and GHRH. Clin. Endocrinol., 27: 727 - 734. Rossmanith, W.G., Mortola, J.F., Laughlin, G.A. and Yen, S.S. (1988) Dopaminergic control of circadian and pulsatile pituitary thyrotropin release in women. J. Clin.Endocrinol. Metab., 67: 560.

29 Salvador, J., Wilson, D.W., Harris, P.E., et al. (1985)Relationships between the circadian rhythms of TSH, prolactin and cortisol in surgically treated microprolactinoma patients. Clin. Endocrinol., 22: 265 - 272. Salvador, J., Dieguez, C. and Scanlon, M.F. (1988) The circadian rhythms of thyrotropin and prolactin secretion. Chronobiol. Intern., 5: 85 - 93. Samuels, M.H., Veldhuis, J.D., Henry, P. and Ridgway, E.C. (1990) Pathophysiology of pulsatile and copulsatile release of thyroid-stimulating hormone, luteinising hormone, folliclestimulating hormone and a-subunit. J. Clin. Endocrinol. Metab., 71: 425. Scanlon, M.F. (1991) Neuroendocrine control of thyrotropin secretion. In: L. Braverman and R.D. Utiger (Eds.), The Thyroid: a Fundamental and Clinical Text, 6th edition, Lippincott, U.S.A. Scanlon, M.F., Weetman, A.P., Lewis, M., et al. (1980) Dopaminergic modulation of circadian thyrotropin rhythms and thyroid hormone levels euthyroid subjects. J. Clin. Endocrinol. Metab., 51: 1251 - 1256. Sheppard, M.C., Kronheim, S. and Pimstone, B.L. (1978) Stimulation by growth hormone of somatostatin release from therat hypothalamusinvitro. Clin. Endocrinol., 9: 583 - 586. Shibasaki, T., Hotta, M., Masuda, A., et al. (1985) Plasma GH responses to GHRH and insulin-induced hypoglycaemia in man. J. Clin. Endocrinol. Metab., 60: 1265 - 1267. Small, M., Cohen, H.N., McLean, J.A., Beastall, G.H. and McCuish, A.C. (1986) Impaired thyrotropin secretion following the administration of thyrotropin-releasing hormone in type I1 diabetes mellitus. Postgrad. Med. J., 62: 445. Souetre, E., Salvati, E., Wehr, T.A., Sack, D.A., Krebs, B. and Darcourt, G. (1988) Twenty-four hour profiles of body temperature and plasma TSH in bipolar patients during depression and during remission and in normal control subjects. Am. J. Psychiatry, 145: 1133. Sowers, J.R., Carlson, H.E., Brautbar, N. andHershman, J.M. (1977) Effect of dexamethasone on prolactin and TSH responses to TRH and metoclopramide in man. J. Clin. Endocrinol. Metab., 44: 237. Sowers, J.R., Catania, R.A. and Hershman, J.M. (1982) Evidence for dopaminergic control of circadian variations in thyrotropin secretion. J. Clin. Endocrinol. Metab., 54: 673. Spencer, C.A., Lun, S.M.C., Wilburn, J.F., Kaptein, E.M. and Nicoloff, J.T. (1983) Dynamics of serum thyrotropin and thyroid hormone changes during fasting. J. Clin. Endocrinol. Metab., 56: 883. Stein, D. and Avni, J. (1988) Thyroid hormones in the treatment of affective disorders. Acta Psychiatr. Scand., 77: 623. Strassman, R.J., Peake, G.T., Qualls, C.R. and Lisansky, E. J. (1988) Lack of an acute modulatory effect of melatonin on human nocturnal thyrotropin and cortisol secretion. Neuroendocrinology, 48: 387. Valcavi, R., Dieguez, C., Azzarito, C., Artioli, C., Portioli, I. and Scanlon, M.F. (1987) Alpha-adrenoreceptor blockade

with thymoxamine reduces basal thyrotrophin levels but does not influence circadian thyrotrophin changes in man. J. Endocrinol., 115: 187- 191. Valcavi, R., Dieguez, C., Page, M.D., et al. (1988) Alpha-2adrenergic pathways release growth hormone via a non-GRFdependent mechanism in normal human subjects. Clin. Endocrinol., 29: 309- 316. Vance, M.L., Kaiser, D.L., Evans, W.S., et al. (1985) Evidence for a limited growth hormone (GH)-releasing hormone (GHRH)-releasable quantityof GH: effects of 6-hinfusions of GHRH on GH secretion in normal man. J. Clin. Endocrinol. Metab., 60: 370- 375. Vance, M.L., Kaiser, D.L., Frohman, L.A., Rivier, J., Vale, W.W. and Thorner, M.O. (1987) Role of dopamine in the regulation of growth hormone-stimulated GH secretion in normal man. J. Clin. Endocrho[. Metab., 6 4 1136- 1141. Van Coeverden, A., Laurent, E., De Coster, C., et al. (1989) Decreased basal and stimulated thyrotropin secretion in healthy elderly men. J. Clin. Endocrinol. Metab., 69: 177. Vanhaelst, L., Van Cauter, E., Degaute, J.P. and Golstein, J. (1972) Circadian variations of serum thyrotrophin levels in man. J . Clin. Endocrinol. Metab., 35: 479. Vierhapper, H., Laggner, A., Waldhausl, W., GrubeckLoebenstein, B. and Kleinberger, G. (1982) Impaired section of TSH in critically ill patients with “low-T, syndrome”. Acta Endocrinol. (Copenh.), 101: 542. Vinik, A.I., Kalk, W.J., McLaren, H., Henrick, S. and Pimstone, B.L. (1975) Fasting blunts the TSH response to synthetic thyrotropin-releasing hormone (TRH). J. Clin. Endocrinol. Metab., 40: 509. Wartofsky, L. and Burman, K.D. (1982) Alterations in thyroid function in patients with systemic illness: the “euthyroid sick syndrome”. Endocrine Rev., 3: 164. Weeke, J. and Laurberg, P. (1976) Diurnal TSH variations in hypothyroidism. J. Clin. Endocrinol. Metab., 43: 32. Wehman, R.E., Gregerman, R.I., Burns, W.H., Saral, R. and Santos, G.W. (1985) Suppression of thyrotropin in the lowthyroxine state of severe non-thyroidal illness. N . Engl. J. Med., 312: 546. Zalaga, G.P., Chernow, B., Smallridge, R.C., et al. (1985) A longitudinal evaluation of thyroid function in critically ill surgical patients. Ann. Surg., 201: 456.

Discussion J.M. Palacios: Pirenzepine, a selectivemuscarinic M 1 antagonist does not cross the blood-brain barrier. Where do you think the interaction between somatostatin and cholinergic muscarinic receptors takes place? M.F. Scanlon: It is likely, I think, that cholinergic mechanisms modulate somatostatin release from somatostatinergic nerve terminals at the level of the median eminence, which lies outside the blood-brain barrier. This anatomical site is clearly of crucial im-

30 portance in the integration of many neuroendocrine and peripheral signals. J.M. Palacios: Are you aware of any study using selective M2 cholinergic muscarinic antagonists? M.F. Scanlon: No, 1 am not aware of any such study. W.A. Scherbaum: Thecatecholaminergic systemmay beinvolved in type 1 diabetes mellitus at the level of the hypothalamus. We have described autoantibodies to adrenal medullary cells in patients with newly diagnosed diabetes mellitus (Scherbaum et al., 1988) and Dr. Brown from the U.S.A. has described the loss of such cells in theadrenal medulla of patients with type 1 diabetes. Does a similar process take place at hypothalamic level? Also, is the excess GH secretion in diabetes due to an involvement of catecholaminergic pathways? M.F. Scanlon: I really cannot say whether any autoimmune mechanism is directed against hypothalamic catecholaminergic systems in diabetes. However, there is evidence pointing to disturbances in the hypothalamic “cholinergic-somatostatinergic” system and its interaction with glucose in diabetes. In my view there are several possible mechanistic hypotheses for this which should be studied before necessarily involving autoimmunity. With regard to your second question, from the available data it is likely that GH hypersecretion in diabetes is secondary to reduced somatostatin release or action, perhaps coupled with reduced peripheral negative feedback via IGF-I . It appears that altered signalling between cholinergic and somatostatinergic systems and glucose is also involved in this process. If catecholaminergic systems are involved then they are probably overactive

(stimulatory to GH) and this is difficult to reconcile with a destructive autoimmune hypothesis. H.P.H. Kremer: Can diabetic autonomic neuropathy explain the excessive GH release in diabetic patients? M.F. Scanlon: This is possible but there are no data. Certainly counterregulatory hormone responses can be grossly disturbed in diabetes and vagal autonomic neuropathy may play a role in this. It is possible that such neuropathic disturbances may extend to certain intrinsic hypothalamic systems in diabetes but this would be difficult to establish at present. G.A. Bray: Can you elaborate further on the control of the menstrual cycle. What is the signal which initiates puberty and how it is related to the Frisch hypothesis which suggests an effect of body weight or body fat? M.F. Scanlon: Puberty in females is highly complex with clear developmental stages which are temporally separated and include thelarche, menarche, pubarche and adrenarche. Many signals are probably involved in such coordinated development. It is equally certain that body weight has a profound deterministic influence on normal pubertal development but unfortunately the mediating signals from the periphery (fat mass or distribution of lean body weight of fat/lean ratio) to the hypothalamic LHRH systems and pulse generator are completely unknown. Indeed, whether such feedback is primarily neural or humoral is unclear. The delineation of such signals is clearly important in the further understanding of normal physiology and has enormous therapeutic implications.

D.F.Swaab, M.A. Hofman, M. Mirmiran, R. Ravid and F.W. van Leeuwen (Eds.) Progress in Brain Research, Vol. 93 0 1992 Elsevier Science Publishers B.V. All righrs reserved.

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CHAPTER 3

Neurologic manifestations of hypothalamic disease Joseph B. Martin’ and Peter N. Riskind2

’Department of Neurology, University of California, San Francisco School of Medicine, San Francisco, CA 94143, U.S.A.; and 2Neurology Service, Massachusetts General Hospital, Boston, MA 02114, U.S.A.

Introduction

Case descriptions

The hypothalamus serves a crucial function in the regulation of anterior and posterior pituitary secretion. In addition, the hypothalamus controls water balance, regulates the ingestion of food, determines body temperature and circadian rhythms, and influences sleep, emotional state and behavior. In 1954 Bauer described the symptoms and signs of hypothalamic disease in 60 autopsy-proven cases (Bauer, 1954). In this series, 43 had sexual abnormalities (hypogonadism or precocious puberty), 21 had diabetes insipidus, 21 had psychic disturbances, 20 obesity or hyperphagia, 18 somnolence, 15 emaciation or anorexia, and 13 disorders of temperature regulation. This breakdown of symptoms has remained an important documentation of the clinical manifestations of lesions in the hypothalamus. Clinical pathologic correlations have been useful in illustrating the importance of the hypothalamus in regulating these various somatic functions. Unfortunately, however, because the hypothalamus is small and lesions often develop slowly, they commonly achieve considerable size before their recognition, making precise local anatomic dissections of the effects of these lesions difficult (Plum and Van Uitert, 1978). Nevertheless, the use of modern noninvasive techniques including computerized tomographic (CT) scans and magnetic resonance imaging (MRI), has provided new information not previously available. This report will include the description of several cases recently seen in the Neuroendocrine Clinic at the Massachusetts General Hospital in Boston.

Case 1 * A 30-year-old black right-handed printer (E.C.) with previous history of cocaine abuse, complained of a 1 year history of weight gain (from less than 200 lb. to 280 lb.), several months of fatigue, forgetfulness, irritability, loss of libido, and 2 months of visual loss (right eye “blurring”), polydipsia (“gallons a day”), polyuria, nocturia 3 - 4 times nightly, and bifrontal headache. He claimed not to have used drugs for several months. Physical examination was notable for childish, inappropriate behavior (he put his arm around the examiner and farted without apparent embarrassment). He was alert, oriented, and attentive, but had poor insight. There was a dense superionasal field cut in the right eye and slight superior depression of the visual field in the left eye. A mild afferent. pupillary defect was present in the right eye, a slight left lower facial droop, mild left pronator drift, lift-sided hyperreflexia, and a positive grasp response on the left. He was markedly obese and had a small testes (12 ml; normally 15 - 20 ml). Endocrine evaluation revealed persistent hyponatremia (Na 136 mEq/l; normally 140 - 145 mEq/l) and panhypopituitarism. CT demonstrated an invasive contrast-enhancing mass involving much of the hypothalamus and extending into the anterior interhemispheric fissure and inferomedial

* Reported in “Case records of the Massachusetts General Hospital” (Reichlin and Vonsattel, 1991).

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frontal lobe on the right, and also involving the pituitary stalk (Fig. 1A). Lumbar puncture showed no cells, moderately increased cerebrospinal fluid protein and normal glucose. Chest CT showed an anterior mediastinal mass. Angiotensin-converting enzyme level (ACE) was modestly increased to 65 (15 - 45 normally). Biopsy of a subcarinal lymph node revealed multiple non-caseating granulomata consistent with sarcoidosis. He was treated with prednisone 60 mg qd with rapid improvements in vision, and left-sided weakness and resolution of the grasp response. Throughout his initial and subsequent hospitaliza-

A

tions he engaged in aggressive “food-seeking behavior”; he was observed to take food from the hospital cafeteria without paying (intimidating cashiers with his sheer size), stole food from his hospital roommates and from the nurses’ lockers, and even learned to go on food-seeking “sprees” when the ward staff were distracted during cardiac resuscitations. He spent much of the day and night sleeping. His thirst, hunger, and drowsiness failed to respond to steroids. His weight increased to 322 lb. and he began to complain of depression. Treatment with fluoxitine (60 mg qd) had no effect on his mood appetite or drowsiness. Repeat CT scan showed no change in the central nervous system mass. He was readmitted 4 month later because of uncontrollable hyperglycemia (to greater than 500 mg%) and weight gain (to 332 lb.). Insulin therapy was started. A trial of prednisone (80 mg/day) was ineffective and he was treated with 2000 rad to the hypothalamic region. ACE level was 79.8. Eight months later weight was reduced to 275 lb (on pred-

Fig. 1. A . CAT scan of hypothalamic sarcoidosis. Contrast-enhanced axial CAT scan prior to treatment, demonstrating an invasive mass involving the hypothalamus, anterior hemispheric fissure, and right inferiomedial frontal lobe. B. Gadolinium-enhanced T1-weighted sagittal MRI lomonths after radiation treatment. Although the patient experienced significant clinical improvement, there is still extensive involvement of the hypothalamus, as well as the infundibulum and sellar contents.

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scanner) demonstrating a mass within the hypothalamus, extending into the infundibulum and sella, with small foci of increased T1 within the pons and temporal lobe (Fig. 1B).

Fig. 2. Contrast-enhanced axial CAT scan of pituitary tumor with suprasellar extension causing compression of the hypothalamus. Biventricular hydrocephalus has been treated by ventriculoperitoneal shunt.

nisone 25 mg qd) and he admitted that his appetite was “slightly reduced”, but complained of inadequate food at the shelter where he was living. He admitted resumption of smoking crack-cocaine. Later that month he noted decreased drowsiness and thirst. One month later his ACE level had declined to 49.4 and the following month the ACE level was 25. At this time he noted a definite reduction of appetite, thirst, and drowsiness. He weighed260lb. He experienced a marked improvement in insight and concern with regard to his illness. Insulin was discontinued because of blood sugars in the low 200’s. He continued to complain of headaches (always following smoking crack-cocaine). MRI scan was obtained (patient could now fit into the

Case 2 J.B. was a previously healthy (except for type I1 diabetes) 61-year-old right-handed engineer. For 4 - 6 months before admission he had become increasingly confused, fatigued, and “absent-minded”. He was asked to “take avacation” from work because of poor job performance. He repeatedly asked his wife to “feed the cat”, although they had not owned a cat for years. He began to nap during the day and developed polydipsia and polyuria. His appetite decreased and he complained of frontal headaches. One month before admission he became unsteady. On admission he had flat affect and was slightly slow in answering, had poor calculations and defective recent memory. There was a positive grasp response on the left and gait ataxia. Serum sodium was 132 mEq/l. Subsequent evaluation was consistent with syndrome of inappropriate antidiuretic hormone secretion (SIADH). CT demonstrated a 3 x 3 x 2 cm suprasellar mass arising from the sella with compression of the hypothalamus and biventricular hydrocephalus (Fig. 2). There was no abnormality of baseline thyroid hormone levels and serum cortisol responded normally to ACTH. Testosterone levels were reduced. His mental status improved after ventriculoperitoneal shunt and appetite transiently improved. His temperature was consistently low (e.g., 36.5”C). Two months after the first admission the tumor was partially debulked by transsphenoidal surgery and immunohistochemistry revealed many ACTH cells, consistent with “cryptic-Cushing’s disease”. He received 4500 Gy without immediate complication. Two months later he complained of a severe loss of appetite, increased fatigue and recurrent headaches. These symptoms progressed, causing a 70 lb. weight loss. He vomited occasionally. Thyroid and adrenal function were normal. CT scan demonstrated tumor in a similar distribution to preoperative studies. Subsequent studies demonstrated

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a large abdominal aortic aneurysm (7.5 cm wide) and mild elevation of liver function studies. Creatinine rose from 1.3 to 3.8 mg/100 ml, presumably as a result of cholesterol emboli to the kidneys. A right frontal craniotomy and radical partial resection of the suprasellar mass were performed because of the patient's increasing headaches, confusion, and anorexia. Following the surgery his body temperature fell to 32.2"C despite a warming blanket but later rose to 36"C, still with a warming blanket. Severaldays after surgery he was more alert and was feeding himself, admitting to an increased appetite. One week later he deteriorated suddenly and died as a result of rupture of the aortic aneurysm.

Case 3 J.S., a 78-year-old man with a history of transitional cell cancer of the bladder 6 years before, was otherwise in excellent health (he regularly played tennis and spoke at political meetings). Over a 4 week period he became increasingly confused, diffusely weak and uncoordinated. He complained of cold intolerance and constipation, increased thirst, and slurred speech. He became acutely stuporous over a 2 day period and was transferred to the hospital. Evaluation 2 weeks before had demonstrated normal thyroid function. Lumbar puncture showed an opening pressure of 160 mm H,O, no cells, a protein of 225 mg/dl (normally less than 45 mg/dl), and a glucose of 41 mg/dl. CT demonstrated a 1.2 x 2 x 2 cm suprasellar mass extending into the hypothalamus. Evaluation later showed a stuporous man who would briefly mutter in response to stimulation. Rectal temperature was 34.4"C. Endocrine studies showed mild hypothyroidism, with a free T4 of 0.6 mg/dl (normally 0.73 - 1.94), prolactin 26.8 ng/ml (normally less than 10 ng/ml), testosterone 16 mg/dl (normally 300- 1100 mg/dl), and cortisol 7.3 mg/dl (with previous normal ACTH stimulation studies). Repeat CT showed biventricular hydrocephalus with a small third ventricle. He had marked improvement in alertness after ventriculoperitoneal shunting. Although he initially had low serum sodium (128 meq/l), he subsequently developed diabetes insi-

pidus requiring treatment with desamino-D-arginine vasopressin (DDAVP). Lumbar puncture 6 days after admission showed six white blood cells (90Vo lymphs; 4% monocytes, 6% non-hemantic), protein 262 mg/dl and glucose 63 mg/dl. Repeat lumbar puncture 2 days later showed 24 white blood cells with abnormal, large lymphocytes having abundant cytoplasm and folded nuclei (consistent with lymphoma). Protein was 97 mg/dl and glucose 74 mg/ dl. His temperature rose to 38.5"C without a clear source of infection; he remained confused, dysarthric and disoriented. His fever spontaneously resolved after several days and he became more alert. He was emotionally labile and cried easily, but was generally pleasant. His recent memory was poor but remote memory was relatively preserved. He had bilateral grasp responses. Proton density MRI demonstrated a diffuse, symmetric bright signal abnormality centered within the hypothalamus, extending into the thalamus, internal capsule and midbrain (Fig. 3). A presumptive diagnosis of primary CNS lymphoma was made and it was elected to treat him with three courses of high-dose methotrexate. MRI scan 2 months after admission showed decreased edema but persistence of two symmetric T2bright lesions in the hypothalamus. MRI scan after the third course of methotrexate showed no demonstrable tumor. Examination 2 months after completion of methotrexate showed improved memory and insight, although his wife noted that his recent memory had not fully recovered. Two years later he developed recurrent transitional carcinoma in the right kidney and expired. At autopsy he was found to have moderate gliosis of the posterior hypothalamus and mammillary bodies bilaterally, consistent with treated lymphoma; he also had a small hamartoma of the posterior hypothalamus and numerous neurofibrillary tangles were seen in the hippocampi.

Case 4 M.W. was a 32-year-old woman librarian who gradually developed increasing fatigue, memory loss, reduced libido, urinary frequency and somnolence in association with cessation of menstrua-

35

tion. Although no change in appetite had been noted, she had gained 15 Ibs. over the year prior to admission. A CT scan was performed at the insistence of her mother, demonstrating a large suprasellar mass with compression of the hypothalamus and obliteration of the third ventricle, but with preservation of normal sellar contents. Stereotactic biopsy demonstrated pituitary adenoma. For 1 week following the stereotactic biopsy there was a marked change in personality, with aggressive unpredictable and impulsive behavior, and she was more irritable than normal for several weeks post-operatively. She was endocrinologically normal except for amenorrhea and chronic hyponatremia, consistent with SIADH. She was treated with 4500 Gy conventional radiation therapy and initially improved with regard to memory loss, energy level and drowsiness as her steroid treatment was tapered. Two months after

completion of the radiation treatment she deteriorated behaviorally, with hyperphagia, belligerence, confusion, hallucinations and disorientation. She confabulated frequently. There was mild left-sided weakness and hyperreflexia. Repeat CT scan did not demonstrate any change in the tumor. Increased prednisone treatment improved urinary frequency but had no beneficial effect on memory loss or other behavioral disturbances. Her appetite increased dramatically, particularly for sweets, and she became much more belligerent. At times she seemed paranoid. These symptoms persisted after a reduction of steroids and a change to every other day therapy. A trial of bromocriptine was associated with partial central necrosis within the tumor but without any reduction in tumor diameter or improvement in mental status. One year after completion of radiation therapy she became

Fig. 3. T1-weighted coronal MRI of presumptive primary CNS lymphoma before treatment with methotrexate. There is diffuse involvement of the hypothalamus, with extension into the thalamus and midbrain bilaterally, and biventricular hydrocephalus.

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acutely stuporous. Temperature was 34.4”C and serum sodium 120 meq/l. CT showed a 4 cm diameter tumor without gross change in size; MRI scan showed increased T2 signal in the white matter surrounding the tumor (edema or radiation effect) and in the temporal lobes bilaterally. Alertness improved with fluid restriction and near-normalization of serum sodium, but she had persistently poor recent memory, confusion, and confabulation. She became progressively less interested in her surroundings and showed progressively less spontaneous emotion. Eight months later she was admitted with acute third ventricular obstruction and received a ventriculoperitoneal shunt. During that hospitalization a urinary tract infection was accompanied by a rise in basic body temperature from 35.2”C to 37.2”C. Over the subsequent 3 years she has had multiple episodes of shunt dysfunction requiring shunt revisions, although there has been no visible growth of the tumor (Fig. 4). Speech has become progressively more sparse and lower in amplitude. On more than three occasions she has had a dramatic transient improvement in response to infusion of hypertonic contrast agents for CT scans. Beginning within 30 min of the infusion she would become much more alert and spontaneous. She asked “am I still married” and “what has happened to me over the past 2 years?” This improvement typically lasted 6 h and gradually diminished over the next 18 h. Treatment with intravenous mannitol did not prolong the response. The patient’s mother has maintained her body temperature by means of an electric heating blanket at night and multiple layers of clothing during the day, monitoring her temperature frequently. About 4 years after completion of radiation therapy she developed a progressively higher requirement for support of her body temperature. This has been recognized by her mother because it is necessary to utilize higher settings on an electric blanket at night than before. She has similarly controlled the patient’s water intake, maintaining a careful fluid restriction and monitoring her serum sodium levels in association with her physician. Food intake has been closely controlled since the patient has lost satiety. Interestingly, the patient’s

memory is best preserved for events relating to meals. For example, she is able to recall particular meals with greater facility than other recent events. She has developed progressive urinary incontinence, which is controlled by regular voiding as instituted by her mother.

Discussion The neurologic manifestations of hypothalamic disease are summarized in Table I. The etiology of hypothalamic disease is illustrated in Table 11.

Fig. 4. Gadolinium-enhanced T1-weighted coronal MRI of a massive suprasellar and intrahypothalamic pituitary tumor. Preservation of normal pituitary fossa structures suggests that the tumor originated outside the sella.

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Disorders of thermal regulation Disturbances of temperature regulation in the human are rather common symptoms of hypothalamic disease. Acute lesions can cause hyperthermia but more commonly chronic, slowly growing lesions induce hypothermia. In a patient presenting with a hypothalamic mass and hypothermia, central thyroid disregulation with hypothyroidism needs to be excluded by the appropriate tests. In euthyroid patients with structural diseases of the brain, hypothermia can occur either as a chronic condition or as periodic hypothermia. Periodic hypothermia with normal body temperature during intervals between attacks is well documented (LeWitt et al., 1983). Autonomic nervous system activities accompany the episodes of hypothermia including vasodilatation, nausea, vomiting, lacrimation, sweating, salivation and cardiovascular effects (bradycardia) (Penfield, 1929; Guihard et al., 1971; Fox et al., 1973; Noel et al., 1973; Solomon 1973). Shivering

may coincide with return of the body temperature toward normal. The episodes last for minutes or hours and the rectal temperature may drop below 30°C. Seizures may occur, probably related to other commonly associated developmental abnormalities in the cortex. Ten of 16 reported cases had agenesis of the corpus callosum. The majority of patients with agenesis of the corpus callosum do not develop spontaneous periodic hypothermiaand it is assumed, based upon relatively limited pathological examinations, that lesions in the hypothalamus coexist with the abnormalities in the development of the corpus callosum. Neuroendocrine disorders may accompany a spontaneous periodic hypothermia. These reports include precocious puberty, diabetes insipidus, growth hormone deficiency and hypogonadism (Guihard et al., 1971; Fox et al., 1973; Noel et al., 1973; Solomon, 1973). Hyperthermia is more commonly a manifestation of acute neurologic damage as occurs with sub-

TABLE I Neurologic manifestations of hypothalamic disease Disorders of temperature regulation Hyperthermia Hypothermia Poikilothermia Disorders of food intake Hyperphagia (bulimia) Anorexia, aphagia Disorders of water intake Compulsive water drinking Adipsia or hypodipsia Essential hypernatremia Disorders of sleep and consciousness Somnolence Sleep-rhythm reversal Akinetic mutism Coma Disorders of psychic function Rage behavior Hallucinations From Martin and Reichlin, 1987.

Periodic disease of hypothalamic origin Diencephalic epilepsy Kleine-Levin syndrome Periodic discharge syndrome of Wolff Narcolepsy Disorders of the autonomic nervous system Pulmonary edema Cardiac arrhythmias Sphincter disturbance Hereditary hypothalamic disease Laurence-Moon-Biedl syndrome de Morsier’s syndrome Kallmann’s syndrome Miscellaneous Prader-Willi syndrome Diencephalic syndrome of infancy Cerebral gigantism

38 TABLE I1 Etiology of hypothalamic disease by age ( I ) Premature infants and neonates Intraventricular hemorrhage Meningitis: bacterial Tumors: glioma, hemangioma Trauma Hydrocephalus, kernicterus

(2) I month to 2 years Tumors Glioma, especially optic glioma Histocytosis X Hemangiomas Hydrocephalus, meningitis “Familial” disorders: Laurence-Moon-Biedl, Prader-Labhart-Willi

(3) 2 - I0 years Tumors Craniopharyngioma Glioma, dysgerminoma, hamartoma, histiocytosis X, leukemia Ganglioneuroma, ependymoma, medulloblastoma Meningitis Bacterial Tuberculous Encephalitis Viral and demyelinating Various viral encephalides and exanthematous demyelinating encephalides Disseminated encephalomyelitis “Familial” disorders: diabetes insipidus, etc. (4) I0 - 25 years Tumors Craniopharyngioma Glioma, hamartoma, dysgerminoma Histiocytosis X, leukemia Dermoid, lipoma, neuroblastoma Trauma Subarachnoid hemorrhage, vascular aneurysm, arteriovenous malformation Inflammatory diseases, meningitis, encephalitis, sarcoid, tuberculosis Associated with midline brain defects; agenesis of corpus callosum Chronic hydrocephalus or increased intracranial pressure (5) 25 - 50 years

Nutritional: Wernicke’s disease Tumors Sarcoma, glioblastoma, lymphoma Meningioma, colloid cysts, ependymoma, pituitary tumors Vascular Infarct, subarachnoid hemorrhage Pituitary apoplexy Inflammation: encephalitis, sarcoid, meningitis From Plum and Van Uitert, 1978, with permission.

39

arachnoid hemorrhage or trauma. Rarely, periodic hyperthermia may occur as in the famous case reported by Wolff (Wolff and Adler, 1964).

Cardiovascular manifestations of hypothalamic disease Acute lesions of the brain, particularly subarachnoid hemorrhage or increased intracranial pressure are commonly accompanied by cardiac disturbances. Supraventricular tachycardia, ectopic ventricular contractions, ventricular flutter and ventricular fibrillation are reported in patients with subarachnoid hemorrhage (Talman, 1985). Electrocardiographic changes include depressed ST segments, flat or inverted T waves, peaked or notched T waves (cerebral T waves), and changes in QRS voltages. Examination of heart tissues in the face of such catastrophic central nervous system lesions have revealed focal destruction of myocardial tissue with cytolysis, myofibrillar degeneration, and histiocytic infiltration (Blum et al., 1982). Coronary artery disease is not an accompanying factor. It is believed that the cause of this condition is the release of catecholamines either systemically or from sympathetic nerve fibers in the heart. Abnormalities of food intake Lesions in the basal hypothalamus commonly result in aberrant food intake behaviors with obesity. These lesions have been demonstrated in experimental animals to involve destruction of the ventral medial nucleus of the hypothalamus (Rabin, 1972). Less commonly hypothalamic lesio& may produce anorexia. It is believed that anorexiain such cases is the result of damage to lateral structures in the basal hypothalamus. The precise mechanisms that underlie disturbances in food intake have been extensively investigated. There are peripheral (gastrointestinal factors) as well as central factors. Postcript. Patient M. W. recently expired during a hospital admission for evaluation of intermittent high fevers of unknown cause. Although previous biopsies has been interpreted as pituitary adenoma, the final pathologic diagnosis, after review of the entire tumor, was meningioma.

Summary and conclusions The hypothalamus, in addition to regulating the anterior and posterior pituitary, controls water balance through thirst, regulates food ingestion and body temperature, influences consciousness, sleep, emotion and other behaviors. Much has been learned of these effects in human disease through the clinical manifestations that occur with hypothalamic lesions. This study reviews the clinical pathologic correlations that have been made in recent years showing that regions of the hypothalamus exert functions in humans that are similar to those identified in experimental animals. Clinical pathologic correlations have not always provided precise analysis of hypothalamic function. The hypothalamus is small and often lesions that come to clinical attention achieve considerable size before their recognition, making local anatomic dissections of the effects of the lesions difficult. Nevertheless, the use of modern non-invasive techniques including CT scans and magnetic resonance imaging (MRI) have provided new information not previously available. This paper reviews several cases of hypothalamic disorder recognized recently. (1) A 33-year-old black man with hypothalamic sarcoidosis. Manifestations of hypothalamic dysfunction included panhypopituitarism, aggressive hyperphagia, polydipsia (partially due to hyperglycemia secondary to diabetes mellitus), drowsiness, depression, and irritability. (2) A 37-year-old woman with a large intrahypothalamic tumor (biopsy showed pituitary adenoma), with drowsiness, poikilothermia, lack of satiety, confusion, and memory loss. She becomes depressed when she is transiently more alert (as after hypertonic contrast-dye infusion). (3) A 60-year-old man with hypothalamic compression by a pituitary tumor, associated with syndrome of inappropriate ADH (SIADH), severe anorexia, memory loss, but preserved thirst. After surgical decompression of the tumor his appetite acutely recovered, but he developed severe hypo(poiki1o)thermia. (4) A 45-year-old woman with a suprasellar

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craniopharyngioma presented with severe drowsiness, hyperphagia, depression, and memory loss post-operatively, which responded to antidepressants (except for the memory loss). She had extremely labile blood pressures and serum Na for about 1 week post-operatively.

References Bauer, H.G. (1954) Endocrine and other manifestations of hypothalamic disease: a survey of 60 cases, with autopsies. J. Clin. Endocrinol. Metab., 14: 13 - 3 I . Blum, B., Israeli, J., Dujovny, M., Davidovich, A. and Farchi, M. (1982) Angina-like cardiac disturbances of hypothalamic etiology in cat, monkey and man. Isr. J. Med. Sci., 18: 127 - 139. Bray, G.A. and York, D.A. (1979) Hypothalamic and genetic obesity in experimental animals: an autonomic and endocrine hypothesis. Physiol. Rev., 59: 719 - 809. Fox, R.H., Wilkins, D.C., Bell, J.A., Bradley, R.D., Browse, N.L., Cranston, W.I., Foley, T.H., Gilby, E.D., Hebden, A,, Jenkins, B.S. and Rawlins, M.D. (1973) Spontaneous periodic hypothermia: diencephalic epilepsy. Br. Med. J., 2: 693 - 695. Guihard, J., et al. (1971) Hypothermie spontanke recidivante avec agenesie du corps calleau. SyndrBme de shapiro nouvelle observation. Ann. Pediatr., 18: 645 - 649. LeWitt, P.A., Newman, R.P., Greenberg, H.S., Rocher, L., Calne, D. and Ehrenkranz, J. (1983) Episodic hyperhydrosis hypothermia and agenesis of the corpus callosum. Neurology, 33: 1122- 1129. Martin, J.B. and Reichlin, S. (1987) Clinical Neuroendocrinology, 2nd edition, F.A. Davis, Philadelphia, PA. Noel, P., Hubert, J.P., Ectors, M., Franken, L. and FlamentDurand, J. (1973) Agenesis of the corpus callosum associated with relapsing hypothermia: a clinicopathological report. Brain, 96: 359- 368. Penfield, W. (1929) Diencephalic autonomic epilepsy. Arch. Neurol. Psychiatry, 22: 358 - 369. Plum, F. and Van Uitert, R:(1978) Non-endocrine diseases and disorders of the hypothalamus. In: S. Reichlin, R.J. Baldessarini and J.B. Martin (Eds.), The Hypothalamus, Vol. 56, Raven Press, New York, pp. 415 - 473. Rabin, B.M. (1972) Ventromedial hypothalamic control of food intake and satiety: a reappraisal. Brain Res., 43: 317 - 336. Solomon, G.E. (1973) Diencephalic autonomic epilepsy caused by a neoplasm. J. Pediatr., 83: 277-282. Talman, W.T. (1985) Cardiovascular regulation and lesions of the central nervous system. Ann. Neurol., 18: 1 - 12. Wolff, S.M. and Adler, R.C. (1964) A syndrome of periodic hypothalamic discharge. Am. J. Med., 36: 956 - 967. Reichlin, S. and Vonsattel, J.P. (1991) Case records of the Massachusetts General Hospital: a 30-year-old man with

polydipsia hypopituitarism, and a mediastinal mass. N . Engl. J. Med., 324: 677 - 687.

Discussion D.F. Swaab: One of your slides contained narcolepsy as a hypothalamic disease. Could you please give the evidence for that? J.B. Martin: I am not aware of any convinving evidence for direct hypothalamic involvement in narcolepsy. The disorder seems to reside in the brain-stem in regions that regulate the phases of sleep. C.B. Saper: I cannot resist one last comment with regard to your challenge for localization of hypothalamic function in humans. Some patients with Shapiro’s syndrome (agenesis of the corpus callosum and periodic hypothermia) also are severely hyponatremic during the periods of hypothermia. We recently obtained a high quality MRI scan on such a patient, which demonstrated that the anterior wall of the third ventricle (pictured on the cover of the program for this meeting) had failed to form. As both the corpus callosum and anterior wall of the third ventricle develop embryologically from the lamina terminalis, the thermoregulatory and serum sodium abnormalities in Shapiro’s syndrome may derive from failure of the rostra1 preoptic area to form normally. J.B. Martin: That is very interesting. N.E. Rance: I just wanted to add a point of clarification to the discussionof “Sheehan’s syndrome”. In 1967,Sheehanpublished a report which described enlargement of neurons in the infundibular nucleus of women suffering from post-partum pituitary necrosis with complete gonadal atrophy. So there are secondary changes in the hypothalamus of patients with Sheehan’s syndrome. J.B. Martin: Thank you for that information. J.M.B.V. De Jong: Twenty one hereditary cerebellar and spinocerebellar syndromes are preceded, accompanied or followed by hypogonadism, hyper- or hypogonadotrophic (see Table A). What evidence is available on the actual control of cerebellum and hypothalamus? J.B. Martin: I am not aware of any anatomic or pathologic relationship. A. Querido: You listed Sheehan’s disease as hypothalamohypophyseal. As far as I recall there is localized destruction of the anterior lobe. Am I right? J.B. Martin: Sheehan’s disease is pituitary necrosis and believed to be due to ischemia occurring with post-partum shock (see also comment by N.E. Rance).

Reference Sheehan, H.L. (1967)Variations in the subventricular nucleus. J. Pathol. Bacteriol., 94: 409 - 416.

41 Table A

Hypogonadotrophic hypogonadism with ataxia (1) Laurence-Moon-Biedl syndrome with spastic ataxia (Ryan, 1961) (2) Spinocerebellar ataxia, areflexia and loss of postural sense (Bernard-Weil and Endtz, 1962) (3) Cerebellar ataxia, pes c a w s and cardiomyopathy (Volp6 et al., 1963) (4) Cerebellar ataxia, retinal degeneration and amenorrhea (Boucher and Gibberd, 1969) (5) Cerebellar ataxia, deafness and eunuchoidism (Matthews and Rundle, 1964). Berciano et al. (1982) have reported evidence for hypothalamic LHRH deficiency in such cases (6) Ataxia oligophrenia, anosmia, retinal degeneration, webbed neck and hypogonadism (Lowenthal et al., 1979). Increased plasma levels of glutamine and alanine; increaed urinary excretion of glutamine and ornithine (7) Hereditary ectodermal dysplasia, ataxia and hypogonadism (Rushton and Genel, 1981) (8) Olivopontocerebellar ataxia with hypogonadism (Berciano et al., 1982) (9) Cerebellar ataxia, mirror movements, anosmia, and hypogonadism (Schwankhaus et al., 1989) Hypergonadotrophic hypogonadism with ataxia (10) Klinefelter’s syndrome with ataxia (Hecht and Ruskin, 1960) (1 1) Marinesco-Sjogren syndrome (cerebellar ataxia, oligophrenia, cataracts) with hypogonadism (Skre et al., 1976) (12) Cerebellar atrophy, dementia, reduced nerve conduction velocities and secondary amenorrhea (De Michele et al., 1987) Normal or unknown gonadotrophins with ataxia (13) Ataxia with diabetes insipidus, diabetes mellitus, optic atrophy and deafness (DIDMOAD) (Cooper et al., 1950) (14) Cerebellar ataxia, areflexia, extensor plantar signs, eunuchoidism and normal gonadotrophin excretion (Spota and Novizki, 1954) (15) Mental deterioration, ataxia, deaf-mutism and neurogenic muscular atrophy (Richards and Rundle, 1959) (16) Ataxia telangiectasia with hypogonadism (Terenty et al., 1978) (17) Xerodermic iodocy with hypogonadism (Reed et al., 1965) (18) Neuroaxonal dystrophy with testicular atrophy (Thibault, 1972) (19) Refsum’s disease with testicular atrophy (Gordon and Hudson, 1959)

(20) Pallido-olivocerebellar degeneration with eunuchoidism (Altschul and Kotlowski, 1956) (21) Behr’s (1909) spastic ataxia, optic atrophy and cryptorchidism Skre and Berg (1977) have demonstrated that, in their patients, two closely linked autosomal recessive genes were responsible for Marinesco-Sjogren syndrome and hypogonadism. Otherwise, the relation between ataxia and hypogonadism remains to be clarified (Howell, 1992). For additional references, see Howell (1992) and De Jong et al. (1992).

References Altschul, R. and Kotlowski, K. (1956) Pallido-cerebello-olivary degeneration with eunuchoidism. J. Nerv. Ment. Dis., 123: 112-116, Behr, C. (1909)Die komplizierte hereditarfamiliare Optikusatrophie des Kindes-alters. Klin. Monatsbl. Augenheilk., 47: 138- 160. Berciano, J., Amado, J.A., Freijanus, J., Rebollo, M. and Vaquero, A. (1982) Familial cerebellar ataxia and hypogonadotropic hypogonadism: evidence for hypothalamic LHRH deficiency. J. Neurol. Neurosurg. Psychiatry, 45: 747 - 751. Bernard-Weil, E. and Endtz, L.J. (1962) Sur un cas familial de degknkration spino-ckrkbelleuse avec eunuchoidisme hypogonado-trophique. Presse Med., 70: 524 - 526. Boucher, B.J. and Gibberd, F.B. (1969) Familial ataxia. Hypogonadism and retinal degeneration. Acta Neurol. Scand., 45: 507 - 510. Cooper, I.S., Rynearson Bailey, A.A. and MacCarty, C.S. (1950) The relation of spinal cord disease to gynecomastia and testicular atrophy. Mayo Clin. Proc., 25: 320- 326. De Jong, J.M.B.V., Bolhuis, J.P. and Barth, P.G. (1992) Differential diagnosis of the patient with hereditary cerebellar and spinocerebellar disorders. In: P.J. Vinken, G.W. Bruyn, H.L. Klawans and J.M.B.V. De Jong (Eds.), Handbookof Clinical Neurology, Vol. 16 (60). Hereditary Neuropathies and Spinocerebellar Atrophies, Elsevier, Amsterdam, pp. 643 - 699. De Michele, G., Filla, A., Iorio, L., Merola, B., De Rosa, M., Lombardi, G. and Campanella, G. (1987) Atassia cerebellare ed ipogonadismo. Descrizione di quattro casi. Riv. Neurol., 57: 328 - 332. Gordon, N. and Hudson, R.E.B. (1959) Refsum’s syndrome heredopathia atactica polyneuritiformis. A report of three cases, including a study of the cardiac pathology. Brain, 82: 41 - 55. Hecht, A. and Ruskin, H. (1960) Seminiferous tubule dysgenesis (Klinefelter’s syndrome) associated with familial cerebellar ataxia. J. Clin. Endocrinol. Metab., 20: 1184- 1190.

42 Howell, D.A. (1992) Ataxia with hypogonadism (MatthewsRundle) (212840). In: P.J. Vinken, G.W. Bruyn, H.L. Klawans and J.M.B.V. De Jong (Eds.), Handbookof Clinical Neurology, Vol. 16 (60). Hereditary Neuropathies and Spinocerebellar Atrophies, Elsevier, Amsterdam, pp. 575 - 580. Lowenthal, A., Bekaert, J., Van Dessel, F. and Van Hauwaert, J. (1979) Familial cerebellar ataxia with hypogonadism. J. Neurol.. 222: 75 - 80. Matthews, W.B. and Rundle, A.T. (1964) Familial cerebellar ataxia and hypogonadism. Brain, 87: 463 - 468. Reed, W.B., May, S.B. and Nickel, W.R. (1965) Xeroderma pigmentosum with neurological complications: the de SanctisCacchione syndrome. Arch. Dermatol., 91: 224- 226. Rushton, M.A. and Genel, M. (1981) Hereditary ectodermal dysplasia, olivopontocerebellar degeneration, short stature and hypogonadism. J. Med. Genet., 18: 335 - 339. Ryan, R.J. (1961) Male hypogonadism. In: Disease-a-Month, Year Book Publ., Chicago, IL, March: 2-36. Schankhaus, J.D., Jaffe, J . J . , Rose, S.R. and Sherins, R.J.

(1989) Neurologic findings in men with isolated hypogonadotrophic hypogonadism. Neurology, 39: 223 - 226. Skre, H. and Berg, K. (1977) Linkage studies on the MarinescoSjogren syndrome and hypergonadotrophic hypogonadism. Clin. Genet., 11: 57-66. Skre, H., Bassoe, H.H., Berg, K. and Frovig, A.G. (1976) Cerebellar ataxia and hypergonadotrophic hypogonadism in two kindreds. Chance occurrence pleiotropism, or linkage? Clin. Genet., 9: 234 - 244. Spoat, B.B. andNovizki, I. (1954) Sindrome adiposo genital and el friedreich. Prensa Med. Argent., 41: 1223 - 1226. Terenty, T.R., Robson, P. and Walton, J.N. (1978) Presumed ataxia-telangiectasia in a man. Br. Med. J., 11: 202. Thibault, J. (1972) Neuroaxonal dystrophy: a case of nonpigmented type and protracted course. Acta Neuropathol. (Bed.)., 21: 232-238. Volpt, R., Metzler, W.S. and Johnston, M.W. (1963) Familial hypogonadotrophic eunuchoidism with cerebellar ataxia. Clin.Endocrinol. Metab., 23: 107 - 115.

Section I11

Technical Potentialities and Pitfalls in the Use of Human Material

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D.F. Swaab, M.A. Hofman, M. Mirmiran, R. Ravid and F.W.van Leeuwen (Eds.) Progress in Brain Research, Vol. 93 0 1992 Elsevier Science Publishers B.V. All rights reserved.

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CHAPTER 4

In situ hybridization histochemistry in the human hypothalamus G. Mengod, E. Goudsmitl, A. Probst2 and J.M. Palacios

'

Department of Neurochemistry, CID-CSIC, Jordi Girona 18 - 26, Barcelona 08034, Spain; Netherlands Institute for Brain Research, Amsterdam, The Netherlands; and Institute of Pathology, Basel, Switzerland

Introduction

Most of the information available until now on the neuropeptide content, cellular localization and alterations with disease and drug treatment in human tissues has been obtained by the application of immunological techniques such as radioimmunoassay and immunohistochemistry (Bjorklund and Hokfelt, 1983). These techniques, however, present some limitations related to the sensitivity and specificity of the antibodies used. Some of these problems are difficult to overcome when human post-mortem tissues are investigated. The recent advances in the application of molecular biology to neurobiology have allowed the introduction of new methodologies into this field. Knowledge of the sequence of the gene coding for a given neuropeptide allows the design of molecular probes that will be useful for detection and visualization of the cells containing mRNA coding for that particular neuropeptide. This technique, known as in situ hybridization histochemistry, is an invaluable tool in the study of the distribution of different neuropeptides in the brain (Valentino et al., 1987). In combination with receptor autoradiography (see Chapter 5 ) in situ hybridization histochemistry allows to study the regulation of gene expression. The purpose of this chapter is to review the application of in situ hybridization histochemistry in

the study of neuropeptide gene expression in human post-mortem brain. Different control experiments carried out in order to ascertain the specificity of the hybridization signal will be discussed with special emphasis, including Northern analysis, use of heterologous and homologous probes, cohybridization assays and thermal stability of the formed hybrids. The influence of the tissue fixation method on the hybridization signal will be considered as well as the influence of parameters such as post-mortem delay, age, gender and disease. We will focus on neuropeptides preferentially expressed in hypothalamus and basal ganglia, such as oxytocin (OT), arginine-vasopressin (VP), preproenkephalin A (ENK), somatostatin (SOM) and neuropeptide Y (NPY). In situ hybridization histochemistry: methodological considerations

Human post-mortem studies When studying human brain tissue there are a number of parameters that could influence the results of in situ hybridization histochemistry. At the present time the information available is still limited. Ante-mortem factors such as agonal state, stress, pre-mortem anoxia, etc., could be responsible for the variability on mRNA levels. Gender and age did

46

not have any systematic effect on the mRNA levels studied so far (Mengod et al., 1990a, 1991; Savasta et al., 1990). Until now, there is not much information available on the influence of post-mortem delay on the stability of mRNA. Post-mortem stability of mRNA has been studied mainly by RNA extraction (Johnson et al., 1986; Perret et al., 1988) and found to be steady for at least 16 h. Freezing and thawing of tissue blocks to room temperature might result in rapid degradation of mRNA (Ragsdale and Miledi, 1991). Visualization and semi-quantification of mRNA by in situ hybridization histochemistry has been possible despite post-mortem delays of longer than 40 h (Mengod et al., 1991). Storage of frozen tissue for more than 6 years appears not to affect the hybridization signal significantly (our own unpublished observations).

we have favored the use of labeled oligonucleotides as hybridization probes for several reasons. They are fast and easy to obtain, provided access to a DNA synthesizer is available, and do not require long cloning and subcloning steps. Obviously, however, their design requires that the mRNA sequence has been published or is available from the investigators who actually cloned the gene or cDNA. The end-labeling and purification of oligonucleotide probes is also a relatively easy and fast procedure which yields probes with highly specific activities. Perhaps the greatest advantage of using oligonucleotide probes is the possibility of obtaining probes highly specific for the mRNA of interest. Another advantage of oligonucleotide probes is that they allow a number of precise control experiments to be performed in order to assess the specificity of the hybridization signal obtained.

Collection of human brain samples. The two cerebral hemispheres including the hypothalamus are separated by a midsagittal cut. Usually samples of the left cerebral and cerebellar hemispheres are collected; the entire right hemisphere is further processed for neuropathological examination. The hemisphere is cut along the frontal plane in 5 mm thick coronal slices. Each slice is trimmed to smaller blocks containing the regions of interest such as to fit into a cryostat holder (maximum size 50 x 40 mm). The whole collection is placed on an aluminum plate and cooled over dry ice. After freezing, the tissue blocks are stored at - 70°C until used. Sections are obtained with a microtome cryostat at -20°C. Twenty micron thick sections are cut, thaw-mounted onto gelatin coated microscope slides and kept at - 20°C until use.

Oligonucleotide synthesis and labeling The oligonucleotides are synthesized on a 380B Applied Biosystem DNA synthesizer, purified on a 20% polyacrylamide/8 M urea preparative sequencing gel and stored at -20°C. They are labeled either at their 3' end with [32P]dATP (>3000 Ci/mmol, New England Nuclear) or with [35S]dATP (> 800 Ci/mmol, Amersham) and terminal deoxynucleotidyltransferase (TdT, Boehringer, Mannheim) to specific activities of 0.9 - 3 x lo4Ci/mmol and the labeled DNA purified through aNACS PREPAC column (BRL, Bethesda, U.S.A) according to the manufacturer's instructions.

Use of oligonucleotides as hybridization probes Several types of molecular probes have been used for in situ hybridization studies: double strand cDNA, single strand cDNA (Mengod et al., 1988), riboprobes (Mengod et al., 1990b), and oligonucleotides(Mengodet al., 1990a, 1991; Savastaet al., 1990). Although cRNA or cDNA probes have potential advantages over others, in our laboratory

Tissue fixation and in situ hybridization histochemistry In order to preserve the morphology of the tissue a fixation step in 4% (wtlvol) paraformaldehyde is included. To increase the penetration of the hybridization probe into the tissue a proteolytic treatment is done before the hybridization. The protocol followed in general is as described (Mengod et al., 1990a, 1991). Briefly, cryostat sections are air dried, fixed 20 min in 4% (wt/vol) paraformaldehyde in 1 x PBS (2.6 mM KCl, 1.4 mM KH,PO,, 136 mM NaC1, 8 mM Na2HP0,). Slides are then

47

washed once in 3 x PBS, twice in 1 x PBS, for 5 rnin each, and incubated in predigested pronase at a final concentration of 24 U/ml in 50 mM Tris-HC1 pH 7.5,5 mMEDTAfor 10min. Theproteolyticactivity is stopped by 30 sec immersion in 1 x PBS containing 2 mg/ml glycine. The slides are rinsed twice in PBS for 2 min each and dehydrated in a graded series of ethanol (60070, 80%, 95% and 100%) for 2 rnin each. Tissue sections are allowed to dry before hybridization. Labeled DNA probe is diluted in a buffer containing 50% formamide, 600 mM NaC1,lO mM Tris-HC1 pH 7.5,l mM EDTA, 0.02% Ficoll, 0.02% polyvinylpyrrolidone, 0.02% BSA, 500 pg/ml yeast tRNA, and 10% dextran sulfate to a final concentration of 0.4-0.8 pmol/ml. The hybridization solution on the slide is covered by a piece of nescofilm to prevent evaporation, and the slides are placed in humid boxes for incubation overnight at 42°C. Nescofilm is removed by floatation in a solution containing 600 mM NaCI, 20 mM Tris-HC1, pH 7.5 and 1 mM EDTA and the slides washed in the same buffer at 60°C for 4 h with four changes. Finally, tissue sections are dehydrated by incubation in 70% ethanol for 5 min andin%% ethanolfor5min, containingboth0.3 M ammonium actetate pH 7.0. We have studied the influence of tissue fixation procedures for the visualization of mRNA transcripts. We routinely use human tissue sections obtained with a microtome cryostat. We have found that hybridization of human paraffin sections can be done by introducing some modifications in the prehybridization steps, as described in the legend to Fig. 1. The two upper panels show toluidine bluestained sections of the same human hypothalamus. The left half of the hypothalamus was fixed in formalin for 2 weeks and embedded in paraffin. The right half was frozen on dry ice and stored at - 20°C. Sections at the same level from both were obtained and hybridized with an oligonucleotide complementary to human vasopressin mRNA. A strong hybridization signal is observed in the PVN and SON following both types of tissue treatment in the two lower panels.

Fig. 1. Effect of different tissue fixation procedures on in situ hybridization histochemistry. The two upper panels show toluidine blue-stained frontal sections of a human hypothalamus at the same rostro-caudal level. The left half of the hypothalamus was fixed in 4% formalin for 2 weeks and embedded in paraffin. Ten micron sections were cut on a microtome. Sections were deparaffinized in xylol and a graded series of ethanol, post-fixed in 4% (wt/vol) paraformaldehyde in PBS, washed once in 3 x PBS, twice in 1 x PBS, for 5 min each, treated with 0.2 N HCl for 15 min and washed twice in 1 x PBS for 5 rnin each. They were then incubated with proteinase K at a final concentration of 100 pg/ml for 10 min at 3 7 T , rinsed twice in PBS for 2 min each and dehydrated. The right half of the hypothalamus was frozen on dry ice and stored at - 20°C. Cryostat sections (10 am) were air dried, fixed for 20 min in 4% wt/vol paraformaldehyde in 1 x PBS. Slides were then washed once in 3 x PBS, twice in 1 x PBS, for 5 min each, and incubated in predigested pronase at a final concentration of 24 U/mlin 50 mM Tris-HCl pH 7.5,5 mM EDTA for 10 min. The proteolytic activity was stopped by 30 sec immersion in 1 x PBS containing 2 mg/ml glycine. The slides were rinsed twive in PBS for 2 mineach and dehydrated in a graded series of ethanol (6O%, 8070,95% and 100%) 2 rnin each. Tissue sections were allowed to dry before hybridization. Both tissue sections were hybridized with an "S-labeled oligonucleotide complementary to the human arginine-vasopressin mRNA (Mohr et al., 1985). A strong hybridization signal is observed in the PVN and SON in the two types of tissue treatment in the two lower panels.

48

tion signal obtained for the localization of neuropeptide gene transcripts (Mengod et al., 1990a, 1991; Savasta et al., 1990).

1 2 3 4 5 6 7 8

-28 S -23s -18 S -16 S

Fig. 2. Northern blot analysis with poly(A)+ mRNA purified from different brain regions of acontrol case of human brain and hybridized with a 32P-labeledoligonucleotide probe complementary to the human NPY mRNA. Total RNA was isolated from different human brain areas according to Chomczynski and Sacchi (1987). Poly (A)+ RNA was selected on oligo(dT)-cellulose (Bethesda Research Laboratories), denatured with glyoxal (McMaster and Carmichael, 1977), run on 1% agarose gel, blotted onto nylon membranes (Hybond N, Amersham), and hybridized with a 32P-labeledoligonucleotide complementary to human NPY mRNA (Minth et al., 1984). Each lane was loaded with 10 pg of poly(A)+ mRNA. Lane I , hippocampus; lane 2, putamen; lane 3 , substantia nigra; lane 4, cerebellum; lane 5, frontal cortex; lane 6, brain-stem; lane 7, tectum; lane 8, hypothalamus. Ribosomal molecular weight markers migration is indicated on the right.

Specificity controls f o r in situ hybridization histochem istry Several control experiments are routinely carried out with all the probes used in our laboratory in order to determine the specificity of the hybridiza-

Northern analysis Northern blots with poly(A)+ RNA purified from several human brain regions were hybridized with the corresponding 32P-labeledoligonucleotide probe. We have found that for neuropeptides the probes hybridized to mRNAs with sizes corresponding to those originally described in the literature, and with a regional distribution correlating well with the areas where the peptides are known to be expressed. An example of such a control for NPY is shown in Fig. 2. Hybridization with the corresponding labeled oligonucleotide probe revealed a discrete band of mRNA with a size corresponding to the reported mRNA length for human NPY. The regional distribution was in agreement with the results of our in situ hybridization histochemistry studies. The highest NPY mRNA content was found in hypothalamus (lane 8), followed by frontal cortex (lane 5 ) ,putamen (lane 2) and hippocampus (lane I ) . A weak hybridizing band could be seen in the tectum (lane 7). Following longer exposure times, still no signal could be detected in substantia nigra (lane 3 ) , cerebellum (lane 4) and brain-stem (lane 6). Use of homologous probes We use at least two different oligonucleotides complementary to distinct regions of the same target mRNA independently as hybridization probes in consecutive tissue sections. The hybridization pattern and regional distribution obtained has always been similar for both probes. Fig. 3 illustrates this for vasopressin (VP) and oxytocin (OT) mRNA distribution. The upper right panels show the hybridization pattern obtained with two oligonucleotides, hVP, and hVP,, complementary to different regions of vasopressin mRNA. Both probes label a large group of cells in the paraventricular nucleus and in the supraoptic nucleus. The lower panels show that the localization of the cells containing oxytocin mRNA is similar using two different oligonucleotide probes, hOT, and hOT2.

49

Fig. 3. Specificity controls for in situ hybridization histochemistry: use of homologous and heterologous probes. Pictures are photographs from film-autoradiograms obtained by hybridizing consecutive sections from the same control case with 35S-labeled oligonucleotide probes. A tissue section of the frontal aspect of the right half of human hypothalamus is shown first stained with toluidine blue. Similar sections were hybridized with two different oligonucleotide probes for human vasopressin mRNA (Mohr et al., 1985), hVP3 and hVP,, and for human oxytocin-neurophysin I mRNA (Mohr et al., 1985), hOT, and hOT,. The film autoradiograms obtained are shown. The VP probes label a large group of cells in the dorsolateral division of the supraoptic nucleus (SON) and a cluster of cells in the paraventricular nucleus (PVN). In contrast, the OT probes label only cells in the medial part of the SON and in the PVN a group of cells overlapping the VP cells. Abbreviations: OT, optic tract; 111, third ventricle.

Use of heterologous probes A different hybridization pattern can be observed when two different oligonucleotide probes are used in consecutive tissue sections. By comparing the localization of VP mRNA containing cells (Fig. 3, upper panels) with that of OT mRNA containing cells, it can be seen that VP probes label a large group of cells in the supraoptic nucleus and a cluster of cells in the paraventricular nucleus, whereas the OT probes label cells in the medial part of the supraoptic nucleus and a group of cells in the paraventricular nucleus overlapping the VP cells. Preproen-

kephalin A transcripts were seen in the supraoptic and paraventricular nuclei. In contrast, no cells containing detectable amounts of cholecystokinin mRNA were observed in these nuclei (data not shown) . Melting curve analysis The thermal stability of the hybrids could be studied during, for example, the washing steps. A sharp decrease in the intensity of the hybridization signal should be observed at a temperature consistent with the theoretical melting temperature (T,)

50

of a perfect hybrid (temperature at which 50% of the hybrids dissociate) (Meinkoth and Wahl, 1984).The upper panels in Fig. 4 illustrate the melting behavior observed for a hybridization experiment with an oligonucleotide probe for OT mRNA in consecutive sections of a human hypothalamus. A sharp decrease in the hybridization signal in both supraoptic and paraventricular nuclei was observed when the washing temperature was increased to 70°C, which is very close to the theoretical Tm of the hybrid formed.

Cohybridization experiments A specific hybridization signal will not be produced when an excess of unlabeled oligonucleotide is included in the hybridization solution, the remaining signal giving an idea of the background levels. No alteration of the specific signal should be observed when the unlabeled oligonucleotide added in excess is complementary to a different region of the same mRNA or to an unrelated mRNA. Fig. 4 illustrates this with a background hybridization signal obtained when an oligonucleotide probe for OT

Fig. 4. Specificity controls for in situ hybridization histochemistry: melting curve analysis and cohybridization experiments. A tissue section of the frontal aspect of the right half of a human hypothalamus is shown stained with toluidine blue in the lower left corner. Tissue sections were hybridized with oligonucleotide probe hOT,. Upper rows show the film-autoradiograms obtained after the hybridized tissue sections were washed at increasing temperatures, resulting in a disappearance of the hybridization signal. The lower right panels show the tissue sections cohybridized with an excess of the same unlabeled probe, hOT, (no hybridization signal could be seen), or with an excess of a heterologous unlabeled probe, i.e., hOT,.

51

Fig. 5. Neuropeptide mRNA visualization in human basal ganglia. The pictures are photographs from film-autoradiogams obtained by hybridizing sections from the same control case with 32P-labeledoligonucleotide probes complementary to the mRNAs coding for: A , preproenkephalin A (Noda et al., 1982); B, somatostatin (Shen et al., 1982); C,cholecystokinin (Takahashi et al., 1985); and D, NPY (Minth et al., 1984). Cd, Caudate nucleus; Put, putamen nucleus; Acc, nucleus accumbens.

52

Fig.4. Alterations in the levels of neuropeptide mRNAs in Parkinson’s and Huntington’s diseases basal ganglia. Levels of SOM mRNi were preserved throughout the striatum in Parkinson’s (A) and Huntington’s (B) diseases, whereas those for ENK mRNA were clear1 decreased in Huntington’s disease striatum (D) as compared to the Parkinson patient (C). Some ENK mRNA can be seen in the ventri part of the Huntington’s disease striatum (D).

53

mRNA (hOT,) is cohybridized with a 50-fold excess of the same unlabeled oligonucleotide. No alterations in the hybridization pattern was obtained when labeled hOT, probe is cohybridized with a 50fold excess of unlabeled hOT3 probe. Neuropeptide mRNA visualization in human basal ganglia We examined the distribution of the cells containing mRNA coding for enkephalin, cholecystokinin, somatostatin and NPY, in several control cases without reported neurological diseases. A heterogeneous distribution of these cells was observed (Fig. 5 ) . Preproenkephalin A mRNA (Fig. 5A) presented a “patchy” distribution in the caudate and putamen nuclei. The overlap of the preproenkephalin A mRNA-rich patches with the zones of low AChE activity (striosomes) (see Graybiel et al., 1981), as examined with the copper thicholine method (Geneser and Blackstad, 1971),was not seen throughout these nuclei (not shown). In contrast, cells containing somatostatin mRNA (Fig. 5D)were scattered throughout the caudate and putamen nuclei. In several striatal areas, they were organized around the patches of high preproenkephalin A mRNA content (data not shown). NPY mRNAcontaining cells (Fig. 5C) were distributed in the basal ganglia in a pattern similar to the one observed for somatostatin mRNA. This codistribution was particularly evident in the basal ganglia but not observed in other areas of the human brain examined. For example, in cells of the anterior thalamus, high levels of somatostatin mRMA contrasted with the apparent absence of a hybridization signal for NPY mRNA. No significant hybridization was observed for cholecystokinin mRNA in the basal ganglia, while a high density of cells expressing this message was observed in adjacent cortical regions and in the claustrum (not shown) (Savasta et al., 1990). Neuropeptide mRNA alterations in Parkinson’s disease and Huntington’s chorea basal ganglia We also examined the expression of neuropeptides in the brain of patients dying from Huntington’s

disease. Fig. 6 illustrates a dramatic decrease in the number of preproenkephalin A mRNA-containing cells in the caudate and putamen nuclei of a Huntington’s disease case (Fig. 6 8 ) . In contrast, in a section from the same case, somatostatin mRNA-containing cells were well preserved (Fig. 6C) as well as NPY mRNA-containing cells (not shown). Summary and conclusions We have seen that mRNA for several neuropeptides can be visualized at the microscopic level in human post-mortem brain tissues using in situ hybridization histochemistry and oligonucleotides as probes. The specificity of the hybridization signal detected in each case is supported by several criteria such as Northern blot analysis, use of at least two oligonucleotides complementary to different regions of the same target mRNA, cohybridization of labeled and excess unlabeled oligonucleotide probes, and melting curve analysis of the formed hybrids. Furthermore, factors such as age, post-mortem delay or gender did not show a significant effect in the levels of hybridization in the control population studied. Hybridization signals comparable to those found in the control population were obtained in frozen tissues, stored for up to 6 years before analysis. The results obtained for the different neuropeptides examined are, in general, in good agreement with the available information on their distribution and cellular localization as determined by radioimmunoassay or immunohistochemistry. The use of in situ hybridization histochemistry has clearly revealed the location of neurons synthesizing these neuropeptides, adding important information to that provided by radioimmunoassay or immunohistochemistry. A typical example is the identification of peptide synthesizing neuronal cell bodies by immunohistochemistry. This requires, in some cases, the use of treatments such as colchicine, obviously impossible with human brain tissues. The abundance of mRNA could be further related to transcriptional activity and, when compared with peptide levels, can provide some clues on peptide turnover rates.

54

Thus in the hypothalamus, the paraventricular and supraoptic nuclei were found to contain cells expressing arginine-vasopressin and oxytocin mRNAs. Their distribution was in good agreement with that determined by immunohistochemistry (Dierickx and Vandesande, 1977). We have also found that these nuclei contain transcripts for neuropeptide genes such as preproenkephalin A, neuropeptide Y and somatostatin, in agreement with previously reported immunohistochemical data (Agid and Javoy-Agid, 1985; Emson et al., 1986). In the basal ganglia, numerous cells heterogeneously distributed throughout the caudate and putamen nuclei were found to contain preproenkephalin A mRNA. This distribution is in good agreement with the proposed localization of enkephalin to spiny projection neurons (Pickel et al., 1980; Graybiel and Ragsdale, 1984; Beal and Martin, 1986). Somatostatin and NPY mRNA-containing cells were less abundant and presented a very similar distribution throughout the striatum, in agreement with the histochemical colocalization of these two neuropeptides to the same neuronal population, the aspiny interneurons (Graybiel and Ragsdale, 1984; Gaspar et al., 1987). No detectable levels of cholecystokinin mRNA could be seen in the striatum, indicating that striatal cholecystokinin immunoreactivity is restricted to processes from cells extrinsic to this nucleus. In Parkinson’s disease we have not detected significant alterations of the mRNA content for somatostatin and NPY. In Huntington’s chorea some neurons, namely the spiny neurons of the caudate nucleus and putamen, are selectively affected (Martin and Reichlin, 1986; Kowall et al., 1987). We have been able to show that in Huntington’s chorea there is a marked decrease in the levels of preproenkephalin mRNA in both caudate and putamen, while in the nucleus accumbens this mRNAis less affected. No alterations were seen in the hybridization signal for somatostatin and NPY mRNA (Mengod and Palacios, 1990). In situ hybridization histochemistry provides a new way to examine neuropeptide gene expression in the human brain. This method allows the visualization of cell bodies expressing the genes for the dif-

ferent neuropeptides. In combination with image analysis, it could be used to study pathological or drug-induced changes in the levels of expression of these genes. Acknowledgements

The authors wish to thank Prof. E. Bird from the Brain Tissue Resource Center, McLean Hospital, Belmont, MA, U.S.A., for kindly supplying the Huntington’s disease tissues. This center is supported in part by PHS Grant number MH/NS 31862. E.G. was supported by the Sandoz Foundation for Gerontological Research (Grant m.SF 194) and the Innovation Fund of the Royal Dutch Academy of Sciences. References Agid, Y. and Javoy-Agid, F. (1985) Peptides and Parkinson’s disease. Trends Neurosci., 1: 30 - 35. Beal, M.F. and Martin, J.B. (1986) Neuropeptides and neurological disease. Ann. Neurol., 20: 547 -565. Bjorklund, A. and Hokfelt, T. (Eds.) (1983) In: Handbook oj Chemicaf Neuroanatomy, Vof. I , Elsevier, Amsterdam. Chomczynski, P. and Sacchi, N. (1987) Single step method 01 RNA isolation by acid guanidinium thiocyanate-phenol chloroform extraction. Anal. Biochem., 162: 156 - 159. Dierickx, K. and Vandesande, F. (1977) Immunocytochemica localization of the vasopressinergic and oxytocinergic neuron: in the human hypothalamus. Cell. Tissue Res., 184: 15 - 21. Emson, P.C., Rossor, M.N. and Tohyama, M. (Eds.) (1986 Progress in Brain Research, Vol. 66, Elsevier, Amsterdam. Gaspar, P., Berger, B., Lesur, A., Borsotti, J.P. and Febvret, A (1987) Somatostatin 28 and neuropeptide Y innervation in thc septa1 area and related cortical and subcortical structures o the human brain. Distribution, relationships and evidence foi differential coexistence. Neuroscience, 22: 49 - 73. Geneser, F.A. and Blackstad, T.W. (1971) Distribution of acety cholinesterase in the hippocampal region of the guinea pig. Z ZeNforsch., 114: 460-481. Graybiel, A.M. and Ragsdale Jr., C.W. (1984) Biochemica anatomy of the striatum. In: P.C. Emson (Ed.), Chemica Neuroanatomy, Raven Press, New York, pp. 427 - 504. Graybiel, A.M., Ragsdale Jr., C.W., Yoneoka, E.S. and Elde R.P. (1981) An immunohistochemical study of enkephalin and other neuropeptides in the striatum of the cat wit1 evidence that the opiate peptides are arranged to form mosail patterns in register with the striosomal compartments visibll by acetylcholinesterase staining. Neuroscience, 6: 377 - 397.

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Johnson, S.A., Morgan, D.G. and Finch, C.E. (1986) Extensive post-mortem stability of RNA from rat and human brain. J. Neurosci. Res., 16: 267 - 280. Kowall, N.W., Ferrante, R.S. and Martin, J.B. (1987) Patterns of cell loss in Huntington’s disease. Trends Neurosci., 10: 24-29. Martin, J.B. and Reichlin, S. (1986) Clinical implications of neuropeptides in neurologic disorders. In: J.B. Martin and S. Reichlin (Eds.), Clinical Neuroendocrinology, Davis, Philadelphia, PA, pp. 607 - 636. McMaster, G.K.and Carmichael, G.G. (1977) Analysis of single and double stranded nucleic acids on polyacrylamide and agarose gels using glyoxal and acridine orange. Proc. Natl. Acad. Sci. U.S.A., 448: 4835 - 4838. Meinkoth, J. and Wahl, G. (1984) Hybridization of nucleic acids immobilized on solid supports. Anal. Biochem., 138: 267 - 284. Mengod, G. and Palacios, J.M. (1990) Molecular neuropathology: the study of neurotransmitter and receptor expression in human post-mortem materials by in situ hybridization and receptor autoradiography. In: W.E. Bunney Jr., H. Hippius, G. Laakmann and M. Schmauss (Eds.), Neuropsychopharmacology,Springer, Berlin, pp. 693 - 703. Mengod, G., Palacios, J.M., Probst, A. and Harris, B. (1988) Regional distribution of the expression of a human stimulatory GTP-binding protein alpha subunit in the human brain studied by in situ hybridization. Mol. Brain Rex, 4: 23 - 29. Mengod, G., Charly, J.L. and Palacios, J.M. (1990a) The use of in situ hybridization histochemistry for the study of neuropeptide gene expression in the human brain. Cell. Mol. Neurobiol., 10: 113 - 126. Mengod, G., Le, H., Nguyen, H., Liibbert, H., Waeber, C. and Palacios, J.M. (1990b) The distribution and cellular localization of the mRNA for the 5-HT1C receptor in the rodent brain examined by in situ hybridization. Comparison with receptor binding distribution. Neuroscience, 35: 577 - 591. Mengod, G., Vivanco, M.M., Cristnacher, A., Probst, A. and Palacios, J.M. (1991) Study of pro-opiomelanocortin mRNA expression in human post-mortem pituitaries. Mol. Brain Res., 10: 129- 137.

Minth, C.D., Bloom, R.S., Polak, J.M. and Dixon, J.E. (1984) Cloning, characterization, and DNA sequence of a human cDNA encoding neuropeptide tyrosine. Proc. Natl. Acad. Sci. U.S.A., 81: 4577-4581. Mohr, E., Hillers, M., Ivell, R., Haulica, I.D. and Richter, D. (1985) Expression of the vasopressin and oxytocin genes in human hypothalami. FEBS Lett., 193: 12- 16. Noda, M., Teranishi, Y . , Takahashi, H., Toyosato, M., Notake, M., Nakanishi, S. and Numa, S. (1982) Isolation and structural organization of the human preproenkephalin gene. Nature, 297: 431 -434. Perret, C.W., Marchbanks, R.M. and Whatley, S.A. (1988) Characterization of messenger RNA extracted post-mortem from the brains of schizophrenic, depressed and control subjects. J. Neurol. Neurosurg. Psychiatry, 51: 325 - 331. Pickel, V.M., Sumal, K.K., Beckley, S.C., Miller, R. J. and Reis, D.J. (1980) Immunocytochemical localization of enkephalin in the neostriatum of rat brain: a light and electron microscopic study. J. Comp. Neurol., 189: 721 -740. Ragsdale, D.S. and Miledi, R. (1991) Expressional potency of mRNAs encoding receptors and voltage-activated channels in the post-mortem rat brain. Proc. Natl. Acad. Sci. U.S.A., 88: 1854- 1858. Savasta, M., Palacios, J.M. and Mengod, G. (1990) Regional distribution of the messenger RNA coding for the neuropeptide cholecystokinin in the human brain examined by in situ hybridization. Mot. Brain Res., 7: 91 - 104. Shen, L-P., Pictet, R.L. and Rutter, W.J. (1982) Human somatostatin I: sequence of the cDNA. Proc. Natl. Acad. Sci. U.S.A., 79: 4575 - 4579. Takahashi, Y., Kato, K., Hayashizaki, Y . , Wakabayashi, T., Ohtsuka, E., Matsuki, S., Ikehara, M. and Matsubara, K. (1985) Molecular cloning of the human cholecystokinin gene by use of a synthetic probe containing deoxyinosine. Proc. Natl. Acad. Sci. U.S.A., 82: 1931-1935. Valentino, K.L., Eberwine, J.H. andBarchas, J.D. (Eds.)(1987) In Situ Hybridization. Applications to Neurobiology, Oxford Univerisity Press, Oxford, New York.

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CHAPTER 5

Receptor localization in the human hypothalamus J.M. Palacios1,2, A. Probst3 and G. Mengod' Department of Neurochemistry, Centro Investigacion y Desarrollo, Consejo Superior de Investigaciones Cienfificas, Jordi Girona, 18 - 26, 08034 Barcelona, Spain; Laboratorios Almirall, Research Institute, Cardener, 68 - 74, 08024 Barcelona, Spain; and Institute of Pathology, Department of Neuropathology, University of Basel, Switzerland

'

Introduction: neurotransmitter receptor structure and function

Our understanding of receptor structure and function has undergone a dramatic change in the recent past. Although the basicconcept of receptor, i.e, the specialized cell component able to recognize with exquisite selectivity messenger molecules, is now more than a hundred years old (Ariens, 1979; Parascandola, 1980), it is only recently that the molecular structure of these proteins has been fully revealed. This development has been made possible by several advances in receptor technology. The first important step was the development of the so-called high affinity binding techniques using radiolabeled ligands. This led for the first time to the direct study of ligand receptor interaction and the kinetic and pharmacological characterization of receptor binding sites (Yamamura et al., 1990). The development of receptor ligand binding was also instrumental in facilitating the purification and isolation of some of the receptor proteins. It was from the information obtained from purified receptor proteins that probes were derived and cloning of the first members of several receptor families was carried out (Barnard et al., 1987; Dohlmanet al., 1987; Hall, 1987). Thanks to these techniques and later to the application of other approaches of molecular biology it has been possible to isolate the genes coding for a large number of receptor proteins.

The main conclusions from these studies can be summarized as follows. (1) Although membrane receptors have been found to belong to at least four different classes of proteins (Seifert, 1991), receptors for neurotransmitter are the products of two large gene superfam'ilies, firstly the family of receptor-ion channels or ligand-gated ion channels (Barnard et al., 1987; Stevens, 1987), and secondlythat of receptors which interact with GTP binding proteins. To the first family belong receptors such as the nicotinic cholinergic, the GABA, and benzodiazepine receptor, several glutamate receptors including the NMDA and AMPA receptor families and the serotonin 5-HT3 receptor. The prototypical receptor of the second family, i.e., that of receptors coupled to GTP binding proteins, is the p2-adrenergic receptor (Dohlman et al., 1987; O'Dowd et al., 1989). The remaining members of the adrenergic receptor family, dopaminergic receptors, muscarinic cholinergics, serotonin lA, lB, lC, ID and 2, several neuropeptide receptors such as those for tachykinin, neurotensin and many others are also members of this family. (2) These molecular biological studies have demonstrated beyond any doubt a high degree of diversity of receptors for a given transmittei. Thus, five different muscarinic receptor proteins have been cloned (Bonner, 1989), and the same is true for the group of dopamine and serotonin receptor

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families, which are formed by at least six different members each (Civelli et al., 1991; Julius, 1991). In the ion channel receptors, the diversity could be even greater. These results are a demonstration of the diversity of receptors for neurotransmitter, suspected from pharmacological binding studies. This increasing understanding of receptor structure and function at the gene and protein levels has had a clear impact on the approaches used to investigate their anatomical, cellular and subcellular localizations. In addition to the now classical radioligand binding techniques it is theoretically possible to develop probes for the protein itself by raising antibodies against synthetic peptides derived from the predicted amino acids sequence of a receptor or obtained from recombinantly produced receptor protein. It is also possible to estimate levels of mRNA for a given receptor protein by producing probes complementary to selected sequences of the mRNA or large fragments from the cDNA or RNA coding for this receptor. The topic of the present paper is the application of these approaches to the study of receptors in the human brain. Techniques for the visualization of receptors in human post-mortem materials. Radioligand binding autoradiography As mentioned above there are at least three different approaches which can be used to visualize receptors in intact tissue sections. The first one is the use of the radioligand binding and autoradiography to study receptor distribution. Historically this is the most widely used technique due to the availability of radioligands labeled at highly specific activities since at least 20 years (Palacios, 1984). Although initially this technique was used to label receptors in the intact living animal, it was only after the development of an in vitro approach that, for obvious reasons, this technique was applicable to study human material (Young and Kuhar, 1979). Interestingly, in recent years the development of techniques such as positron emission tomography

(PET) and single photon emission tomography (SPECT), has allowed for the visualization of receptors in the living human brain, which could be considered as a non-invasive in vivo “receptor autoradiography” (see for example Wagner et al., 1983).

The principles and applications of receptor autoradiography have been extensively reviewed (Kuhar et al., 1986). Basically in receptor autoradiography the principle of radioligand binding techniques is applied to intact tissue sections. Radioactively labeled compounds bind under appropriate conditions with high affinity to the receptor under study. For studies in the human brain, sections from tissues obtained at autopsy are incubated with labeled compounds in vitro. Autoradiograms are generated by apposing dry photographic emulsions in the form of sheetfilms or emulsion-coated coverslips to these labeled tissues. Boththedensityandthedistributionof binding sites can be determined quantitatively in microscopic areas enriched in the receptor under study and can, therefore, be orders of magnitude more sensitive than biochemical assays using tissue homogenates.

Factors influencing receptor autoradiography in human post-mortem materials Tissue preparation. The application of receptor autoradiographic techniques to human postmortem material has encountered problems of stability, variability, etc., of neurochemical and morphological parameters already known from previous studies (Perry and Perry, 1983; Swaab and Uylings, 1988) and, in addition, problems specificto the use of in vitro autoradiographic techniques (Palacios et al., 1986). The preparation and storage of human tissue for autoradiography present a number of difficulties. Our usual procedure is to overcomethem as follows. Immediately after autopsy, brain hemispheres are separated on ice. One cerebral hemisphere and one cerebellar hemisphere are fixed for routine neuropathological examination. The remainder of the brain as well as the entire brain-stem and upper

59

spinal cord are kept at 4°C for 60-90 min. Large blood vessels and meninges are then removed, and brain tissue is promptly cut into several blocks containing the different regions and nuclei, their size being compatible with standard microtome requirements (50 x 20 x 10 mm, approximately). Tissue blocks are immediately frozen to minimize ice-crystal artefacts, and are then placed in plastic bags, identified and stored at - 80°C. Tissue sections (10-20 pm) are cut, using a microtomecryostat, and are then mounted on gelatin-coated microscope slides and stored at - 20°C. Labeling of different neurotransmitter receptors is subsequently carried out according to standard protocols (Palacios et al., 1988). An important technical problem relates to the large size of the human brain compared to those of laboratory animals. The acquisition of whole-hemisphere human brain sections would avoid the process of cutting it into small blocks, with the added benefit of the visualization of receptors over many

related structures in the same sample. In this regard, the availability of large-stage microtomes represents a useful tool to obtain such sections. It is possible to hold the sections during cutting by means of flexible tapes glued on one side. However, this method has been found to seriously affect the binding properties of many neurotransmitter receptors. Although this problem has not been rigorously investigated, it is probably due to the solvent used to maintain the glue together with the physical support. During the freeze-drying of these tissues this solvent could act on membrane lipids affecting binding properties of receptors. Another procedure is to roll the hemisphere sections onto gelatin-coated lanterntype glass slides (Palacios et al., 1986).

Ante-mortem factors. Many factors, difficult to account for experimentally, have to be considered when interpreting results obtained with human postmortem material. Ante-mortem factors such as data on the functional state of the patient’s medication

Fig. 1 . Effects of age ( A ) ,post-mortem delay (B) and gender (C) on benzodiazepine receptor densities. Individual values of [3H] FLU binding in the nucleus accumbens (squares) and substantia nigra (circles) are plotted against the age and post-mortem delay of the subjects. Panel C shows the densities of benzodiazepine receptorsin the putamen (diamonds) and nucleus accumbens (squares) in females (closed symbols) and males (open symbols) separately. No significant changes in binding site densities are observed in relation to the investigated parameters. (From Zezula et al., 1988.)

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and other relevant clinical details are often incomplete or missing. Therefore, correlations between pathophysiological states of patients and levels of neurochemical parameters are difficult to establish. The influence of agony on a number of neurochemical parameters has been reported (Perry and Perry, 1983); to our knowledge, there is no systematic study on the influence of these parameters on binding sites. Gender did not have any significant effect on the binding sites examined until now. Age-related effects on binding sites appear to be most marked during the first 2 - 3 decades of life (CortCset al., 1987,1989;Zezulaet al., 1988;Camps et al., 1989). Fig. 1 illustrates the influence of age and gender for benzodiazepine receptors.

Post-mortem factors. At the present time there is abundant information available on the influence of post-mortem delay on the stability of binding sites. Binding sites have been reported to be relatively stable in animal studies simulating human postmortem conditions (Whitehouse et al., 1984). In human tissue delays up to 24 h (or more) did not affect in a significant way the binding sites examined until now (see CortCs et al., 1987,1989; Zezulaet al., 1988, as examples). The influence of post-mortem delay on benzodiazepine receptors is illustrated in Fig. 1. Receptors in human hypothalamus

The technique of receptor autoradiography has been extensively used to study receptors in human brain. It is interesting that few studies have addressed specifically the analysis of receptors in the human hypothalamus. A possible reason could be that in general the hypothalamus is not an area presenting high densities of the classical neurotransmitter receptors. Neverthelessmany different neurotransmitter receptors have been mapped in the hypothalamus as a part of detailed studies in the human CNS. Examples are muscarinic cholinergic (CortCset a1.,1987), dopamine D, (CortCs et al., 1989) and D, (Camps et al., 1989), serotonin 5-HT1(Pazos et al.,

1987a) and 5-HT, (Pazos et al., 1987b), somatostatin (Reubi et al., 1986), adenosine A, (Fastbom et al., 1987), benzodiazepines (Zezula et al., 1988) and galanin (Bonnefond et al., 1990), while no significant densities of adenosine A, (Martinez-Mir et al., 1991) or CCK (Diet1 et al., 1987)binding sites have been reported. Table I compares the densities of four of these receptors in several nuclei of the human hypothalamus. Figs. 2 - 4 illustrate the localization of some receptors at selected levels of the human hypothalamus. These results show that neurotransmitter receptors are very heterogeneously distributed in the hypothalamus. Table I shows that the highest levels of muscarinic cholinergic and somatostatin binding sites were seen in the tuberal nuclei while adenosine and benzodiazepine were enriched in the mammillary bodies. The comparison of the distribution of receptors in the human hypothalamus with that seen in experimental animals, e.g., in the rat, reveals interesting differences. An example could be the enrichment of some receptors in the human tuberal system. The use of receptor autoradiography in human post-mortem material allows also the analysis of (1) the pharmacological characteristics of different receptor binding sites in the same brain region (in this case the hypothalamus) and (2) the study of the correlation of the distribution of binding sites with that of the pre-synaptic markers. An example of the first point is shown in Fig. 3 where the characteristics of benzodiazepine binding sites in the tuberal formation are illustrated and compared with those in the amygdala. Using the triazolopyridazine CL 218872 it was possible to show that the tuberal nuclei were particularly enriched in type 1 benzodiazepine binding sites (Zezula et al., 1988). Another example is binding of [3H]Nmethylscopolamine (NMS), a non-selective muscarinic cholinergic ligand (CortCs et al., 1987)to the tuberal nuclei. [3H]NMS binding was poorly displaced by both pirenzepine (a so-called M, preferring ligand) and carbachol (a M, ligand), in-

61

Fig. 2. A , B . Examples of the localization of neurotransmitter receptors in the human hypothalamus. A . In the autoradiogram the highest densities of muscarinic cholinergic are seen in the nucleus tuberales (Tub) followed by the anterior nucleus in the thalamus. The other structures contain moderate levels of receptors. B. A section close to the one used for receptor autoradiography was stained for acetylcholine esterase activity, a “putative” pre-synaptic cholinergic marker. Acetylchofinesterase activity is higher in the reticular nucleus of the thalamus (Ret) and cell bodies of the nucleus paraventricularis hypothalami (PVH). Note weak activity of acetylcholinesterase in the mamillary bodies. Other abbreviations: A, nucleus anterior thalami; VA, nucleus ventralis anterior thalami; Ro, nucleus paramedianus rotundocellularis thalami; PH, nucleus posterior hypothalami; PVH, nucleus paraventricularis hypothalami; PeH, nucleus periventricularis hypothalami; ALH, area lateralis hypothalami; fx, fornix; CMm, corpus mamillare, nucleus medialis. Bar, 3 mm. (From CortCs et al., 1987.) C. Somatostatin receptors. Autoradiogram of a coronal section through the diencephalon. In the thalamus low densities of somatostatin receptors are present in the anterior (A) and ventral anterior (VA) nuclei; the nucleus paramedianus rotundocellularis (Pm) contains somewhat higher levels of binding. In the hypothalamus somatostatin receptors are heterogeneously distributed, densities being very low in the mamillary bodies (MB) and the lateral nucleus (LH), somewhat higher in the posterior (Po) and dorsomedial (DM) nuclei and markedly high in the tuberal nuclei (Tub). Bar, 5 mm. (From Reubi et al., 1986.)

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TABLE 1 Examples of neurotransmitter receptor distribution in human hypothalamus Hypothalamic nucleus

Lateral area Ventromedial nucleus Posterior nucleus Infundibular nuclei (arcuatus) Tuberal nuclei Mammillary bodies Preoptic area

Receptor Benzodiazepine’

Muscarinic cholinergic’

235 408 264 172 358 635 277

254 329 246 393 802 251 241

k 18 k 65

12 72 f 34 k 50 f 52

f k

i 17

f 19 i 11 i 51 i 40 f 16 k 27

~omatostatin~

26.8 t- 11.3 62.9 f 18.5 -

161.2 f 11.4 6.0 f 1.9 -

Adenosine A t

167 f 9.7 167 k 5.8 171 k 35.3 -

254 f 89.0 -

From Zezula et al. (1988). From Cortts et al. (1987). From Reubi et al. (1986). From Fastbom et al. (1987). Results are the mean & S.E.M. from 3 to 12 cases and expressed in fmol/mg protein, except for somatostatin receptors which are in dpm . 10-3/mg protein. The brain areas presenting the highest densities are given: benzodiazepine = hippocampus dentate gyrus, 784 f 41; muscarinic cholinergic = island of Calleja, 1225 f 75; somatostatin = entorhinal cortex, 244 f 5; adenosine A, = hippocampus, CAI, stratum radiatum, 820 f 71. - , Not measured.

dicating that this site could belong to a non-M1nonM2 class (Bonner, 1989). Examination of the correlation between the distribution of pre-synaptic markers and postsynaptic markers is illustrated in Fig. 4. In this study (Bonnefond et al., 1990) consecutive sections from the human hypothalamus were incubated with 1251 galanin to label receptors for the neuropeptide, and alternating sections were hybridized with a probe complementary to a selected region of the mRNA coding for this neuropeptide. The autoradiograms revealed the relationship between galanin expressing neurons and the distribution of galanin binding sites.

The combination of in situ hybridization histochemistry with receptor autoradiography is favored by the similarityand compatibility of tissue preparation in both techniques. The result is also produced in films that can be analyzed using the same type of equipment (Palacios et al., 1991). Combination of receptor autoradiography with other radiohistochemical, immunohistochemical or histochemical procedures is sometimes also possible, in particular if consecutive sections are used. Examples of these approaches could be the labeling of monoamine presynaptic terminals with antidepressants (CortCs et al., 1988), substance P immunoreactivity and its receptors (Diet1 et al., 1989) or acetylcholine

Fig. 3. Distribution of benzodiazepine receptor subtypes in the amygdala and hypothalamus. In the amygdala (panel A ) receptor concentrations are very high in the lateral (La) and granular (Gr) nuclei, high in the accessory basal nucleus (AcB) and moderate in the basal nucleus (Ba). Note that most of the benzodiazepine receptors in the amygdala belong to the type 11, since C l 218872 weakly displaces [3H]FLU binding as shown in panel B. [3H]FLU binding to the anterior part of the hypothalamus (panel C) demonstrates the presence of high densities of BZR in the ventromedial nucleus (VMH), intermediate densities in the nuclei tuberales (Tub) and low densities in the nucleus infundibularis (Inf) and area lateralis hypothalami (ALH). Panel D shows that in the nucleus anterior thalami (A) [3H]FLU binding is very sensitive to C1 218872. In contrast, in hypothalamic nuclei the displacement is only weak, especially in the tuberal nuclei. Bars, 3 mm. (From Zezula et al., 1988.)

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esterase and cholinergic receptors (Cortes et al., 1987). Double labeling attempts have not been carried out at the present time. The limitations of the autoradiographic technique have been discussed extensively (Kuhar et al., 1986). The most important is probably the lack of cellular resolution which is a problem intrinsic to the detection method used, ligand diffusion and scatter of radiation. Other limitations are related to the non-selective nature of some of the tools used. For example, many ligands that are described as selective for a receptor population have been shown to be less selective than expected once the multiple nature of the population of labeled receptors has been demonstrated by the use of cloned receptors. Parallel application of in situ hybridization techniques to establish the pharmacological nature of the receptor being labeled has been found very useful (Vilaro et al., 1992). Thus, by demonstrating the nature of the receptor being expressed by a particular neuronal population, the specificity of the binding sites examined can be clarified using receptor autoradiography with non-selective ligands (Palacios et al., 1991). Traditionally the approach to solve this problem has been the use of non-selective radioligands in combination with more selective unlabeled displacers (Cortes et al., 1987; Zezula et al., 1988, for examples).

Other techniques for receptor visualization

As mentioned in the introduction, recent advances in the biochemistry and molecular biology of receptors have stimulated the development of new tools useful in chemical neuroanatomy. Thus, antibodies against receptors and nucleic acid probes for the mRNA coding for these receptors have become available and allowed the application of immunohistochemistry and in situ hybridization techniques to the study of receptors in human postmortem materials.

Immunohistochemistry of receptors The number of antibodies against receptor proteins is still limited and very few have been applied to the study of human materials. There are, however, examples of the applicability of this technique. Richards et al. (1987) have demonstrated the distribution of benzodiazepine receptors in the human brain using a monoclonal antibody against purified benzodiazepine receptors. In general the results obtained with this technique are in very good agreement with those obtained using radioligand binding autoradiography. However, because of the higher resolution of immunohistochemistry it has been possible to demonstrate the association of these receptors sites with different parts of the

Fig. 4. Distribution of galanin mRNA and galanin binding sites in the human hypothalamus. Panels a, b and c show photographs of staining and autoradiograms generated either with 1251-galaninor with 32P-labeled G A L , probe in the anterior hypothalamus. a. Cresyl violet staining. b. Galanin binding sites. c. Galanin mRNA. Neurons expressing galanin mRNA were observed within the paraventricular and supraoptic nuclei. In those nuclei, the densities of galanin binding sites were low when compared with the ones g, h and i show photographs of staining and autoradiograms of the medial observed in the preoptic area. Panels d, e, hypothalamus. Panels d and g: cresyl violet staining; e and h: galanin binding sites; f and i: galanin mRNA. Neurons containing galanin mRNA were found in the anterior part of the arcuate nucleus and in the paraventricular nucleus. In contrast, a high density of galanin binding sites was found in the ventromedial nucleus. Panels j , k and / show photographs of staining and autoradiograms of the posterior hypothalamus. j . Cresyl violet staining. k. Galanin binding sites. /. Galanin mRNA. Neurons expressing galanin mRNA were observed in the arcuate nucleus and in scattered cells of the posterior nucleus whereas the highest density of galanin binding sites were found in the lateral hypothalamic nucleus. Abbreviations used: 3V, third ventricle; AHA, anterior hypothalamic area; ARC, arcuate nucleus; DMN, dorsomedial nucleus of the hypothalamus; f, fornix; IM, intermediate nucleus; LHN, lateral hypothalamic nucleus; ME, median eminence; mt, mammillothalamic tract; oc, optic chiasm; ot, optic tract; PHN, posterior hypothalamic nucleus; POA, preoptic area; PVN, paraventricular nucleus of the hypothalamus; SON, supraoptic nucleus; VMN, ventromedial nucleus of the hypothalamus. Scale bar, 5 mm. (From Bonnefond et al., 1990.)

66

neuron, including labeling of processes as well as cell bodies (Somogyi et al., 1989): Antibodies against serotonin 1A, dopamine or nicotinic receptors have been produced although the application of these antibodies to human post-mortem material is not described yet.

In situ hybridization of receptor mRNAs The application of in situ hybridization to human post-mortem materials is discussed in the contribution by Mengod et al. (this volume). As is mentioned there, the application of this technique has suffered from the same limitations as those described for receptor binding, namely the influence of postmortem factors, the pre-mortem factors, type of storage, etc. In particular in the case of the application of in situ hybridization for receptor messenger mRNA, an important parameter is the relatively low level of abundance of the message (mRNA) for these particular proteins which make in some cases the use of this technique very difficult because of the lack of adequate (low degree) sensitivity. However, this technique has been successfully applied in our laboratory in human materials to several receptors including dopaminergic (Mengod et al., 1991a,b), serotoninergic (Jin et al., 1992) and excitatory amino acid receptors (Garcia-Ladona, Palacios and Mengod, to be published). In situ hybridization permits the visualization of the perikarya where receptor mRNAs are synthesized, whereas receptor autoradiography reveals the localization of the receptor polypeptides themselves on their way to or at their final location (cf., Young et al., 1980). When comparing both techniques, an overlap of the signal patterns can be seen when receptors found in a given brain nucleus are synthesized by cells intrinsic to this nucleus. In contrast, if receptors visualized in one nucleus are synthesized by afferent neurons, whose perikarya are located in distant brain areas, the signal patterns will be complementary rather than overlapping (Palacios et al., 1991; Pompeiano et al., 1992).

Summary and conclusions In summary, we have illustrated and discussed the applicability of different techniques to the study of neurotransmitter receptors in the human brain. Because of the availability of these techniques it is possible today to examine in detail the alterations in density produced by different physiological or pathological conditions. The use of these techniques will in the future, without any doubt, be of help in understanding the chemical neuroanatomy of the human hypothalamus.

References Ariens, E.J. (1979) Receptors: from fiction to fact. Trends Pharmacol. Sci., 1: 11 - 15. Barnard, E.A., Darlison, M.G. and Seeburg, P. (1987) Molecular biology of the GABA, receptor: the receptor/channel superfamily. Trends Neurosci., 10: 502 - 509. Bonnefond, C., Palacios, J.M., Probst, A. and Mengod, G. (1990) Distribution of galanin mRNA containing cells and galanin receptor binding sites in human and rat hypothalamus. Eur. J. Neurosci., 2: 629- 637. Bonner, T.I. (1989) The molecular basis of muscarinic receptor diversity. Trends Neurosci., 12: 148 - 151. Camps, M., CortCs, R., Gueye, B., Probst, A. and Palacios, J.M. (1989) Dopamine receptors in human brain: autoradiographic distribution of D, sites. Neuroscience, 28: 275 - 290. Civelli, O . , Bunzow, J.R., Grandy, D.K., Zhou, Q.-Y. and Van Tol, H.H.M. (1991) Molecular biology of the dopamine receptors. Eur. J. Pharmacol., 207: 277 - 286. CortCs, R., Probst, A. and Palacios, J.M. (1987) Quantitative light microscopic autoradiographic localization of cholinergic muscarinic receptors in the human brain: forebrain. Neuroscience, 20: 65 - 107. Cortes, R., Soriano, E., Pazos, A., Probst, A. and Palacios, J.M. (1988) Autoradiography of antidepressant binding sites ,in the human brain: localization using [3H]-imipramine and [3H]-paroxetine. Neuroscience, 27: 473 - 496. CortCs, R., Gueye, B., Pazos, A., Probst, A. and Palacios, J.M. (1989) Dopamine receptors in human brain: autoradiographic distribution of D, sites. Neuroscience, 28: 263 - 273. Dietl, M.M., Probst, A. and Palacios, J.M. (1987) On the distribution of cholecystokinin receptor binding sites in the

67 human brain: an autoradiographic study. Synapse, 1 : 169- 183. Dietl, M.M., Sanchez, M., Probst, A. and Palacios, J.M. (1989) Substance P receptors in the human spinal cord: decrease in amyotrophic lateral sclerosis. Brain Res., 483: 39 - 49. Dohlman, H.G., Caron, M.G. and Lefkowitz, R.J. (1987) A family of receptors coupled to guanine nucleotide regulatory proteins. Biochemistry, 26: 2657 - 2664. Fastbom, J., Pazos, A., Probst, A. and Palacios, J.M. (1987) Adenosine A1 receptors in the human brain: a quantitative autoradiographic study. Neuroscience, 22: 827 - 839. Hall, Z.W. (1987) Three of a kind: the P-adrenergic receptor, the muscarinic acetylcholine receptor, and rhodopsin. Trends Neurosci., 10: 99 - 101. Jin, H . , Oksenberg, D., Askenazi, A., Peroutka, S.J., Rozmahel, R., Mengod, G., Yang, Y., Palacios, J.M. and O’Dowd, B.F. (1992) Identification and characterization of the human 5-hydroxytryptamine 1B receptor. J. Biol. Chem., in press. Julius, D. (1991) Molcular biology of serotonin receptors. Annu. Rev. Neurosci., 14: 335 - 360. Kuhar, M.J., De Souza, E.B. and Unnerstall, J.R. (1986) Neurotransmitter receptor mapping by autoradiography and other methods. Annu. Rev. Neurosci., 9: 27 - 59. Martinez-Mir, M.I., Probst, A. and Palacios, J.M. (1991) Adenosine A, receptors: selective localization in the human basal ganglia and alterations with disease. Neuroscience, 42: 697 - 706. Mengod, G., Vilaro, M.T., Landwehrmeyer, G.B., MartinezMir, M.I., Niznik, H.B., Sunahara, R.K., Seeman, P., O’Dowd, B.F., Probst, A. and Palacios, J.M. (1991a) Visualization of dopamine D,, D, and D, receptor mRNAs in human and rat brain. Neurochem. Int., in press. Mengod, G., Vilaro, M.T., Niznik, H.B., Sunahara, R.K., Seeman, P., O’Dowd, B.F. and Palacios, J.M. (1991b) Visualization of a dopamine D, receptor mRNA in human and rat brain. Mol. Brain Res., 10: 185-191. O’Dowd, B.F., Lefkowitz, R.J. and Caron, M.G. (1989) Structure of the adrenergic and related receptors. Annu. Rev. Neurosci., 12: 61 - 83. Palacios, J.M. (1984) Receptor autoradiography: the last ten years. J. Receptor Res., 4: 633 - 644. Palacios, J.M., Probst, A. and Cortes, R. (1986) Mapping receptors in the human brain. Trends Neurosci., 9: 284 - 289. Palacios, J.M., Cortes, R. and Dietl, M.M. (1988) A laboratory guide for the in vitro labelling of receptors in tissue sections for autoradiography. In: F.W. van Leeuwen, C.W. Buijs and 0. Pach (Eds.), Molecular Neuroanatomy, Elsevier, Amsterdam, pp. 95- 109. Palacios, J.M., Mengod, G., Vilaro, M.T. and Ramm, P. (1991) Recent trends in receptor analysis techniques and instrumentation. J. Chem. Neuroanat., 4: 343 - 353. Parascandola, J. (1980) Origins of the receptor theory. Trends

Pharmacol. Sci., 1: 189- 192. Pazos, A., Probst, A. and Palacios, J.M. (1987a) Serotonin receptors in the human brain, 111. Autoradiographic mapping of serotonin-1 receptors. Neuroscience, 21: 97 - 122. Pazos, A., Probst, A. and Palacios, J.M. (1987b) Serotonin receptors in the human brain, IV. Autoradiographic mapping of serotonin-2 receptors. Neuroscience, 21: 123 - 139. Perry, E.K. and Perry, R.H. (1983) Minireview: human brain neurochemistry - some post-mortem problems. Life Sci., 33: 1733- 1743. Pompeiano, M., Palacios, J.M. and Mengod, G. (1992) Distribution and cellular localization of mRNA coding for 5HT,, receptor in the rat brain: correlation with receptor binding. J. Neurosci., 12: 440-453. Reubi, J.C., CortCs, R., Maurer, R., Probst, A. and Palacios, J.M. (1986) Distribution of somatostatin receptors in the human brain: an autoradiographic study. Neuroscience, 18: 329 - 346. Richards, J.G., Schoch, P, Haring, P., Takacs, B. and Mohler, H. (1987) Resolving GABAA/benzodiazepine receptors: cellular and subcellular localization in the CNS with monoclonal antibodies. J. Neurosci., 7: 1866 - 1886. Seifert, G. (Ed.) (1991) Cell Receptors. Morphological Characterization and Pathological Aspects, Springer, Berlin. Somogyi, P., Takagi, H., Richards, J.G. and Mohler, H. (1989) Subcellular localization of benzodiazepine/GABAA receptors in the cerebellum of rat, cat and monkey using monoclonal antibodies. J. Neurosci., 9: 2197- 2209. Stevens, C.F. (1987) Channel families in the brain. Nature, 328: 198 - 199. Swaab, D.F. and Uylings, H.B.M. (1988) Potentialities and pitfalls in the use of human brain material in molecular neuroanatomy. In: F.W. van Leeuwen, C.W. Buijs and 0. Pach (Eds.), Molecular Neuroanatomy, Elsevier Science Publishers, Amsterdam, pp. 403 - 416. Vilaro, M.T., Wiederhold, K.H., Palacios, J.M. and Mengod, G. (1992) Muscarinic Miselective ligands also recognise M,receptors in the rat brain: evidence from combined in situ hybridization and receptor autoradiography. Synapse, in press. Wagner Jr., H.N., Burns, H.D., Dannals, R.F., Wong, D.F., Langstrom, B., Duelfer, T., Frost, J.J., Ravert, H.T., Links, J.M., Rosenbloom, S.B., Lukas, S.E., Kramer, A.V., and Kuhar, M.J. (1983) Imaging dopamine receptors in the human brain by positron tomography. Science, 221: 1264- 1266. Whitehouse, P.J., Lynch, D. and Kuhar, M.J. (1984) Effects of post-mortem delay and temperature on neurotransmitter receptor binding in a rat model of the human autopsy process. J. Neurochem., 43: 553 -559. Yamamura, H.I., Enna, S.J. and Kuhar, M.J. (1990) Methods in Neurotransmitter Receptor Analysis, Raven Press, New York. Young 111, W.S. and Kuhar, M.J. (1979) Autoradiographic

68 localization of benzodiazepine receptors in the brains of humans and animals. Nature, 280: 393 - 395. Young, 111, W.S., Wamsley, J.K., Zarbin, M.A. and Kuhar, M.J. (1980) Opioid receptors undergo axonal flow. Science, 210: 76-78. Zezula, J., CortCs, R.,Probst, A. and Palacios, J.M. (1988) Benzodiazepine receptor sites in the human brain: autoradiographic mapping. Neuroscience, 25: 771 - 795.

Discussion B.S. McEwen: Given the scatter of data, age and post-mortem delay, how do you tell what factors are involved? With the age, there must be different post-mortem delays; and with postmortem delay, there must be different patients ages? How do you sort this out? J.M. Palacios: One can use statistical procedures to examine age/gender/post-mortem effects and the influence of more than one of these factors. Until now we have not seen “important” changes in receptor densities (particularly for the last four decades of life) that can be traced to one (or more than one) of those parameters. D.F. Swaab: I agree with Bruce McEwen that we need more systematic studies on the importance of post-mortem interval both by comparing left and right halves with different times between death and fixation and by animal experiments (cf. Van Zwieten et al., 1991). J.M. Palacios: I agree more results are necessary to carefully analyze all these parameters. However, it is my personal impression, after years of work, that, at least for receptors, only small changes will be found. W.A. Scherbaum: You mentioned the possible influence of drugs upon receptor binding, but you did not give us data on that. I would expect that most people who die in hospital have obtained some drugs, so that it may be difficult to sort this out. J.M. Palacios: Quite a remarkable number of different drugs are given to people dying in hospitals. A “definitive” study of the influence of drugs in receptor binding will require a large, well-documented cohort of patients. This study has not been done until now. However, we have examined the effects of, e.g., chronic neuroleptic administration in schizophrenic or Huntington’s chorea patients and found no change when compared to control populations in several receptors.

N. Kopp: How long can you store sections at -20°C for autoradiography? J.M. Palacios: There is no systematic study on this topic. The evidence in our laboratory is that receptors in sections appear to be stable for years. We have used sections of up to 3 years old. H.P.H. Kremer: Regarding the receptor autoradiography data on muscarin and benzodiazepine receptors in the lateral tuberal nucleus (of which so little is known): could you confirm these data by in situ hybridization of the appropriate receptor subtype mRNA and discriminate between receptor expression on neuronal cell bodies or processes? J.M. Palacios: We have not done hybridization studies for these receptors in the hypothalamus. D.F. Swaab: I was struck by the magnificent staining of the intermediate nucleus (sexually dimorphic nucleus; SDN) by the galanin probe, but it seems as if its localization was more caudally than usual. Therefore, I wonder whether this was the intermediate nucleus of Braak (i.e., the SDN) or of Feremutch (1948) (i.e., vasopressin oxytocin-containing neurons between the SON and PVN? J.M. Palacios: These results were published in 1990 (Bonnefond et a]., 1990). We followed the description of Braak and Braak (1987).

References Bonnefond, C., Palacios, J.M., Probst, A. and Mengod, G (1990) Distribution of galanin mRNA containing cells anc galanin receptor binding sites in human and ra hypothalamus. Eur. J. Neurosci., 2: 629-637. Braak, H. and Braak, E. (1987) The hypothalamus of thc human adult: chiasmatic region. Anat. Embryol., 176 315 - 330. Feremutch, K. (1948) Die Variabilitat der cytoarchitektonischer Struktur des menschlichen Hypothalamus. Monatschr Psychiatr. Neurol., 116: 257 -283. Van Zwieten, E.J., Ravid, R., Van der Sluis, P.J., Sluiter A.A., Pool, Chr.W., Smyth, D. and Swaab, D.F. (1991) In creased vasopressin immunoreactivity in the rat brain after 2 post-mortem interval of 6 hours. Brain Res., 550: 263 - 267.

D.F. Swaab, M.A. Hofman, M. Mirmiran, R. Ravid and F.W. van Leeuwen (Eds.) Progress in Brnin Research, Vol. 93 0 1992 Elsevier Science Publishers B.V. All rights reserved.

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CHAPTER 6

Human hypothalamic and pituitary neuroendocrine function during in vitro perifusion Dennis D. Rasmussen Department of Reproductive Medicine, University of California, Sun Diego, La Jolla, C A 92093-0802, U.S.A.

Introduction

In vitro perifusion of hypothalamic and pituitary tissue is a technique which has been used extensively in animal studies, where it has been clearly demonstrated to complement and enhance other in vivo and in vitro methodologies for investigating neuroendocrine regulation. This technique has recently also been adapted for investigations with human tissue. In order to assess the utility of this methodology in the investigation of human hypothalamic and pituitary function, we will discuss (1) basic in vitro perifusion technology, (2) examples of the application of this technique to human hypothalamic and pituitary neuroendocrine regulation, and (3) the.strengths and limitations of this experimental approach. Methods

General Adult or fetal human hypothalamic or pituitary tissue is maintained in perifusion chambers at 37”C, as illustrated in Fig. 1. Oxygenated artificial cerebrospinal fluid flowing through the chamber bathes the tissue and is then collected in intervals into sample tubes. Changes in the tissue rate of release of a compound over time are then determined by assaying (e.g., radioimmunoassay) the content of the compound in the sequentially timed perifusate fractions.

Chambers The perifusion chambers currently in use in our laboratory are constructed from the barrels of plastic disposable 3 ml (to provide a chamber containing approximately 0.5 ml medium for perifusion of hypothalamic tissue) and 1 ml (for an approximately 0.1 ml chamber volume; pituitary tissue) syringes. As illustrated in Fig. 1, the perifusion medium (i.e., artificial cerebrospinal fluid) is delivered (A) into the original syringe outlet which is now at the bottom of the chamber. The medium flows up and around the tissue (C) which rests on a stainless steel screen, and then exits the chamber through a 22-gauge needle pierced through the rubber seal (from the disposable syringe plunger) which closes the chamber. We prefer these “home-made” perifusion chambers over those currently available commercially because the volume of the chamber can be easily regulated by adjusting the position of the effluent needle. In this way each chamber is individually “tailored” so that the medium just surrounds the tissue, minimizing dead space. Temperature regulation The most inexpensive method of maintaining the perifusion chamber and tissue at 37°C is to suspend them, as well as the tubing leading to the chamber, in a heated water bath. Alternatively, a commercially available perifusion system (e.g., ACUSYST-S Perifusion Culture System, Endotronics, Inc., Menneapolis, MN, U S A . ) provides convenient

70 A-=

r=-D

Y

E

Fig. 1. Perifusion chamber. A, Inlet; B, injection port or alternate inlet; C, mediobasohypothalamus (MBH) or pituitary tissue; D, outlet; E, 37°C water bath or incubation chamber.

temperature regulation in an enclosed incubation cavity. We monitor the temperature of the tissue chamber throughout the experiment by means of a YSI Model 43 Tele-Thermometer (Yellow Springs Instrument Co,, Yellow Springs, OH, U.S.A.) with a thermoprobe attached directly to the side of the chamber.

Perifusion medium We have employed perifusion media (artificial cerebrospinal fluid) based on either a bicarbonate buffer system (e.g., Medium 199, Grand Island Biological Co., Grand Island, NY, U.S.A., oxygenated with 95% 0 , - 5 % COJ or a phosphate buffer system (Robinson, 1949). In both cases, 0.1% crystalline bovine serum albumin (Pentex, Miles Laboratories, Elkhart, IN, U.S.A.), 5 pg insulin/ml (approximately 120mIU/ml), 50 units streptomycin sulphate/ml, and 50 units penicillin G sodium/ml are added and the glucose concentration is adjusted to 10 mM if necessary (i.e., for the phosphate medium). The pH is then adjusted to 7.4 and the medium is positive pressure filter-sterilized. During the perifusion a Micro Flow Through pH Monitor (LAZAR Research Laboratories, Inc., Los Angeles, CA, U.S.A.) placed in line immediately “down-

stream” from a perifusion chamber which does not contain tissue is used to confirm that the medium perfusing the chamber is maintained at pH 7.3 - 7.4. We have not detected differences in the levels of peptides or proteins released by hypothalamic or pituitary tissue due to the use of bicarbonate-based vs. phosphate-based buffer systems. However, phosphate-buffered media do maintain a stable pH in the sample tubes after effluent fraction collection (i.e., while exposed to air), which can help to avoid some problems during subsequent radioimmunoassay quantification. Oxygenation The perifusion medium can be oxygenated in several ways. The simplest and still most common is to constantly bubble the medium with 95% 0, - 5 % CO, (for a bicarbonate-based buffer) or air (for a phosphate-based buffer). However, we have found that this method tends to produce inconsistent pH changes in the medium, especially when a bicarbonate buffer system is used. An inexpensive and effective alternative is to deliver the medium through 3 ft. of gas-permeable 0.020 inch i.d. X 0.037 inch 0.d. silastic medical grade tubing (Dov Corning Corp., Midland, MI, U.S.A.) which ir enclosed in a chamber flushed with 95% 0, - 5 % CO, or air (depending on the buffer in the medium: as described by Takahashi et al. (1980). A variatior on this method is used in the ACUSYST-S Perifu, sion Culture System, in which both the perifusior chamber and silastic tubing leading to the chambei are maintained in an enclosed chamber which is con stantly flushed with 95% 0, - 5% CO,. This desigr eliminates the necessity for freshly oxygenate( medium to flow through additional exposed tubin! before delivery into the incubation chamber, whicl is important because it has been shown that 0, cai be lost through the walls of plastic (e.g., poly ethylene) tubing and that the resulting decreasec PO, in the perifusion medium can decrease thj amount of peptide released from hypothalami1 tissue (Joanny et al., 1989). For this reason, we hav adopted the ACUSYST-S perifusion system for ou studies. Furthermore, whenever perifusion condi

71

tions are altered (e.g., a change in flow rate or medium composition) we confirm that 0, saturation has not been compromised with the aid of an in line Micro Flow Through PO, Electrode (LAZAR Research Laboratories, Inc., Los Angeles, CA, U.S.A.) connected to the effluent from a perifusion chamber which does not contain tissue.

Flow regulation The pump which is used to deliver the medium to the chamber must be of a low pulsation design, such as the Rainen Rabbit Peristaltic Pump (Rainen Instrument Company, Inc., Woburn, MA, U.S.A.) or the pump component of the ACUSYST-S perifusion system. This is necessary in order to minimize disturbance of the tissue, which can induce artefactual release of secretory products and decrease tissue viability. We have further minimized exposure of the tissue to the effects of pump pulsations by moving the pump to a position “downstream” from the perifusion chamber (i.e., between the perifusion chamber and the fraction collector) and elevating the medium reservoir so that medium is delivered to the perifusion chamber at an appropriate rate by gravity alone. In this way the pump simply serves as a flow regulator.

Administration of test substances An injection port in the medium delivery tubing (B in Fig. 1)can allow bolus administration of small volumes (e.g., 10- 25 ~ 1 of ) test substances into the flow of the medium before it enters the perifusion chamber. However, this must be done carefully, since the change in flow resulting from an abr‘upt injection of even these small amounts can induce artefactual release of secretory products by the perifused tissue. A much safer method, and one which allows convenient administration of repetitive or more prolonged pulses without artefactual tissue stimulation, utilizes a microprocessorbased timer (Chron Trol, Lindburg Enterprises, San Diego, CA, U.S.A.) and DC power supply to regulate solenoid-activated stream switching valves (Rainen Instrument Company, Inc., Woburn, MA, U.S.A.) which switch the flow between media of dif-

ferent composition in milliseconds. Alternatively, a commercially available automatic pulsatile delivery system (APS10, Endotronics, Inc., Minneapolis, MN, U.S.A.) can be used to deliver a test substance in pulses of varying wave forms in the medium flow, as described by Negro-Vilar and Culler (1986).

Sample collection The effluent is delivered (D) by Teflon tubing through a port in the side of a chromatography refrigerator to an automated fraction collector (FC80 Microfractionator, Gilson Medical Electronics, Middleton, WI, U.S.A., or Model 21 10 Fraction Collector, BIO-RAD Laboratories, Richmond, CA, U.S.A.) which is maintained at 4°C. Preservatives Many investigators add peptidase inhibitors to the perifusion medium in order to minimize enzymatic degradation of peptides which are secreted by the tissue. However, peptidase activity appears to be a mechanism by which in vivo processing and secretion of hypothalamic peptides are regulated (Adviset al., 1983; Pierottiet al., 1991), so weprefer to omit peptidase inhibitors from the medium and thus avoid the possibility of artefactually altering secretory functions. Instead, we maintain a relatively high perifusion rate relative to chamber size in order to quickly remove secretory products from further exposure to the tissue, minimize the length of tubing between the perifusion chamber and the refrigerated fraction collector so that the effluent medium is refrigerated as quickly as possible, and add peptidase inhibitors such as bacitracin (Sigma, St. Louis, MO, U.S.A.) or Trasylol (Research Plus, Inc., Bayonne, NJ, U.S.A.) to the fraction collection tubes. Under these conditions we have detected no decrease in detected levels of peptides in the effluent medium compared to levels with these same peptidase inhibitors added directly to the perifusion medium. Hypothalamus

We initially investigated gonadotropin-releasing hormone (GnRH) and 0-endorphin (@END)release

12

from hypothalami of adult men and women obtained at autopsy within 24 h post-mortem. The mediobasohypothalamus (MBH) was removed with sagittal cuts in the lateral sulci, transverse cuts through the rostra1 edges of the mammillary bodies and optic chiasm, and a horizontal cut at a depth of 3 - 4 mm. The optic chiasm was removed, the MBH was bisected at the midline, each half-MBH was cut into four coronal sections, and all four pieces of an individual half-MBH were perifused in a single chamber, separated by stainless steel screens. Adult human MBH tissue obtained within 12 h post-mortem initially released relatively large and variable quantities of both GnRH (Rasmussen et al., 1986b) and @END(Rasmussen et al., 1987) and this large initial release declined within 1.5 h to basal levels which were not consistently correlated with age, sex or latency from death (within 12 h) (Fig. 2). The release of both peptides was stimulated in response to 56 mM potassium (Fig. 2), and since equimolar sodium did not alter the release of either peptide (Fig. 2) this response to potassium was apparently due to specific membrane depolarization

I

rather than non-specific response to monovalent ions or osmotic changes. Release of both peptides was also stimulated by veratridine (Fig. 2), an alkaloid which depolarizes cells by inactivating the sodium conductance mechanism of functional membrane sodium channels, thereby increasing sodium permeability (Ohta et al., 1973; Conn and Rogers, 1980). In addition, the stimulation of both peptides by 56 mM potassium was suppressed during perifusion with calcium-free medium (Fig. 2), consistent with the requirement for calcium in the normal active secretory response to depolarization in many neuronal systems (Kelly et al., 1979). Thus, the increased release of both GnRH and @ENDin response to 56 mM potassium but not sodium, the stimulation by the sodium channel activator veratridine, and the suppressed response to potassium in the absence of calcium demonstrated that the fundamental secretory properties of the human MBH tissue obtained within 12 h post-mortem were satisfactorily preserved. In contrast, tissue obtained more than 12 h post-mortem did not reliably respond to these physiological challenges in a consistent fashion, but responded erratically or released undetectable levels of these peptides. In additional studies with this human hypothalamic tissue obtained within 12 h post-mortem, we demonstrated that dopamine stimulated the release of both GnRH and @END release and that this stimulation was mediated by a dopamine receptormediated mechanism, since the responses to dopamine were prevented by administration of the dopamine receptor antagonist haloperidol but not the adrenergic receptor antagonist phentolamine (Rasmussen et al., 1986b, 1987). These results demonstrate that membrane receptor mechanisms in this tissue remained functional. We have also used this methodology to investigate release of GnRH and PEND from human fetal hypothalamic tissue (Rasmussen et al., l983,1986a, 1989; Rasmussen, 1989). This hypothalamic tissue was obtained at autopsy immediately after prostaglandin FZa-and urea-induced termination and delivery at 21 - 23 weeks gestation. Initial studies determined that in vitro GnRH release tended to

73

be erratic or below detection level if more than 6 h had elapsed between induction and delivery. So, hypothalamic tissue was subsequently perifused for experimental studies only if the time between induction and delivery was less than 6 h. In the initial studies the MBH was removed with sagittal cuts in the hypothalamic sulci, coronal cuts through the rostra1 edges of the mammillary bodies and optic chiasm, and a horizontal cut at a depth of 3 mm, and the optic chiasm and tracts were trimmed from the tissue. Each MBH was quartered and the two pieces of each half-MBH were perifused together in one chamber, separated by stainless steel screens. Perifusion was initiated within 60 - 90 min of delivery. As with the adult hypothalamic tissue, functional viability of the fetal tissue was confirmed by a rapid release of GnRH in response to a depolarizing dose of KC1 but no response to equimolar administration of NaCl (Rasmussen et al., 1983). Also similar to adult tissue, dopamine administration stimulated GnRH release in a dose-dependent and dopamine receptor-mediated fashion (Rasmussen et al., 1986a). Furthermore, the fetal hypothalamic tissue responded to administration of the opiate receptor antagonist naloxone with increased release of GnRH, and this response to naloxone was inhibited by simultaneous administration of equimolar PEND (Rasmussen et al., 1983). This increased release of hypothalamic GnRH in response to opiate receptor blockade is consistent with a great deal of compelling evidence (see Rasmussen, 1.991, for review) which supports the hypothesis that endogenous hypothalamic opioid-mediated mechanisms have an inhibitory role in the physiological regulation of GnRH secretion. Thus, even though the neuroendocrine activity of the fetal hypothalamus may differ from that of the adult, these results are consistent with a variety of evidence which suggests that the human hypothalamic-pituitary system forms a well-differentiated functional unit by midgestation (Kaplan et al., 1976; Decherney and Naftolin, 1980; Gluckman et al., 1981). Accordingly, these results suggest that the human fetal MBH may provide a useful model for the investigation of hypothalamic neuronal and neuroendocrine function in humans.

Although these studies indicated that perifused adult and fetal human hypothalamic tissues remain functionally viable with at least fundamental secretory capacity as well as receptor-mediated mechanisms intact under the conditions which have been described, they do not resolve whether the tissue is functioning in a relatively “normal” physiological manner. Since in vitro release of GnRH from the isolated MBH of the rat and guinea pig has been demonstrated to be pulsatile (Levine. and Ramirez, 1986; McKibbin and Belchetz, 1986; Bourguignon et al., 1987) and in vivo deafferentation of the rat and monkey MBH allows pulsatile LH secretion (which is dependent upon pulsatile secretion of hypothalamic GnRH into the hypothalamo-hypophyseal portal system) to be maintained (Blake and Sawyer, 1974; Krey et al., 1975; Ferin et al., 1977), it appears that all of the necessary neural elements comprising the GnRH “pulse generator” are not only resident within the MBH but capable of functioning as an intrinsic pacemaker independent of neural innervation from the remainder of the brain. Thus, if the human MBH tissue which is perifused in vitro is indeed functioning in a “normal” physiological manner, it should be possible to demonstrate that the in vitro MBH releases GnRH in a pulsatile manner. We have addressed this issue by modifying the perifusion conditions to accomodate resolution of acute changes in GnRH release from the human fetal hypothalamus, i.e., the intact MBH (as opposed to several MBH pieces, trimmed more closely to extend laterally only approximately 2 mm from the midline and only approximately 2 mm in depth) was perifused in a chamber containing only 150 pl of medium (as opposed to approximately 300 pl) in addition to the tissue, the flow rate of the medium perfusing the chamber was increased from 100 to 150 pl/min, the fraction collection interval was decreased from 15 to 10 min, the perifusate fractions were lyohilized so that when reconstituted in a smaller volume of assay buffer the entire fraction could be utilized for radioimmunoassay determinations, and these reconstituted perifusate fractions were assayed in triplicate instead of duplicate. Under these conditions, GnRH release during

74

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perifusion of four fetal MBHs (3 females, 1 male) with medium alone was distinctly and consistently pulsatile throughout an entire 13 h experimental period, with a mean ( & S.E.) pulse interval of 58 f 4 min (Fig. 3) (Rasmussen et al., 1989). In order to confirm that the fluctuations in GnRH levels during these perifusions represented pulsatile GnRH release rather than fluctuations arising from experimental procedures or assay variability, we then conducted another perifusion with GnRH added to the medium perfusing an empty 150 pl chamber. When the perifusate fractions were collected, frozen, lyophilized, reconstituted, assayed and analyzed identically to the experimental samples, no significant GnRH pulses were detected (Rasmussen et al., 1989). Furthermore, addition of the membrane calcium channel blockers verapamil(50 pM) and nifedipine (5 pM) dramatically suppressed this pulsatile GnRH release during another perifusion (Rasmussen et al., 1989). Although GnRH release during perifusion of an MBH obtained 6 h postmortem from a 39-year-old woman was somewhat erratic, there were several pulses that were determined to be significant, most commonly with an

80 - 90 min pulse interval (Fig. 4). When an MBH obtained 3.25 h post-mortem from a 65-year-old man was bisected longitudinally at the midline of the median eminence, each half-MBH released GnRH in a pulsatile manner with significant pulses at intervals of 63 & 8 and 100 t 10min (Fig. 4).Thus fetal human MBHs released GnRH in a pulsatile and calcium-dependent manner with a periodicity of approximately 1 h, and adult human MBHs also released GnRH in a pulsatile manner, with a periodicity of 60- 100 min. These results indicate that the human hypothalamic GnRH pulse-generating mechanism is located entirely within the MBH, and this pulse generator can maintain intrinsically pulsatile GnRH release independent of all innervation from outside this site. Furthermore, the periodicity of pulsatile GnRH release from the human MBH in vitro is similar to the periodicity of pulsatile LH, and presumably GnRH, secretion in vivo (Yen et al., 1972; Marshall and Kelch, 1986). In another study (Rasmussen et al., 1989) five fetal MBHs (3 males, 2 females) were perifused with

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15

medium alone for the first 5 h, with 10 pM morphine sulphate added to the medium for the next 6 h, and with both 10 pM morphine sulphate and 10 pM naloxone hydrochloride added to the medium for the final 2 h. Addition of morphine to the medium midway during the perifusion reduced the frequency of GnRH pulses whereas subsequent addition of the opioid receptor antagonist naloxone restored the frequency (Fig. 5). These results indicate that human hypothalamic pulsatile GnRH release can be modulated by an opiate receptor-mediated mechanism within the MBH. This opiate receptor-mediated reduction of pulsatile GnRH release is similar to opioid suppression of pulsatile LH, and presumably GnRH, secretion in humans in vivo (Ropert et al., 1981).Furthermore, these results are consistent with in vivo experimental evidence in other species: (1) 15

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morphine administration to monkeys suppressed hypothalamic electrophysiological activity associated with the pulsatile release of LH, and this suppression was reversible with naloxone (Kesner et al., 1986); (2) microinjection of antiserum to PEND and dynorphin into the arcuate nucleus of rats stimulated LH release (Schulz et al., 1981); and (3) administration of naloxone increased the amplitude and frequency of GnRH pulses in the hypophyseal portal blood of castrated rams (Caraty et al., 1987). So, these results suggest that the opioid modulation of pulsatile GnRH release from these human MBHs maintained in vitro is similar to that which has been demonstrated in vivo. Overall, these results suggest that perifusion of the human MBH can provide a good model for investigating some basic mechanisms of neuroendocrine regulation within the human hypothalamus.

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Pituitary

The perifusion of human pituitary tissue provides a methodology for investigating the direct regulation of human pituitary hormone secretion. For example, we have used in vitro perifusion to examine luteinizing hormone (LH) release from human fetal (19 - 24 weeks gestation) anterior pituitaries in response to repetitive GnRH stimulations at concentrations comparable to those found in human portal blood (Antunes et al., 1978)and intervals consistent with those of in vitro pulsatile GnRH release from mid-gestational human fetal MBHs (Rasmussen et al., 1989), administered throughout a 24 h period (Rossmanith et al., 1990b). Pituitaries (5 male, 4 female) were dissected into halves, and one hemipituitary of each pair was stimulated with 10 min pulses of 1 nM GnRH administered at 60 min intervals over 24 h, whereas the matching hemipituitary received pulses of medium alone. As shown in Fig. 6, the repetitive pulses of GnRH elicited increased LH release throughout the 24 h perifusion of female hemipituitaries, simulating normal in vivo pulsatile LH secretion. Also, early in the perifusions these hemipituitariesreleased more LH in response to successive GnRH pulses, consistent with a “priming”

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effect of pulsatile GnRH stimulation which has been demonstrated invivo in both the human (Hoff et al., 1979) and rat (Fink et al., 1976). Hourly pulses of GnRH did not significantly alter LH release from male hemipituitaries (Rossmanith et al., 1990b). This sex difference in the response to a physiological pattern of GnRH stimulation in vitro is consistent with the higher serum LH levels, pituitary LH content and concentration, and pituitary LH release in response to GnRH stimulation which have previously been demonstrated in the human female vs. male fetus and mid-gestation (Dumesic et al., 1987). In other similar studies we have demonstrated that perifused human fetal pituitaries also respond to stimulation by physiological concentrations of CRF with large increases in both PEND and adrenocorticotropin (ACTH) release, consistent with the in vivo response (Gambacciani et al., 1987a; Rossmanith et al., 1988). So, it appears that the human fetal pituitary responds to at least GnRH and CRF stimulation in a relatively physiological manner when maintained by in vitro perifusion. An advantage of this technique which had not been fully appreciated until recently is the ability to

investigate very rapid and transient changes in hormone secretion, i.e., changes which would be unresolvable when diluted in the large circulating plasma and tissue distribution volume. We initially investigated these changes with human fetal anterior pituitaries perifused at a high flow rate (0.4 ml/min) in a small chamber (50 pl) with perifusate fractions collected at 2 min intervals. These studies showed that LH, ACTH and PEND release were all characterized by high-frequency (i.e., at intervals of 9 - 12 min)/low-amplitude pulses, independent of hypothalamic stimulation (Gambacciani et al., 1987a,b; Rossmanith et al., 1988). Furthermore, the high-frequency/low-amplitude secretion of each of these hormones was suppressed by addition of the membrane calcium channel blocker verapamil hydrochloride and the calcium chelator EGTA to the medium (Gambacciani et al., 1987a,b; Rossmanith et al., 1988). In addition, when adult anterior pituitaries were quartered and each quarter was perifused individually, these adult anterior pituitary quarters also released LH, ACTH and PEND in an intrinsically pulsatile pattern, with pulse intervals of 11 - 15 min (Gambacciani et al.,

77

1987a,b; Rossmanith et al., 1988). This spontaneous calcium-mediated pulsatile release of ACTH, PEND and LH suggests the activity of intrinsic intrapituitary pulse-generating mechanisms for these hormones. The similarity of the periodicities of ACTH, PEND and LH release suggested that the basal (i.e., non-stimulated) low-amplitude pulsatile release of these hormones might be entrained by a common mechanism. We addressed this issue by using an even higher resolution perifusion system employing very frequent (30 sec) perifusate sample collections to simultaneously assess both LH and immunoreactive BEND release from individual adult human anterior pituitaries (Rossmanith et al., 1990a). Each of six anterior hemipituitaries released LH and PEND in a distinctly pulsatile fashion (Fig. 7). The

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periodicities of the LH and PEND pulsatile release were remarkably similar, with a pulse occurring every 3.2 & 0.1 min for LH and every 3.2 f 0.2 min for PEND. The decreased pulse interval during perifusions of the adult pituitaries with sample collection every 30 sec (approximately 3.2 min) relative to the pulse interval determined with sample collections every 2 min (9 - 12 min) probably reflects increased ability to resolve acute peaks. This hypothesis is consistent with previous in vivo studies which have demonstrated that intensified rates of venous blood sampling unmask the presence of high-frequency LH pulsations in man (Veldhuis et al., 1984). Of the 152 LH and 149 BEND pulses detected in all six perifusions, 92 of each were synchronized, i.e., the hormone peaks were coincident or occurred within one time lag (30 sec). Cross-

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78

correlation analysis revealed that changes in LH and @ENDlevels within individual perifusions exhibited highly significant ( P < 0.001) positive correlations at zero time lags, indicating that the correlation between these hormone levels was high only when the LH and PEND data series were not time-shifted against each other, and confirming that LH and PEND pulsatile release tended to be synchronized. Power spectral analysis revealed that both LH and PEND were released in significantly rhythmic patterns during four of the six perifusions, with periodicities of 3.1 0.4 min and 3 . 2 f 0.4 min, respectively. In another perifusion LH release was determined to be rhythmic (period of 3.3 min) whereas PEND was not, and in the final perifusion neither LH nor @ENDrelease was determined to be rhythmic. In the four perifusions in which both LH and @ENDrelease were rhythmic, the periodicity of the LH and PEND rhythms were correlated (r = 0.97). These results confirm that spontaneous release of both LH and @END from the human anterior pituitary in vitro is pulsatile. In addition, they demonstrate that the LH and @ENDpulses tended to be rhythmic and occur in synchrony with each other, suggesting a common intrapituitary pulse generating mechanism. Since these pulses occur with a very high frequency and low amplitude it is unlikely that this phenomenon could be resolved reliably with in vivo methods, since the small frequent release of these hormones would be confounded by mixing and dilution in the large circulating plasma volume. Strengths and weaknesses of the perifusion methodology This methodology offers several advantages. For example, interactions between neuroendocrine factors directly and exclusively at pituitary or hypothalamic sites without confounding intermediary responses (e.g., other brain site, pituitary, adrenal, ovarian, blood flow or metabolic responses) can be readily investigated. Furthermore, the concentration, pattern (pulsatile, constant, gradient) and duration (acute, chronic) of administration of one

or several potentially interacting modulators can be easily controlled. The metabolic stability of the compound of interest and the administered modulators is also much greater in the defined culture medium than when released or injected in vivo, with no interference of anesthetics, and isolation and analysis of a secreted compound is easier and more reliable than when the compound is secreted into the cerebrospinal fluid or the blood. Rapid changes in transmitter or hormone release can also be resolved, since the total amount released is collected (rather than being diluted in circulating plasma) and sample collection can be frequent. In contrast to cell culture techniques, in situ neuronal networks and cell-cell contacts are maintained and secretory products and metabolites are removed without prolonged contact with the cells, thus more accurately simulating in vivo conditions. Finally, as opposed to in vivo brain or pituitary microdialysis or push-pull cannulation, or hypothalamohypophyseal portal blood sampling, this technique allows direct investigation of human physiological mechanisms. There are also significant limitations on the utility of this technique. The first is common to all in vitro techniques, i.e., it is difficult to confirm whether the activity of the tissue in vitro accurately reflects activity in vivo. This limitation must be considered to be particularly important in these studies with human tissue in which there is necessarily a delay between time of death and subsequent removal and perifusion of the tissue. In particular, it must be assumed that populations of cells that are different in terms of either function or their position within the piece of perifused tissue most probably also have different viability or extent of altered activity under these in vitro conditions. Furthermore, isolation of a piece of brain tissue from other inputs may alter the function of cell populations within the tissue. It is for this reason that we have limited our investigations of hypothalamic function to that of the GnRH- and PEND-producing cells, whose entire populations or perikarya as well as many terminals are located entirely within the MBH. Finally, the presence of a compound in the perifusate effluent

79

reflects overall release from the tissue, and does not resolve the potentially inconsistent activity of individual subpopulations within the tissue which may have contrasting regulation and function. Thus, even though the function of small pieces of postmortem hypothalamic and pituitary tissue appear to be remarkably “physiological” in our studies, it must be assumed that functions demonstrated with this technique reflect only the presence of mechanisms capable of mediating the function, and not that this is necessarily how the tissue normally functions in vivo. For this reason in vitro perifusion results must always be considered in the context of results obtained with other complementary experimental methods. Acknowledgements

This work was supported by NIH grants HD-22608 and HD-12303, Unit 4. References Advis, J.P., Krause, J.E. and McKelvy, J.F. (1983) Evidence that endopeptidase-catalyzed luteinizing hormone releasing hormone cleavage contributes to the regulation of median eminence LHRH levels during positive steroid feedback. Endocrinology, 112: 1147 - 1149. Antunes, J.L., Carmel, P.W., Housepian, E.M. and Ferin, M. (1978) Luteinizing hormone-releasing hormone in human pituitary blood. J. Neurosurg., 49: 382- 386. Blake, C.A. and Sawyer, C.H. (1974) Effects of hypothalamic deafferentation on the pulsatile rhythm in plasma concentrations of luteinizing hormone in ovariectomized rats. Endocrinology, 94: 730 - 736. Bourguignon, J., Gerard, A., Debougnoux, G., Rose, J . and Franchimont, P. (1987) Pulsatile release of gonadotropinreleasing hormone (GnRH) from the rat hypothalamus in vitro: calcium and glucose dependency and inhibition by superactive GnRH analogs. Endocrinology, 121: 993 - 999. Caraty, A., Locatelli, A. and Schanbacher, B. (1987) Augmentation par la naloxone de la frequence et de l’amplitude des pulses de LH-RH dans le sang porte hypothalamohypophysaire chez le belier castre. C.R. Acad. Sci. Paris, 305: 369- 374. Conn, P.M. andRogers, D.C. (1980)Gonadotropinreleasefrom pituitary cultures following activation of endogenous ion channels. Endocrinology, 107: 2133 -2134. Decherney, A. and Naftolin, F. (1980) Hypothalamic and pituitary development in the fetus. Clin. Obstet. Gynecol., 23: 749 - 761.

Dumesic, D.A., Goldsmith, P.C. and Jaffe, R.B. (1987) Estradiol sensitization of cultured human fetal pituitary cells to gonadotropin-releasing hormone. J. Clin. Endocrinol., Metab., 65: 1147-1153. Ferin, M., Antunes, J.L., Zimmerman, E., Dyrenfurth, I., Frantz, A.G., Robinson, A. and Carmel, P.W. (1977) Endocrine function in female rhesus monkeys after hypothalamic disconnection. Endocrinology, 101: 1611 - 1620. Fink, G., Chiappa, S.A. and Aiyer, M.S. (1976) Priming effect of luteinizing hormone releasing factor elicited by preoptic stimulation and by intravenous infusion and multiple injections of the synthetic decapeptide. J. Endocrinol., 69: 359- 372. Gambacciani, M., Liu, J.H., Swartz, W.H., Tueros, V.S., Rasmussen, D.D. and Yen, S.S.C. (1987a) Intrinsic pulsatility of ACTH release from the human pituitary in vitro. Clin. Endocrinol. (Oxf.), 26: 557 - 563. Gambacciani, M., Liu, J.H., Swartz, W.H., Tueros, V.S., Yen, S.S.C. and Rasmussen, D.D. (1987b) Intrinsic pulsatility of luteinizing hormone release from the human pituitary in vitro. Neuroendocrinology, 45: 402 - 406. Gluckman, P.D.,Grumbach, M.M. andKaplan,S.L. (1981)The neuroendocrine regulation and function of growth hormone and prolactin in the mammalian fetus. Endocr. Rev., 2: 363 - 395. Hoff, J.D., Lasley, B.L. and Yen, S.S.C. (1979) The functional relationship between priming and releasing actions of luteinizing hormone-releasing hormone. J. Clin.Endocrinol. Metab., 49: 8 - 11. Joanny, P., Steinberg, J., Zamora, A.J., Conte-Devolx, B., Millet, Y. and Oliver, C. (1989) Corticotropin-releasing factor release from in vitro superfused and incubated rat hypothalamus. Effect of potassium, norepinephrine, and dopamine. Peptides, 10: 903 - 91 1. Kaplan, S.L., Grumbach, M.M. and Aubert, M.L. (1976) The ontogenesis of pituitary hormones and hypothalamic factors in the human fetus: maturation of central nervous system regulation of anterior pituitary function. Recent Prog. Horm. Res., 32: 161-243. Kelly, R.B., Deutsch, J.W., Carlson, S.S. and Wagner, J.A. (1979) Biochemistry of neurotransmitter release. Annu. Rev. Neurosci., 2: 299 - 321. Kesner, J.S., Kaufman, J.M., Wilson, R.C., Kuroda, G. and Knobil, E. (1986) The effect of morphine on the electrophysiological activity of the hypothalamic luteinizing hormonereleasing hormone pulse generator in the rhesus monkey. Neuroendocrinology, 43: 686 - 688. Krey, L.C., Butler, W.R. and Knobil, E. (1975) Surgical disconnection of the medial basal hypothalamus and pituitary function in the rhesus monkey. Gonadotropin secretion. Endocrinology, 96: 1073- 1087. Levine, J.E. and Ramirez, V.D. (1986) Measurement of neuropeptide release: in vitro and in vivo procedures. Methods Enzymol., 124: 466 - 494.

80 Marshall, J.C. and Kelch, R.P. (1986)Gonadotropin-releasing hormone: role of pulsatile secretion in the regulation of reproduction. N. Engl. J. Med., 315: 1459- 1468. McKibbin, P.E. and Belchetz, P.E. (1986)Prolonged pulsatile release of gonadotropin-releasing hormone from the guinea pig hypothalamus in vitro. Life Sci., 38: 2145-2150. Negro-Vilar, A. and Culler, M.D. (1986)Computer-controlled perifusion system for neuroendocrine tissues: development and applications. Methods Enzymol., 124:67 - 19. Ohta, M., Narohashi, T. and Keeler, R.F. (1973)Effects of veratrum alkaloids on membrane potential and conductance of squid and crayfish giant axons. J. Pharmacol. Exp. Ther., 184: 143 - 145. Pierotti, A.R., Lasdun, A., Ayala, J.M., Roberts, J.L. and Molineaux, C.J. (1991) Endopeptidase-24.15 in rat hypothalamic/pituitary/gonadal axis. Mol. Cell. Endocrinol., 16:95 - 103. Rasmussen, D.D. (1989)Pulsatile release of immunoreactive 0endorphin (END) from the human mediobasal hypothalamus (MBH) in vitro. Proc. Soc. Gynecol. Invest. Mtg., 87 (abstract). Rasmussen, D.D. (1991)The interaction between mediobasohypothalamic dopaminergic and endorphinergic neuronal systems as a key regulator of reproduction: a hypothesis. J . Endocrinol. Invest., 14:323 - 352. Rasmussen,D.D.,Liu, J.H., Wolf,P.L.andYen,S.S.C.(1983) Endogenous opioid regulation of gonadotropin-releasing hormone release from the human fetal hypothalamus in vitro. J. Clin. Endocrinol. Metab., 57:881 - 884. Rasmussen, D.D., Liu, J.H., Swartz, W.H., Tueros, V.S. and Yen, S.S.C. (1986a)Human fetal hypothalamic GnRH neurosecretion: dopaminergic regulation in vitro. Clin. Endocrinol. (Oxf.), 25: 127- 132. Rasmussen, D.D., Liu, J.H., Wolf, P.L. and Yen, S.S.C. (l986b)Gonadotropin-releasing hormone neurosecretion in the human hypothalamus: in vitro regulation by dopamine. J . Clin. Endocrinol. Metab., 62:419 - 483. Rasmussen, D.D., Liu, J.H., Wolf, P.L. and Yen, S.S.C. (1987) Neurosecretion of human hypothalamic immunoreactive beta-endorphin: in’vitro regulation by dopamine. Neuroendocrinology, 45: 197 - 200. Rasmussen, D.D., Gambacciani, M., Swartz, W.H., Tueros, V.S. and Yen, S.S.C. (1989)Pulsatile GnRH release from the human mediobasal hypothalamus in vitro: opiate receptor mediated suppression. Neuroendocrinology, 49: 150- 156. Robinson, J.R. (1949)Someeffects of glucoseand calcium upon the metabolism of kidney slices from adult and newborn rats. Biochem. J., 45: 68 - 74. Ropert, J.F., Quigley, M.E. and Yen, S.S.C. (1981)Endogenous opiates modulate pulsatile luteinizing release in humans. J. Clin. Endocrinol. Metab., 52: 583 - 585. Rossmanith, W.G., Gambacciani, M., Liu, J.H., Swartz, W.H., Tueros, V.S., Yen, S.S.C. and Rasmussen, D.D. (1988)

Pulsatile P-endorphin release from the human pituitary in vitro. Gynecol. Endocrinol., 2: 1 - 10. Rossmanith, W.G., Swartz, W.H., Tueros, V.S., Yen, S.S.C. and Rasmussen, D.D. (1990b)Pulsatile GnRH-stimulated LH release from the human fetal pituitary in vitro: sex-associated differences. Clin. Endocrinol. (Ox&), 33: 719 - 127. Rossmanith, W.G., Yen, S.S.C. and Rasmussen, D.D. (1990a) Synchronous pulsatile release of luteinizing hormone and immunoreactive beta-endorphin from the human pituitary in vitro. J. Neuroendocrinol., 2:91 - 94. Schulz, R., Wilhelm, A., Pirke, K.M., Gramsch, C. and Herz, A. (1981)6-Endorphin and dynorphin control serum luteinizing hormone level in immature female rats. Nature, 294: 757 - 159. Takahashi, J.S., Hamm, H. and Menaker, M. (1980)Circadian rhythms of melatonin release from individual superfused chicken pineal glands in vitro. Proc. Natl. Acad. Sci. U.S.A., 77:2319-2322. Veldhuis, J.D., Evans, W.S., Rogol, A.D., Drake, C.R., Thorner, M.O., Merriam, G.R. and Johnson, M.L. (1984)Intensified rate of venous sampling unmasks the presence of spontaneous, high-frequency pulsations of luteinizing hormone in man. J. Clin. Endocrinol. Metab., 59: 96- 102. Yen, S.S.C., Tsai, C.C., Naftolin, F., Vandenberg, G . and Ajabor, L. (1972)Pulsatile patterns of gonadotropin release in subjects with and without ovarian function. J. Clin. Endocrinol. Metab., 34: 671 -675.

Discussion G.A. Bray: Since LH and P-endorphin (PEND) are released from different pituitary cells, how do you think their pulsatile co-secretion is synchronized during perifusion after removal from hypothalamic control? D.D. Rasmussen: The mechanism by which the timing of the LH and immunoreactive PEND pulses were correlated is unknown. It is plausible that independent secretory mechanisms could be entrained by paracrine factors associated with the release of one hormone that could induce the release of the other. For example, angiotensin I1 has been localized in gonadotropes (Steele et al., 1982;Deschepper et al., 1986) and has been shown to stimulate @ENDrelease from pituitary cells in vitro (Kraft et al., 1984). Additionally, GnRH, which we have demonstrated can stimulate not only LH but also PEND release from the rat anterior pituitary (Gambacciani et al., 1988), has been localized in non-gonadotrope pituitary cells (Li et al., 1984) and may thus function to stimulate both LH and PEND release. Alternatively, the intrinsically pulsatile LH and PEND release could be independently entrained to another factor, such as calcium fluxes, or could feasibly be coordinated between cells by spreading depolarization (Schlegel et al., 1987). W.A. Scherbaum: You showed in your talk that a pulsatile pat-

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tern of hormone release is inherent to hypothalamic and pituitary cells. I would even go further and say that this appears to be a feature of all endocrine cells that have been studied in their microanatomical structure so far. Winfried Rossmanith from our university showed that isolated corpus luteum secretes progesterone in a pulsatile manner (personal communication) and we were able to show that isolated thyroid follicles release T3 and T4 hormone in a pulsatile manner (Scherbaum et al., 1991), but the odd observation was that the release was quickly and reversibly increased by removal of calcium from the medium. I recall that Dr. Rossmanith has made a similar observation with pituitary explants in vitro (personal communication). What is your comment on that? Do you have data on the calcium concentration within the cells in this situation? D.D. Rasmussen: In regard to your first statement, it does appear that most, if not all, endocrine cells secrete in a pulsatile rather than constant pattern. With regard to your observations of apparently irregular responses to removal of calcium, when Winfried Rossmanith was working on my laboratory we did indeed also obtain some “odd” results. When calcium was M verapamil (memremoved from the medium or when brane calcium channel blocker) and 4 mM EGTA (calcium chelator) were added to medium-containing calcium, LH and PEND release was often increased, although this increase often followed an initial suppression. Furthermore, subsequent perifusion with normal control medium reversed this increase. I do not have a very satisfying explanation for this increased release in response to removal of available extracellular calcium, which is probably why we were unable to publish these nonetheless interesting results. However, it can be hypothesized that the removal of extracellular calcium suppressed the activity of an undetermined inhibitory mechanism, possibly of a paracrine nature, allowing increased LH and PEND secretion maintained by intracellular calcium stores. Finally, in regard to your question about the calcium concentration in these cells during perifusion with calcium-free medium, we unfortunately do not have this information. D.R. Repaske: First, does finely dividing or mincing the tissue before perfusion increase the amount of hormone released due to increased number of cells in contact with perifusion medium? Secondly, does the pulsatile release of LH from the pituitary tissue persist if the pituitary tissue is minced? Must the cells remain in direct contact to maintain synchronized hormone release? D.D. Rasmussen: We have not perifused minced tissue, so I cannot confidently answer your questions. However, I suspect that release would be higher after mincing, due to an increased surface area and increased viability of a larger proportion of the

cells. In our preparation it must be assumed that cells in the center of the tissue are probably not maintained adequately. Since we showed that pituitary quarters each released LH in a pulsatile manner (Gambacciani et al., 1987) it is apparent that the entire pituitary is not required for pulsatile secretion to be expressed. We do not know whether it is necessary for the cells to remain in direct contact to maintain synchronized release of LH and PEND. However, the putative mechanisms which I proposed in response to Dr. Bray’s question would suggest that the cells would indeed have to be in relatively close contact, allowing paracrine regulation or direct cell-cell communication.

References Deschepper, C.F., Crumrine, D.A. and Ganong, W.F. (1986) Evidence that the gonadotrophs are the likely site of production of angiotensin I1 in the anterior pituitary of the rat. Endocrinology, 119: 36 - 43. Gambacciani, M., Liu, J.H., Swartz, W.H., Tueros, V.S., Yen, S.S.C. and Rasmussen, D.D. (1987) Intrinsic pulsatility of luteinizing hormone release from the human pituitary in vitro. Neuroendocrinology, 45: 402 - 406. Gambacciani, M., Yen, S.S.C. and Rasmussen, D.D. (1988) GnRH stimulates ACTH and immunoreactive betaendorphin release from the rat pituitary in vitro. Life Sci., 43: 755 -760. Kraft, K., Lang, R.R., Gaida, W., Unger, T. and Ganten, D. (1984) Angiotensin stimulates P-endorphin release from anterior pituitary gland cell cultures of rats. Neurosci. Lett., 46: 25-31. Li, Y.K., Knapp, R.J. and Sternberger, L A . (1984) Immunocytochemistry of a “private” luteinizing-hormonereleasing hormone system in the pituitary. Cell Tissue Res., 235: 263 - 266. Scherbaurn, W.A., Morgenthaler, N.G. and Rossmanith, W.G. (1991) Episodic secretion of thyroxine (T4) and triiodothyronine (T3) from porcine thyroid fragments in vitro. Horm. Metab. Res., 23: 92 - 93. Schlegel, W., Winiger, B.P., Mollard, P., Vacher, P., Wuarin, F., Zahnd, G.R., Wollheim, C.B. and Dufy, B. (1987) Oscillations of cytosolic Ca2+ in pituitary cells due to action potentials. Nature, 329: 719 - 721. Steele, M.K., Brownfield, M.S. and Ganong, W.F. (1982) Immunocytochemical localization of angiotensin immunoreactivity in gonadotropes and lactotropes of the rat anterior pituitary gland. Neuroendocrinology, 35(3): 155 - 158.

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D.F. Swaab, M.A. Hofman, M. Mirmiran, R. Ravid and F.W. van Leeuwen (Eds.) Progress in Brain Reseorch, Vol. 93

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@ 1992 Elsevier Science Publishers B.V. All rights reserved.

CHAPTER 7

Brain banking and the human hypothalamus match for, pitfalls and potentials

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factors to

R.Ravid, E.J. Van Zwieten and D.F. Swaab Netherlands Institute f o r Brain Research, I I05 AZ Amsterdam 20, The Netherlands

Introduction

Ante-mortem factors

The development of sophisticated neurobiological techniques which can be applied on the human brain causes an increased demand for post-mortem tissue for research. Brain bank organizations serve as an important link between the clinician, the neuropathologist and the basic scientist by supplying clinically and neuropathologically well-documented specimens for research. The various enzymes, transmitter systems and other active substances in the brain have their specific cellular localization. Therefore, data obtained by biochemical assays in homogenates or tissue extracts have only a limited value and brain banks collecting human brain specimens for research purposes should also strive to develop techniques which leave the morphology of the tissue intact (Swaab et al., 1986, 1989; Swaab and Uylings, 1988). The various techniques which are applied on human brain have one drawback in common: many patient-related factors introduce a huge variation and systematic errors, which have to be corrected or carefully matched for. There are several factors one should be aware of when collecting and handling human specimens and some of the data accumulated in recent years on the human hypothalamus may serve as a good example for the wide variety of potentialities and pitfalls in the use of post-mortem human brain.

Age Age-related changes occur in many structures in the hypothalamus, both in normal aging and in Alzheimer’s disease (AD). A decrease in volume and cell number was observed in the suprachiasmatic nucleus (SCN) in senescence (80 - 100 years) and was even more pronounced in Alzheimer’s disease (Fig. 1; Swaab et al., 1987). Another hypothalamic nucleus which shows clear age-related changes is the sexually dimorphic nucleus of the preoptic area (SDNPOA; Swaab and Hofman, 1988; Hofman and Swaab, 1989). The SDN cell number reaches a peak value at the age of 2 - 4 years and only after this age sexual differentiation becomes manifest. The nucleus decreases greatly in volume and in cell number with age (Fig. 2; Swaab and Fliers, 1985). Age must be also taken into consideration when studying monoamines, their metabolites and enzyme activities in the human hypothalamus (Adolfsson et al., 1979). Hypothalamic concentrations of monoamines were also reported to be decreased in brains from patients suffering from AD (Hardy et al., 1982) and in vascular dementia (Wallin and Gottfries, 1990; Wallin et al., 1991). Sex

There is an increasing amount of data concerning sex differences in the various nuclei of the hypo-

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Fig. 1. Number of AVP cells (left panel) and total number of cells in the SCN (right panel). Note the low values in the 81 - 100-year-old 5 years of age. The vertical lines denote the S.E.M. The extremely high group and in the Alzheimer patients (DEM) that were 78 values for the two transsexual objects (TI and T2) and one case of Prader-Willi syndrome (P) are not included in the group means and are only given as individual values. (With permission from Swaab et al., 1987.)

thalamus. Morphometric analysis revealed that the shape of the SCN is sexually dimorphic (Swaab et al., 1985) and that there is a striking sexual dimorphism in the size and cell number in the SDN-POA (Swaab and Hofman, 1984; Swaab andFliers, 1985; Hofman and Swaab, 1989). The sexual differentiation of the human SDN-POA occurs after 4 years post-natally and only after this age the nucleus differentiates according to sex, due to a decrease in both volume and cell number in women, whereas in men it remains unaltered up to the fifth decade after which a marked decrease in cell number was observed as well (see Swaab et al., this volume). Sexual differentiation has also been reported (Allen et al., 1989) for two other cell groups in the preoptic-anterior-hypothalamus (INAH 2 and 3). According to this report, these areas were larger in males than in females and this was later partly confirmed by LeVay (1991). Another brain region

having a larger volume in males than in females is the bed nucleus of the stria terminalis (BNSTdspm), described by Allen and Gorski (1990). Sex differences in hypothalamus have been reported in the concentration of Met-enkephalin in cases of sudden infant death victims (Bergstrom et al., 1984) and in the levels of monoamines, monoamine metabolites and related enzymes (Gottfries et al., 1974, 1980). Brain weight Recent studies made it clear that in relation to brain weight, allometric measures and sex differences should be taken into account (Swaab and Hofman, 1984). Already in 1880, Bischoff reported that sex differences in absolute brain weight are present at birth, but nowadays we know in addition that one should also take into account the differences in age, body weight and length (Fig. 3)

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Fig. 2. I. Thionin-stained frontal sections (6 pm) of the hypothalamus of ( A )a 28-year-old man and (B)a 10-year-old girl. Arrows show the extent of the sexually dimorphic nucleus of the preoptic area (SDN-POA). 11. ( A )Volume and (B)cell number of the human SDNPOA (means and S.E.M.). Points represent individual values. (With permission from Swaab and Fliers, 1985.)

(Dekaban and Sadowsky, 1978; Voigt and Pakkenberg, 1983; Haug et al., 1984; Swaab and Hofman, 1984; Hofman and Swaab, 1989). Brain weight is significantly influenced by various fix-

atives and the duration of fixation; it is known to increase during fixation in formalin and this increase is directly correlated to the fresh brain weight (Skullerud, 1985).

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Fig. 3. Brain weight as a function of body height. Data have been compiled from Voigt and Pakkenberg (1983). Note the sex difference in relative brain weight from the age of 2 years onwards. (With permission from Swaab and Hofman, 1984.)

Agonal state The agonal effects associated with death may influence the pH and a number of chemical substances in the brain. Subjects who died after a long terminal illness have a lower pH in the brain, CSF and blood, and this acidosis corresponds to increased lactic acid concentrations (Perry et al., 1982; Hardy et al., 1985b). Lower levels of pH were found throughout the brain in cases of death following protracted illness, as compared to sudden death (Spokes, 1979). Various enzymatic activities were found to be related to pH and lactate in post-mortem brain in Alzheimer’s disease and Down’s syndrome as well as other dementias (Yates et al., 1990). These authors found that lactate levels were higher and phosphate-activated glutaminase and glutamic acid decarboxylase (GAD) levels were lower in the hypothalamus of agonal controls than in the sudden death controls. Phosphate-activated glutaminase and GAD activities were correlated with tissue pH and lactate and were also reduced by in vitro acidification, suggesting that the low enzyme activities in agonal controls were directly due to the decreased pH. Strong positive correlations were obtained between the concentration of tryptophan, another putative agonal status marker of post-mortem brain tissue and the concentration of gamma-amino bu-

tyric acid (GABA) in all brain areas (Korpi et al., 1987). The regional distribution and tissue levels of neuropeptide Y-like immunoreactivity (NPY-IR) in the human hypothalamus was also found to be elevated by chronic respiratory failure; (Fig. 4;Corder et al., 1990). In view of the data mentioned before, measuring the pH in CSF makes it possible to match the samples for agonal state and has therefore been introduced as a routine procedure in The Netherlands Brain Bank. In order to check whether the pH was not affected by post-mortem time, we investigated whether there is any correlation between pH in brain tissue and the post-mortem interval, in both rat and human brain. Thirty-five female Wistar rats were decapitated and the brains left intact in the skull at room temperature for various time intervals. At each time point, five brains were dissected out and separately homogenized. Subsequently, pH of each homogenate was measured and the mean of these measurements was used as representative pH value. From the rat data (Fig. 5 ) it became obvious that there was no significant change in the pH within a

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87 CONTROLS

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Fig. 5 . Effect of post-mortem delay on rat brain. Thirty-five female Wistar rats were decapitated and the skulls left at room temperature for 0, 2, 4, 6, 10, 16 and 24 h. At each time point, each brain was homogenized in five volumes of aquadest and the pH was subsequently measured. Each point represents the mean value of five homogenized brains. Since all standard errors were below 0.04 pH units, they were not plotted in this figure. Statistical analysis of the data was performed by applying oneway ANOVA. No significant differences were found. (F = 0.2756).

post-mortem interval of 24 h. This is well within the range of most of the post-mortem intervals of rapid autopsies in our brain bank. Following decapitation, there is an initial rise in lactate levels which reaches a steady state after 10min (Swaab and Boer, 1972). The higher variation in our data at point 0 may thus be due to rapid changes in pH immediately after decapitation. Comparable observations have been made on human autopsy material collected by The Netherlands Brain Bank in the past 4years. The pH values measured in CSF obtained by autopsy of non-demented controls ( A )and Alzheimer's disease patients (B),did not change significantly with postmortem delay (Fig. 6). From our own data and others we concluded that measuring the pH of either the brain tissue or CSFobtained at autopsy is influenced by agonal state and not by post-mortem delay and thus is of crucial importance for brain banking routine procedures. A striking difference in increase in brain weight after fixation was observed by Skullerud (1985) in patients dying either after a protracted illness or in those who had a sudden death.

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To prevent the use of unsuitable tissue for research we always note whether we have information on sudden and unexpected death or protracted illness and verify this at autopsy by measuring the pH in CSF. In human studies, prolonged diseases such as respiratory distress may influence a number of biochemical parameters. Whenever possible, subjects should thus be matched for premorbid state. This is a particularly difficult criterium to satisfy in studies of aging, since most young donors die from accidents, suicides or drug overdose whereas older donors die from various chronic disease states. A

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Fig. 7, Level of 5-HT in human hypothalamus examined postmortem, in relation to month of death. Shown are the means + S.E.M. (n) of pooled values of two consecutive months. Statistics: Student's t-test. (With permission from Carlssonet al., 1980a.)

similar problem exists in studies of Alzheimer's disease patients, who frequently suffer from pneumonia and cachexia. Seasonal variation Seasonal alterations have been found in the level of hypothalamic 5-HT with a minimum during the months December - January and a maximum during October-November (Fig. 7; Carlsson et al., 1980a). Hypothalamic dopamine also showed seasonal variation, with two peaks, i.e., during January Fkbruary and August - September, and two nadirs; i.e., during March- June and October-

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December. In other brain regions the influence of biorhythms on dopamine levels was less evident. A striking seasonal variation was observed in the volume and cell number of the human SCN; the volume was 2.5 times larger in October - November than in May- June and contained 2.4 times as many cells. Similar annual oscillations were found in the number of vasopressin-expressing neurons in the SCN (see Hofman and Swaab, this volume). The month of death is consequently a factor to consider in studies. Circadian variation Clock time of death has been found to be a significant factor for the levels of hypothalamic monoamines. Circadian changes in the hypothalamus were observed in noradrenaline (NA), 5-HT and dopamine (DA) and their metabolites (Fig. 8; Carlsson et al., 1980a). The hypothalamic levels of 5-HT were found to be low between 6 a.m. and 3 p.m. and a rapid fall occurred between 5 a.m. and 8 a.m. The hypothalamic NA levels showed no seasonal variation but were found to fluctuate significantly during the day in a manner similar to 5-HT. Also the level of DA in the hypothalamus showed significant circadian variation, with a nadir between 6 and 9 a.m. Circadian variation in dopamine levels in brain regions other than the hypothalamus were detected only in a few instances. There is evidence of diminished concentrations of 5-HT and DA in various brain regions in Alzheimer's disease (Hardy et al., 1985a), suggesting reduced activity of both systems. In a recent study, the hypothalamic concentrations of monoamines and various peptides in Alzheimer's disease and vascular dementia were measured (Wallin et al., 1991). The authors suggest that these changes may be important for the changes in circadian symptoms in dementia. A sleep centre in the hypothalamus and the neighboring portions of the mesencephalon has been postulated already long ago (Kleitman, 1963) and more recently it has been suggested that changes in sleep/wake rhythms may be based upon the degenerative changes in the SCN (Swaab et al., 1985, 1987). 5-HT has been claimed to play an important role as a sleep-inducing

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neurotransmitter (Koella, 1974) and the high nocturnal activity of hypothalamic 5-HT favors its active role in sleep. The fact that hypothalamic neurotransmitters have marked circadian variations with high nocturnal activity pleads for an active and specific role of the hypothalamus in the control of sleep/wakefulness patterns. Since a clear-cut circadian pattern was found to be present in the levels of various transmitters in the human hypothalamus with higher values around midnight (Gottfries et al., 1980), time of death should be considered as a factor to match for when collecting hypothalamic specimens for research.

Lateralization Fixing one hemisphere and freezing the other is current practice in many brain banks. It prevents, however, the recognition of possibly existing leftright differences of various systems in the brain. Several functions and transmitters are asymmetrically represented in the left or right hemisphere; lateralization of norepinephrine has been demonstrated in the human hypothalamus (Oke et al., 1978) and there is evidence for a left prominence in the distribution of thyroid-releasing hormone (TRH) in discrete nuclei of the hypothalamus, i.e., in the ventromedial dorsal and paraventricular nuclei (Borson-Chazot et al., 1986) with higher concentrations on the left side. Consequently it is preferable to sample bilaterally and if not possible, mention on which hemisphere the measurements have been performed.

Many neurochemical substances which are also present in the hypothalamus, arevery stable. Neuropeptides and several receptors seem to belong to the latter group. Correlating the post-mortem delay to various chemical variables in the hypothalamus revealed that there was a significant negative correlation between this time variable and noradrenaline (NA) and normetanephrine (NM) levels. On the other hand, a positive correlation was found for the levels of the amino acids tryptophane and tyrosine (Gottfries et al., 1980). Immunocytochemical (ICC) procedures have shown that peptides are less sensitive to the postmortem delay; an excellent staining of vasopressin neurons in the supraoptic nucleus (SON) and paraventricular nucleus (PVN) is obtained on tissue which was fixed 48 h after death. A similar stability up to post-mortem time of more than 60 h was found for extrahypothalamic vasopressin fibers (Swaab, 1982; Fliers et al., 1986). Immunocytochemical studies on rat brain showed that the amount of stainable vasopressin in the suprachiasmatic nucleus (SCN) even doubled during a 6-h post-mortem interval (Van Zwieten et al., 1991). Incubating rat brain slices in medium for 6 h we found enhanced ICC staining and levels of hypothalamic VP-containing neurons in the SON, AVP

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Post-mortem delay The time between death and fixation or freezing of the tissue is important not only from a neurochemical point of view but also for several morphological parameters. Some substances in the brain are very unstable. Rat studies showed a striking effect of ischemia on various substrate levels in the cortex and in the hypothalamus. For P-creatine and ATP it is a matter of seconds for a significant drop to take place (Swaab and Boer, 1972).

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Fig. 9. Mean integrated optical density of arginine-vasopressin (AVP), c-terminal glycopeptide (CPP) and neurophysin (NF) in thesuprachiasmatic nucleus ofthe Wistarrat, inimmediately fixed brain, in brain incubated for 6 h in Ringer medium and in brain left intact in the skull for 6 h. Bars represent mean values of five animals; for each antigen the values of the five animals were analyzed on the same gel. Vertical lines represent S.E.M. (With permission from Van Zwieten et al., 1991.)

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PVN and SCN as compared to brain tissue fixed immediately after death (Fig. 9). These results clearly indicate that a post-mortem interval of several hours is not neccessarily detrimental for ICC studies or peptide measurements on brain material which in turn enlarges the feasibility of routinely obtained autopsy material for research. Quantitative autoradiography has been used to study the localization and regional distribution of enzymes and receptor binding sites in the hypothalamus, in post-mortem human brain in both normal and diseased tissue. This approach appears to be particularly valuable as most binding sites appear to be quite stable in post-mortem tissue (Hardy et al., 1983). Binding studies of imipramine and desmethylimipramine (DMI) have been proven to be stable in post-mortem human brain with the highest density found in the hypothalamus (Langer et al., 1981; Cortes et al., 1988; Gross-Isseroff and Biegon, 1988; Gross-Isseroff et al., 1988). Post-mortem delay does not influence the distribution of highaffinity somatostatin receptors in the tuberal nuclei of the hypothalamus obtained at routine autopsies (Reubi et al., 1986). It is very well possible to use post-mortem tissue to examine the ante-mortem expression of a human neuropeptide gene. Neuropeptides are known to play a major role in neural transmission in the brain and changes in these compounds occur in various neurodegenerative disorders. To be able to interpret the dynamics of neuropeptides, it is often important to investigate neuropeptide messenger RNA (mRNA) in human brain in addition to the neuropeptide levels themselves. Modern molecular biological techniques allow the study and quantification of those mRNA's by using various hybridization techniques (Kobayashi et al., 1990). RNA stability has been investigated in several studies, with some discrepancy between results. An extensive postmortem stability of total RNA has been reported in rat and human brain up to 48 h and 36 h, respectively. Both the yield and the integrity of RNA stayed unchanged during the post-mortem period (Johnson et al., 1986). Vasopressin mRNA has been detected in rat hypothalamic nuclei by quantitative in situ hybridiza-

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Fig. 10. Post-mortemchanges in rRNA and AVP mRNA (mean i S.E.M.) are expressedas percentagesof respective RNA level at 0 h post-mortem. n = 4 - 6 except in case of the data at 48 h post-mortem. *P < 0.05, **P < 0.01 compared with data at 0 h post-mortem (Student's t-test). (With permission from Noguchi et al., 1991.)

tion (Rivkees et al., 1989). The hybridization signals had a modest rate of loss within 12 h of post-mortem time. This technique has been also applied for the study of other neuropeptide gene expression in postmortem tissue. No significant correlations are found between the density of the hybridization signal and parameters such as post-mortem delay, age and sex (Mengod et al., 1990). However, when the post-mortem stability of arginine-vasopressin (AVP) messenger RNA (mRNA) in the rat brain was measured it appeared to be degraded post-mortem more rapidly than rRNA (Fig. 10; Noguchi et al., 1991). The splicing pattern of mRNA has been investigated in rat and human post-mortem brain tissue, using a method based upon the polymerase chain reaction (Granneman and Bannon, 1991). There may thus be some differences between mRNA species in post-mortem degradation; total RNA recovery does not decrease with post-mortem time, whereas rRNA and vasopressin mRNA signals show a tendency for reduction. The use of the polymerase chain reaction (PCR) in the analysis of mRNA allows to measure

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simultaneously various types of mRNA which have a different susceptibility to pre- and post-mortem variables. (Burke et al., 1991). Surprisingly, prolonged death - refrigeration interval was positively correlated to increase in specific RNA. Some recent results plead thus for the need for rapid autopsies and suggest that autopsied human brain should be used for vasopressin mRNA studies within a short post-mortem time. Alterations in neurotransmitter and drug receptors in various neuro-degenerative disorders can be studied on autopsy material. The effect of postmortem delay and prolonged storage of the tissue prior to performing binding assays may limit the interpretation of the disease-related changes in receptor populations. The understanding of receptor changes associated with the disease may have implications for the development of therapeutic strategies by using drugs that modify receptor function. In order to be able to claim that changes in receptors found in human brain result from the disease process being studied, it is important to exclude other factors which may affect ligand binding. Autolysis of tissue due to the post-mortem delay and the freezing process of the tissue may both contribute to such changes, alter the receptor and affect the binding of a ligand. The post-mortem delay is consequently a very essential variable. Post-mortem delay of the human brain tissue obtained by autopsy can not be much shorter for obvious reasons. It would therefore be an important development for brain banking if more neurobiological techniques would be adapted such that they are suitable for tissue with a relatively long post-mortem delay.

Freezing procedures, fixation and storage time Changes in such factors may affect many of the parameters used to assess changes in the brain and the potentialities of staining procedures considerably. On the other hand, some tissue components are not very sensitive to these factors. Human brain tissue used for biochemical studies is usually rapidly frozen and slowly thawed. However, to isolate synaptosomes which are morpholo-

gically well-preserved and have retained their metabolic performance one should use the opposite procedure as snap-freezing generally yields metabolically and functionally inactive preparations (Hardy et al., 1983). It is noteworthy that a large number of metabolic and functional processes as well as binding capacity of various receptors are retained surprisingly well in frozen tissue. That way it becomes possible to study regional variations, distribution and comparative activities of various transmitters or drugs in normal and diseased brain and correlate them to the anatomical changes. Fixation in formalin causes an increase in brain weight and the subsequent washing in water introduces a systematic error in brain weight, e.g., larger brains gain more weight than small brains. However, brains from younger individuals do not gain more in weight than older ones when the difference in fresh brain weight between the two groups was taken into account. Similarly, the increase in brain weight during fixation is not sexually dimorphic when the fresh brain weight is taken into account (Skullerud, 1985). Conventional formaldehyde fixation for 1 month results in excellent vasopressinand oxytocin-staining of the hypothalamic SON, PVN and SCN neurons (Fliers et al., 1985; Swaab et al., 1985). However, this procedure was not suitable for studying the extrahypothalamic fibers of these peptidergic neurons for which thick glutaraldehydeparaformaldehyde sections are preferable. Our experience with rat material is that storage in glutaraldehyde-paraformaldehyde fixative preserves immunoreactivity of vasopressin, oxytocin and alfaMSH for more than a year (R. Ravid, unpublished results). Since immersion in this fixative does not fully penetrate a human brain, smaller tissue blocks have to be fixed by immersion in 2.5% glutaraldehyde and 1Yo paraformaldehyde for 1 week. Subsequently the blocks can be frozen and stored in sealed plastic at - 80°C and cryostat sections can be made for immunocytochemistry. This procedure gave good staining of VP fibers in the human brain (Fliers et al., 1986). On the other hand, vasopressin immunoreactivity of hypothalamic neurons was still

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present in material which had been fixed and stored for more than 50 years (Swaab, 1982). All the observed changes in post-mortem brain mentioned in this section have clear consequences for brain banking procedures. Our group had used in the past 10 years a constant procedure for the human hypothalamus in order t o minimize the systematic errors and variables. Brains obtained at autopsy are fixed for 1 month in 10% buffered formalin at room temperature before further washing and embedding in paraffin blocks (Fliers et al., 1985). Summary and conclusions The study of an increasing number of processes occurring in the human brain can be carried out on autopsy material. The availability of this material, whether fresh, frozen or fixed, makes it possible to develop methodologies for studying the neuroanatomical and neurochemical aspects of the human brain. It has also become possible in recent years to correlate functional changes with neurochemical changes and with neuroanatomical abnormalities in disease states. Some compounds and structures are damaged irreversibly within minutes after death and some brain components are known to disintegrate within seconds. This led to the widespread idea that autopsy material would not be suitable for basic research purposes and would not supply the neccessary answers on the various fundamental questions regarding processes occurring in normal or diseased brain. However, from data published in recent years in which autopsy material has been routinely used, it becomes more and more evident that this is a misconception. There is an increasing number of reports based on the use of normal and pathological human brain tissue obtained by autopsies in spite of the fact that there is a worrying continuous decline in autopsy rate which causes serious concern among scientists world-wide (Anderson and Hill, 1989). It also became evident that when using the proper fixation procedures, sufficient structural integrity is

retained in the tissue to allow morphological and morphometrical studies (Swaab and Uylings, 1988). Electron microscopic examination of synaptosomal preparations from post-mortem human brain showed them to be only slightly less pure than preparations from fresh tissue although there was some degree of damage (Hardy et al., 1982). Agonal state effects the stability of brain compounds and causes brain hypoxia. This again forms a tremendous difficulty for the study of human neurological and psychiatric diseases as one of the frequent causes of death is bronchopneumonia which leads to brain hypoxia and results in pronounced lactic acidosis. The Netherlands Brain Bank has succeeded to partly circumvent some of the serious problems encountered in providing human tissue for research by performing rapid autopsies with an average post-mortem delay of 2 - 4 h. This has become possible by a close collaboration of numerous nursing homes in Amsterdam and its vicinity and with the neuropathologists of the Free University in Amsterdam. We also measure the pH of the tissue as indicator of agonal state in order to reveal unsuitable specimens. The human hypothalamus contains various nuclei manifesting a wide variety of changes in different conditions. Studies of the hypothalamic nuclei may provide more insight into the development of different systems of this important brain structure in health and disease. The analysis of postmortem human brain data nevertheless remains extremely difficult. The interpretation of the various results must be done with great care to exclude confounding factors due to the heterogeneity of the material with respect to the various factors mentioned above in detail. It is evident that numerous possible pitfalls remain to be encountered when human brain tissue is studied with the conventional neuroanatomical techniques. A concerted effort is needed to ensure that the samples would not differ systematically. Matching for the various ante- and postmortem factors is an essential step towards obtaining meaningful results. Without it, differences observed between groups of samples may be wrongly attributed to the disease process.

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Acknowledgements

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human brain: an autoradiographic study. Neuroscience, 18: 329 - 346. Rivkees, S.A., Chaar, M.R., Hanky, D.F., Maxwell, M., Reppert, S.M. and Uhl, G.R. (1989) Localization and regulation of vasopressin mRNA in human neurons. Synapse, 3: 246 - 254. Simpson, J., Yates, C.M., Watts, A.G. andFink, G. (1988) Congo red birefringent structures in the hypothalamus in senile dementia of the Alzheimer type. Neuropathol. Appl. Neurobiol., 14: 381 - 393. Skullerud, K. (1985) Variations in the size of the human brain. Acta Neurol. Scand., 71: 14- 15. Sokal, R.R. and Rohlf, F.J. (1969) In: J. Wilson and S. Cotter (Eds.), Biometry, 2nd edition, Freeman, San Francisco, CA. Spokes, E.G.S. (1979) An analysis of factors influencing measurements of dopamine, noradrenaline, glutamate decarboxylase and choline acetylase in human post-mortem brain tissue. Brain, 102: 333 - 346. Swaab, D.F., (1982) In: V. Chan-Palay and S.L. Palay (Eds.), Cytochemical Methods in Neuroanatomy, Alan R. Liss, New York, pp. 423-440. Swaab, D.F. and Boer, K. (1972) The presence of biologically labile compounds during ischemia and their relationship to the EEG in rat cerebral cortex and hypothalamus. J. Neurochem., 19: 2843 - 2853. Swaab, D.F. and Fliers, E. (1985) A sexually dimorphic nucleus in the human brain. Science, 228: 1 1 12 - 1 1 15. Swaab, D.F. and Hofman, M.A. (1984) Sexual differentation of the human brain; a historical perspective. In: G.J. de Vries et al. (Eds.), Progress in Brain Research, Vol. 61, Elsevier, Amsterdam, pp. 361 - 374. Swaab, D.F. and Hofman, M.A. (1988) Sexual differentiation of the human hypothalamus; ontogeny of the sexually dimorphic nucleus of thepreoptic area. Dev. Brain Rex, 44: 314- 318. Swaab, D.F. and Hofman, M.A. (1990) An enlarged suprachiasmatic nucleus in homosexual men. Brain Res., 537: 141 - 148. Swaab, D.F. and Uylings, H.B.M. (1988) Potentialities and pitfalls in the use of human brain material in molecular neuroanatomy. In: F.W. Van Leeuwen, R.M. Buys, C.W. Pool and 0. Pach (Eds.), MolecularNeuroanatomy, Elsevier, Amsterdam, pp. 403 -416. Swaab, D.F., Fliers, E. and Partiman, T.S. (1985) The suprachiasmatic nucleus of the human brain in relation to sex, age and senile dementia. Brain Res., 342: 37 - 44. Swaab, D.F., Fliers, E., Goudsmit, E. and Uylings, H.B.M. (1986) Brain sampling and preservation of specimens from Alzheimer patients and controls for morphology. Modern trends in aging research. Colloque INSERM-EURAGE, Vol. 147, Libbey Eurotext Ltd., pp. 413-420. Swaab, D.F., Roozendaal, B., Ravid, R., Velis, D.M., Gooren, L. and Williams, R.S. (1987) Suprachiasmatic nucleus in aging, Alzheimer’s disease, transsexuality and Prader-Willi syndrome. In: E.R. De Kloet, W.M. Wiegant and D. De Wied

95 (Eds.), Neuropeptides and Brain Function - Progress in Brain Research, Vol. 72, pp. 301 - 3 10. Swaab, D.F., Hauw, J.-J., Reynold, G.P. and Sorbi, S. (1989) Tissue banking and EURAGE. J. Neurol. Sci., 93: 341 - 343. Van Zwieten, E.J., Ravid, R., Van der Sluis, P.J., Sluiter, A.A., Pool, Chr.W., Smyth, D. and Swaab, D.F. (1991) Increased vasopressin immunoreactivity in the rat brain after a postmortem interval of 6 hours. Brain Res., 550: 263 - 267. Voigt, J. and Pakkenberg, H. (1983) Brain weight of Danish children. A c t a h a t . , 116: 290-301. Wallin, A. and Gottfries, C.G. (1990) Biochemical substrates in normal aging and Alzheimer’s disease. Pharmacopsychiatry, 23 (Suppl.): 37-43. Wallin, A., Carlsson, A., Ekman, R., Gottfries, C.G., Karlsson,

I., Svennerholm, L. and Widerlov, E. (1991) Hypothalamic monoamines and neuropeptides in dementia. Eur. Neuropsychopharmacol., 1: 165 - 191. Widerlov, E., Wallin, A., Gottfries, C.G. andEkman, R. (1991) Hypothalamic neuropeptide in neuropsychiatric illnesses. In: A.R. Genazzani, G. Nappi, F. Petraglia and E. Martignoni (Eds.), Stress and Related Disorders from Adaptation to Dysfunction, The Parthenon Publishing Group, Casterton Hall, Carnforth, pp. 167 - 178. Yates, C.M., Butterworth, J., Tennant, M.C. and Gordon, A. (1990) Enzyme activities in relation to pH and lactate in postmortem brain in Alzheimer type and other dementias. J. Neurochem., 55(5): 1624- 1630.

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SECTION IV

Biological Rhythms

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The fourth C.U. Ariens Kappers lecture

Dr. R.Y. Moore was invited to deliver the fourth C.U. Ariens Kappers lecture during the 17th International Summer School of Brain Research, on 27 August 1991. The first three lectures in this series were given by Dr. P. Rakic (Yale University School of Medicine, New Haven, CT, USA, 1987), Dr. A. Bjorklund (Institute of Histology, University of Lund, Sweden, 1988) and Dr. M. Mishkin (Laboratory of Neuropsychoiogy, National Institute of Mental Health, Bethesda, MD, USA, 1989). The first director of the Netherlands Institute for Brain Research, C.U. Ariens Kappers, was born in 1877 at Groningen. During his medical training Ariens Kappers was inspired by the neurologist Prof. C . Winkler to take up brain research. Prof. Winkler was one of the founders of the Netherlands Institute for Brain Research. When Ariens Kappers was still a student at the University of Amsterdam, his research abilities were honored with a gold medal for a study on myelin sheets. In I904he obtained his PhD with a thesis on a comparative neuroanatomical subject and he continued to work in this field of research ever after. This choice was strongly reinforced by his appointment in 1906 as “Abteilungsvorsteher” (i.e., head of a department) in the institute of the famous neurologist and comparative neuroanatornist Prof. Dr. L. Edinger in Frankfurt am Main. Meanwhile, the International Association of Academics had decided that brain research should be placed on an international footing. In 1904 this resulted in the formation of the International Academic Committee for Brain Research, which claimed that “the time is not far distant when the study of the millions of brain cells will have to be divided amongst researchers in the way that astronomers have been obliged to divide the millions of stars into various groups” and proposed “to organize a network of institutions throughout the civilized world, dedicated to the study of the structure and functions of the central organ . . .”. The first country to respond to this ambition was The Netherlands, where the “Netherlands Central Institute for Brain Research” was opened in 1909. Ariens Kappers became the first director and held this position until his death in 1946. He made the institute into aninternationally renowned place by his excellent work (the book he wrote together with G.C. Huber and E.C. Crosby, entitled “The Comparative Anatomy of the Nervous System of Vertebrates, including Man” (1936), is still well cited), he traveled all over the world and received a visiting professorship at Peking Union Medical College in China from 1923 to 1924. The international character of the institute is underlined by the fact that during Ariens Kappers’ directorship 69 foreign scientists paid a working visit to the Amsterdam Institute as well as by the Honorary Doctorate of Sciences he received from Yale University in 1928 (see photograph). In 1929 Ariens Kappers held his inaugural lecture as “extraordinary professor” at the medical faculty of the University of Amsterdam. We are glad that Dr. R.Y. Moore accepted our invitation to deliver the C.U. Ariens Kappers lecture in honour of this exceptional scientist. Dr. Moore had a genuine multidisciplinary training in neurosciences with graduate training resulting in a BA in Zoology, a MD and a PhD in Biopsychology, and a post-graduate train-

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C.U. Ariens Kappers

ing in clinical neurology. Three of his already classical papers (Brain Res. 42, 201 - 206, 1972; J. Comp Neurol. 146,l- 15,1972; Brain Res. 49,403 - 409,1973) deal with aspects that also had the attention of C.U Ariens Kappers, viz. the structure and function of the suprachiasmatic nucleus (SCN) and its comparativc aspects. In the thesis of R.B.W.F.M. Diepen (1941), entitled “The hypothalamic nuclei and their ontogenetic development in ungulates (OVIS ARIES)”, which was supervised by C.U. Ariens Kappers, these topics werr already dealt with. Diepen stated: “In rabbits and carnivores Spiegel and Zweig (1919) describe a triangulai nucleus of small polymorphous cells lying dorsally to the chiasm on both sides of the ventricle. To this nucleu! they gave the topographical name of nucleus suprachiasmaticus. As stated by Spiegel and Zweig, the cell! of this nucleus can hardly be distinguished from the central grey mass round the third ventricle of which i is only a local thickening. Its boundaries and density show great variations and sometimes it is difficult tc speak of a real nucleus in this region. In the human hypothalamus a nucleus suprachiasmaticus is not ever mentioned by most authors (Malone 1910, Greving 1925, Gage1 1928, Grunthal 1933). In lower mammals however, it is usually described. We have already called the attention to a similar difference in the centra grey substance of lower mammals and primates. Grunthal, in his minute analysis of the hypothalamus o man and mammals (1929- 1931, 1933) emphasizes this conclusion and states that the hypothalamus in thc ascending series of mammals acquires a simpler nuclear pattern and that particularly the suprachiasmatic nucleus in man is rudimental. (For references see Diepen’s thesis.) We are very glad that Dr. R.Y. Moore accepted our invitation to deliver an excellent fourth Ariens Kapper! lecture on the “rudimental” human suprachiasmatic nucleus. D.F. Swaat Source: B. Brouwer (1946) In Memoriam Prof. Dr. Cornelius Ubbo Ariens Kappers, Psychiatrische er Neurologische Bladen, pp. 1 - 16.

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0 1992 Elsevier Science Publishers B.V. All rights reserved.

CHAPTER 8

The organization of the human circadian timing system Robert Y. Moore Departments of Psychiatry, Neurology and Behavioral Neuroscience and the Center for Neuroscience, University of Pittsburgh, Pittsburgh, PA 15261, U.S.A.

Introduction

The solar cycle of light and dark is the most pervasive cyclic stimulus in our environment. Thus, it is hardly surprising that the human, like other animals, exhibits an adaptation to this cyclic environmental event. This adaptation is expressed as circadian rhythms. The most evident circadian rhythm is the daily cycle of sleep and wakefulness but this is accompanied by a multiplicity of behavioral and physiological rhythms, and all of the rhythms are timed so as to provide maximal adaptation to the environment. How is this accomplished? The answer to this question has gradually unfolded over the last 20 years and we now have a very substantial understanding of the neural mechanisms

that mediate circadian timing in mammals. Circadian rhythms have two principal properties; they are endogenously generated and, hence, free-run in the absence of a light-dark cycle but are normally entrained to the light-dark cycle (Fig. 1). These properties permit us to infer the major components of a system which is responsible for the generation and regulation of circadian rhythms. This system, designated the circadian timing system (CTS), can be defined as a set of related neural structures whose function is the temporal organization of physiological processes and behavior into a circadian pattern. The endogenous generation of circadian rhyth s indicates the presence of pacemakers in the system and the property of entrainment necessitates the presence of photoreceptors with visual pathways cou-

T

LD LIGHl

Y

EYE

ENTRAINING PATHWAY(S1

PACEMMER

COUPLING PATHWAYS

Fig. 1. The left panel shows aschematized circadian rhythm. Each line represents one24-h period. h a light-dark cycle (LD), the rhythm is entrained with the function (dark line) present only during the dark period. In constant darkness (DD), the rhythm free-runs with the function onset occurring later each day. The right panel is a simplified diagram showing the necessary components of a circadian timing system to account for the properties of circadian rhythms shown on the left.

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pling the photoreceptive elements to the pacemakers. Finally, the pacemakers must be coupled by efferent pathways to the multiple effector systems expressing circadian function (“slave oscillators”, Fig. 1). The intent of this paper is to review our understanding of the functional organization of the human CTS. This will be accomplished by reviewing the data available on the most intensively studied species, rodents, and then that on non-human primates to place the human data into an appropriate context. Functional organization of the rodent CTS

The suprachiasmatic nucleus, a circadian pacemaker The discovery of a direct retinohypothalamic projection (RHT; Hendrickson et al., 1972; Moore and Lenn, 1972;Moore, 1973)was the crucial step in beginning the elucidation of CNS structures involved in circadian timing. The RHT terminates in the suprachiasmatic hypothalamic nucleus (SCN) and, virtually immediately after demonstration of the RHT, two studies demonstrated the functional importance of the SCN by showing that its ablation abolishes circadian function (Moore and Eichler, 1972; Stephan and Zucker, 1972). These studies were widely confirmed (cf., Rusak and Zucker, 1979; Moore, 1983; Rosenwasser and Adler, 1986, for reviews), and it is now generally accepted that the SCN is the principal pacemaker in the CTS. The evidence for this conclusion can be summarized as follows. As noted above, ablation of the SCN results in a loss of circadian rhythms. The SCN exhibits a rhythm in neuronal firing rate in vivo that is preserved after isolation from the rest of the brain in a small hypothalamic island (Inouye and Kawamura, 1979, 1982). A similar rhythm is present in glucose utilization (Schwartz and Gainer, 1977; Schwartz et al., 1980) and both rhythms persist in the SCN in hypothalamic slices in vitro (Green and Gillette, 1982; Groos and Hendriks, 1982; Shibata et al., 1982; Newman and Hospod, 1986; Shibata and Moore, 1988). Perhaps the most powerful evidence ~

for the role of the SCN as a circadian pacemaker is that the restoration of circadian rhythmicity induced by transplantation of fetal anterior hypothalamus into arrhythmic hosts requires the presence of SCN (Drucker-Colin et al., 1984; Sawaki et al., 1984; Lehman et al., 1987; DeCoursey and Buggy, 1989), and the period of the restored rhythmicity is a function of donor, not host, parameters (Ralph et al., 1990).

Entrainment path ways Entrainment pathways are those that render the phase and period of the endogenous pacemaker appropriate to the environmental light-dark cycle. Thus, entrainment pathways are visual projections. Two entrainment pathways have been demonstrated. As noted above, the first to be demonstrated was a direct projection from the retina to the SCN. The RHT appears to arise from a class of retinal ganglion cells within the general group termed Wcells (Murakami et al., 1989). It seems likely that these cells respond predominantly to changes in luminous flux (cf., Meijer and Rietveld, 1989, for review). The RHT is sufficient to maintain the entrainment of circadian rhythms; section of the RHT eliminates entrainment (Johnson et al., 1988a) whereas section of all other visual pathways does not alter stable entrainment (Klein and Moore, 1979). There are two patterns of RHT projection in rodents (Johnson et al., 1988b). In both albino and hooded rats, the projections are bilateral with that to the contralateral SCN approximately twice as dense as that to the ipsilateral SCN. Within the SCN, the projection is predominantly to the ventrolateral portion of the nucleus. There are sparse projections to the adjacent anterior hypothalamic area but the major projections outside the SCN are to the lateral hypothalamic area and the retrochiasmatic area. In contrast, the hamster has RHT projections that extend widely over the anterior hypothalamic area in addition to those to the SCN, lateral hypothalamus and retrochiasmatic area and small projections to basal forebrain and anterior thalamus (Johnson et al., 1988b). A recent study indicates, however, that the projections in the rat are more widespread than we

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had appreciated (Levine et al., 1991), but still not as extensive as those in the hamster. The second entrainment pathway is a secondary visual projection from the intergeniculate leaflet (IGL) of the lateral geniculate complex to the SCN. The intergeniculate leaflet is a small group of cells, intercalated between the dorsal lateral geniculate and ventral lateral geniculate, receiving a bilateral projection from the retina, apparently from the same retinal ganglion cells projecting to the SCN (Pickard, 1985). The projection of the lateral geniculate to SCN was first shown by anterograde transport (Swanson et al., 1974; Ribak and Peters, 1975). Subsequent studies demonstrated that this was a projection, at least in part, from neuropeptide Y (NPY)-producing neurons (Card and Moore, 1982; Moore et al., 1984; Harrington et al., 1985, 1987). The IGL also contains a population of enkephalin (ENK)-producing neurons (Mantyh and Kemp, 1983) and recent work has shown that the ENK neurons project exclusively to the contralateral IGL in a commissural pathway (Card and Moore, 1989). The projection of NPY neurons in the IGL is exclusively to the ventrolateral SCN where it overlaps the RHT projection (Card and Moore, 1982,1989). If the RHT is sufficient to maintain entrainment, what is the function of the IGL-SCN projection, the geniculohypothalamic tract (GHT)? This question has been studied using three techniques: lesions, stimulation and in vitro electrophysiology. Ablation of the IGL alters the period of freerunning rhythms and alters entrainment to changes in light cycles (Harrington and Rusak, 1986, 1988; Pickard et al., 1987; Johnson et al., 1989). Stimulation of the IGL produces time-dependent phase changes with a phase-response curve very similar to that of dark pulses (Johnson et al., 1989). Application of NPY to the SCN region in intact hamsters (Albers and Ferris, 1984), or to the SCN in slices (S. Shibata and R.Y. Moore, unpublished results), produces phase changes with a very similar phase-response curve. Thus, it seems very likely that the GHT participates in entrainment but the interaction of the GHT with the RHT, and non-visual inputs to the SCN, remain to be elucidated.

SCN organization The SCN, in virtually all mammals, is a cytoarchitectonically distinct cell group lying above the optic chiasm separated from the ependymal wall of the ventral third ventricle by a thin rim of periventricular nucleus. It is distinctive both because of its location and its population of small, tightly compacted neurons. There are two subpopulations of neurons in the SCN. The region of the nucleus receiving RHT and GHT input is characterized by a population of vasoactive intestinal polypeptide (VIP)-producing neurons (Fig. 2; Card et al., 1981; Van den Pol and Tsujimoto, 1985; Cassone and Speh, 1988). The remainder of the nucleus is characterized by a population of vasopressin (VP)producing neurons (Swaab et al., 1975: Sofroniew Rostra1

VP

Mid-

1'

a

VIP

RHT

u

NPY

I-

Fig. 2. Drawings showing the organizationof the rat SCN at three levels from rostra1 to caudal. The distribution of VP and VIP neurons and fibers, and RHT and GHT (NPY)fibers is shown for each.

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and Weindl, 1978; Van den Pol and Tsujimoto, 1985; Cassone and Speh, 1988). Although the VP and VIP populations predominate in the rat SCN, there are a number of other peptides (e.g., somatostatin, enkephalin, angiotensin 11, gastrin releasing peptide, atrial natriuretic peptide) found in small numbers of SCN neurons (Van den Pol and Tsujimoto, 1985; Watts and Swanson, 1987). The rat SCN has a complex intrinsic structure and many intrinsic neurons appear to give off axons with extensive collateralization within the nucleus (Van den Pol, 1980). Recent work suggests that most, if not all, SCN neurons are GABA-producing (Okamura et al., 1989). This would indicate that the peptides demonstrated in SCN neurons are colocalized with GABA. In summary, the rodent SCN has two subdivisions: (1) a visual component which receives RHT and GHT input and contains a population of VIP neurons; and (2) a separate zone receiving limited visual input and characterized by a population of VP neurons. As noted above, SCN neurons exhibit very uniform and stable rhythms in firing rate (Shibata et al., 1982; Shibata and Moore, 1988), and it seems likely that they are born as individual circadian oscillators that are coupled during development into a neural network that functions as a pacemaker (Moore and Bernstein, 1989)or, in selected circumstances, as sets of pacemakers (Moore et al., 1989). SCN efferents SCN efferents were first demonstrated in the rat using the autoradiographic tracing method (Swanson and Cowan, 1975) and recently were shown using anterograde transport of plant lectin (Watts et al., 1987). There are five principal projection pathways but most of these are quite small (Watts et al., 1987).Thesesmallprojectionsareas follows: (1) projections from the nucleus rostrally into the anterior hypothalamic area and medial preoptic area; (2) projections laterally into the lateral hypothalamic area; (3) projections dorsally and rostrally into the lateral septum and into the paraventricular nucleus of the thalamus and extending caudally into the rostra1 periaqueductal gray. The remaining pro-

jections are essentially intrahypothalamic and largely in the vicinity of the SCN. These take two routes, directly caudally into the retrochiasmatic area and dorsally and caudally into the adjacent anterior hypothalamic region, particularly in an areaimmediately beneath the paraventricular nucleus, the “subparaventricular zone” (Watts et al., 1987). From these pathways projections extend into the paraventricular nucleus and caudally into the dorsomedial nucleus, ventromedial nucleus, arcuate nucleus, periventricular zone, dorsal hypothalamic area and the ventral tuberal area. This pattern of lectin transport is mimicked to a large extent by the pattern of VP immunoreactivity (Sofroniew and Weindl, 1978) and VIP immunoreactivity (Moore et al., 1985) arising from SCN neurons. The general features of the organization of the rodent CTS are shown in Fig. 3. Organization of the primate CTS

The primate CTS will be described in two parts, nonhuman primate and human. Non-human primates are generally accepted as the best model for studying brain organization and function relevant to the human brain. And, because experiments can be done on these primate brains, they can provide information that might only be obtained from the rare, circumscribed lesion produced by disease in the human that permits an analysis of brain organization. Our work has been done on the macaque monkey, and human brain obtained from routine post-mortem examination, and this will form the basis for the descriptions that follow.

Non-human primate CTS (monkey) Monkey entrainment pathways The monkey SCN, like that in other mammals, is made up of parvicellular neurons in a tightly-compacted nucleus lying dorsal to the optic chiasm and lateral to the ventral third ventricle. The organization of the monkey SCN demonstrated by immunocytochemistry will be described below.

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OTHER BRAIN AREAS

BASAL FOREBRAIN EYE THALAMUS

D-

HYPOTHALAMUS PERIAQUEDUCTAL GRAY

0 ".:

"OTHER PACEMAKERS

Fig. 3. Schematic diagram showing the organization of the CTS in the rodent brain. The diagram indicates that there are two visual entraining pathways, RHT and GHT, terminating in the ventrolateral SCN. This region functions as a circadian oscillator and is normally coupled to the dorsomedial SCN. Other inputs to the SCN are neural (other brain areas) and hormonal (e.g., melatonin). Outputs are principally to hypothalamus but also to basal forebrain, thalamus and periaqueductal gray.

RHT. The monkey RHT was first shown in autoradiographic material (Moore, 1973). Subsequent studies have confirmed this and demonstrated that the monkey RHT is similar in most features to that in other species (Tigges and O'Steen, 1974). In our material the RHT is predominantly an ipsilatera1 projection (Fig. 4). It is evident in the ventral portion of the SCN rostrally and is largest in the mid-portion of the nucleus where it forms an extremely dense plexus over the ventral SCN. There are scattered fibers in the remainder of the SCN and these extend beyond its borders into the adjacent anterior hypothalamic area, particularly dorsal to the SCN. The dense plexus in the SCN continues to the caudal border of the nucleus and extends into the retrochiasmatic area. In addition to these projections, there is a projection to the lateral hypothalamic area. With the exception of the feature of a predominantly ipsilateral projection, this pattern of RHT projection is nearly identical to that seen in the rat (Johnson et al., 1988b). The predominance of the ipsilateral projection has been

described previously (Tigges and O'Sheen, 1974; Magnin et al., 1989).

GHT. Although there are no experimental data demonstrating a projection from the geniculate to the SCN in the monkey, it has been shown that the monkey has a population of NPY neurons in the geniculate complex in a region believed to be a homologue of ventral lateral geniculate and a dense axonal plexus in the ventral SCN in the same distribution as the RHT (Fig. 4; Moore, 1989). This plexus is essentially identical to that in rodents and it seems reasonable to conclude that there is a NPYproducing GHT in the monkey as in other mammals. One point will be discussed further in considering the human CTS. The monkey and human have a very similar organization of the lateral geniculate complex. The major component is the dorsal lateral geniculate with its typical lamination. This is surrounded medially, and dorsally, by a pregeniculate nucleus, generally believed to be the homologue of the ventral lateral geniculate. This

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contains a large population of NPY neurons, and is likely to be comprised of both ventral lateral geniculate and intergeniculate leaflet, but the basis for making this division is not yet evident (Fig. 5 ) . There is another group, with neurons larger than those in the pregeniculate nucleus, that is scattered laterally and dorsally to the dorsal lateral geniculate. No NPY neurons are present in this group which is continuous with the reticular nucleus of the thalamus and corresponds to the perigeniculate nucleus of non-primate mammals (Fig. 5 ; Jones, 1985).

Monkey SCN organization As in other mammals, the monkey SCN has two Rostra1

Mid-

Caudal

VP

distinct subdivisions. The zone receiving RHT input is characterized by a large population of VIP neurons. There is a dense VIP axon plexus in this zone extending into the adjacent SCN (Fig. 4). The VIP neuron area is surrounded by an area of VP neurons. These neurons are small and produce an extensive plexus. The V P neuron area is continuous with the large VP neurons of the paraventricular nucleus dorsally and those of the supraoptic nucleus laterally. The entire SCN is covered by an extensive plexus of GAD-containing axons. There are very scattered NT neurons in the monkey SCN but no NPY neurons are evident (R.Y. Moore and J.C. Speh, unpublished results).

Monkey SCN efferents No experimental studies of SCN efferents have been carried out on the monkey. Thus, the only basis for examining efferents is analysis of the pattern of VIP-containing projections. In our material, this appears quite similar to that in the rat. There are VIP axons that run rostrally into the anterior hypothalamic area and medial preoptic area. Axons ascending dorsally form an extensive plexus in the anterior hypothalamic area and the zone adjacent to the paraventricular nucleus. This is more pronounced than that in the rat and, in addition, there are projections into the paraventricular nucleus. SCN efferents also project in a small bundle into the lateral hypothalamus and the paraventricular thalamic nucleus. There are dense projections into the retrochiasmatic nucleus with some continuing into the tuberal periventricular area, ventral tuberal area, dorsomedial nucleus and dorsal hypothalamic area. Thus, the monkey pattern of SCN efferents appears very similar to that described in the rat with the exception of a more extensive distribution of projections to the anterior hypothalamic area (R.Y. Moore and J.C. Speh, unpublished results). Human CTS

Fig. 4. Diagrams showing the organization of the monkey SCN at three levels from rostra1 to caudal. The distribution of VP and VIP neurons and fibers, and RHT, GHT (NPY) and NT fibers is shown for each.

The human nervous system cannot be studied by invasive experimentation and the hypothalamus is not accessible to non-invasive functional techniques

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A

Fig. 5. Diagrams of coronal sections through the mid-portion of the lateral geniculate nucleus (LGN) drawn at the same magnification for the monkey (A) and human (E) brains. The largest component of the LGN is the dorsal lateral geniculate nucleus (LGd). Adjacent to that nucleus medially is a large nucleus that contains numerous NPY neurons (dots) and axons (stipple). The nucleus is usually designated the pregeniculate nucleus and it probably contains both intergeniculate leaflet (IGL) and ventral lateral geniculate (LGv). It is continuous with the zona incerta (ZI) medially and lies dorsal and medial to the cerebral peduncle (CP). Dorsal and lateral to the LGd is a perigeniculate nucleus (PG) which does not contain NPY neurons or axons and is continuous with the reticular nucleus of the thalamus.

because of its small size and location. Hence, the organization of the human CTS must be inferred at this time from study of the normal human brain, analysis of the consequences of spontaneous pathology and a direct study of the animal nervous system. The focus of the mammalian CTS is the SCN. The SCN has been recognized as a distinct component of the human hypothalamus for most of this century (cf., Braak and Braak, 1987; Saper, 1990, for reviews). It is not always a distinct nucleus in Nissl material and some authors have questioned its existence on that basis (Griinthal, 1930; Le Gros Clark, 1936). However, a number of recent studies using immunocytochemical methods have confirmed that the SCN is a standard feature of the human hypothalamus (Dierickx and Vandesande, 1977; Stopaet al., 1984; Swaabet al., 1985; Hofmanetal., 1988). I will review the organization of the human SCN as we have viewed it in studies of a series of

human hypothalami, obtained from individuals of both sexes, aged 6 - 9 0 years, at routine postmortem examinations. In material stained using the Nissl method, the SCN is usually evident as a compact group of small cells lying dorsal to the optic chiasm and lateral to the third ventricle. It is usually elongated in the dorsoventral axis and frequently has a fusiform or crescent shape. The boundaries of the nucleus are often difficult to discern, particularly the rostra1 and caudal boundaries. In addition, there are individual brains where the nucleus is quite indistinct. Indeed, the most characteristic feature of the human SCN is its variability from brain to brain. The shape of the human SCN is sexually dimorphic, more elongated in women and more spherical in men (Swaab et al., 1985). All of the brains inour studies were processed for immunocytochemistry with antisera against VP, VIP, NPY and neurotensin (NT). It is noteworthy

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also that we have examined a series of Nissl-stained sections from fetal and neonatal brains, prepared in our laboratory or in the Yakovlev Collection (Armed Forces Institute of Pathology, Washington, D.C., U.S.A.), and found that the SCNis a distinct and readily identifiable nucleus in virtually all of these young brains. The development of VP neurons in the human brain has been described recently as a largely post-natal event (Swaab et al., 1990). In the material prepared for immunohistochemical analysis, the SCN is clearly recognizable in all brains (Fig. 6). The SCN, with the periventricular nucleus, is the most rostral nucleus identifiable in the human brain. It first appears as a set of scattered neurons lying lateral to the rostral third ventricle in a thin zone of hypothalamic tissue ventral to the lamina terminalis and dorsal to the rostral optic Rostra1

Mid-

Caudal

NT

Fig. 6. Diagrams showing the organization of the human SCN. The distribution of VP, VIP, NT and NPY neurons and fibers is shown at three levels from rostral to caudal.

chiasm. Neurons immunoreactive to VP, VIP, NPY and NT antisera are present in this rostral SCN. As in the remainder of the SCN, these appear to be distinct populations of neurons. There are axonal plexuses within the region occupied by neuronal perikarya and extending into adjacent neuropil of chiasmatic hypothalamus, including the rostral periventricular nucleus, and into the lamina terminalis. This rostral extension of the SCN is quite long but the nucleus assumes its more characteristic appearance as the rostral hypothalamus forms as two pillars of tissue extending Iateral to the third ventricle between the optic chiasm and the basal forebrain. Three levels of SCN, from rostral through caudal, are shown in Fig. 6. As in the other mammals, the human SCN has populations of VP- and VIP-containing neurons forming subdivisions of the nucleus. VIP neurons are present in the ventral and central part of the nucleus. Rostrally a number of VIP neurons are embedded within the fibers of the dorsal chiasm. VIP neurons form an extensive axonal plexus within the SCN and this extends well beyond its boundaries (see below). The VIP neurons are scattered among V P neurons rostrally, but caudally, in the main portion of the nucleus, they are surrounded by VP neurons (Fig. 7). The V P neurons also form a dense axonal plexus within the nucleus and this plexus overlaps the region of the main concentration of VIP neurons. It seems likely from the animal data that this region is the major zone of RHT projection. Using a method intended to show long-lasting axonal debris, Sadun and coworkers (Sadun et al., 1984; Schaechter and Sadun, 1985) have reported the presence of RHT fibers in the human SCN and paraventricular nucIeus in individuals blinded many months before death. On the basis of the monkey data, one would expect RHT projections in the human to be distributed to the anterior and lateral hypothalamic areas and the retrochiasmatic area as well as the SCN. A detailed analysis of SCN organization in the human was recently published by Mai and coworkers (Mai et al., 1991). The human SCN has two features that differ significantly from the monkey and other mammals.

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Fig. 7. Photomicrographs of coronal sections through the mid-portion of the human SCN. Adjacent sections stained with Nissl stain ( A ) and antisera to NPY ( B ) , NT (C) and VP (0). The SCN is designated by the arrows in A . In B , there are numerous NPY neurons within the SCN and a modest axonal plexus. The SCN is surrounded by a dense NPY plexus. In C , the NT neurons and plexus fill the entire SCN and extend into adjacent hypothalamus. In D, the VP neurons surround the central region of the SCN, presumably the locus of RHT terminals. Marker bar, 500 Fm.

First, the human SCN has avery large population of NT-containing cells. NT neurons extend from the rostral border of the SCN through its caudal extent. These were first noted by Mai et al. (1987). NT neurons are the largest population in the human SCN. There are NT neurons in the adjacent anterior hypothalamus but these are much more scattered than the SCN neurons. The NT neurons in the SCN extend over the entire nucleus and have a very dense axonal plexus. This plexus is contiguous with a plexus in the adjacent anterior hypothalamus. It should be noted that the monkey and rat SCN have only very scattered NT neurons with a very limited plexus. Thus, the human SCN differs significantly

from the SCN in those species. Second, the human SCN contains a large population of NPY neurons. These also extend from the rostral portion of the SCN to its caudal border. They are predominantly located in the center of the nucleus where they largely overlap the distribution of the VIP neurons. Since there are no NPY neurons in the rodent or monkey SCN, this population in the human nucleus is a distinctive feature (Moore, 1989). The NPY neuron group differs from the other peptide-containing groups in that it is associated only with a scattered axonal plexus. The plexus appears to have two components, very fine fibers with small varicosities and larger fibers with

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coarse varicosities (Moore, 1989). Since this population is so different from that found in other animals, its functional significance cannot be inferred from animal studies. In addition, the SCN is surrounded dorsally and laterally by a very dense NPY plexus arising from local anterior hypothalamic neurons, and the projections of the intrinsic SCN neurons cannot be determined directly. It is possible that the NPY neurons project within the SCN as a local plexus which might function in a manner similar to the GHT (Moore, 1989). It is unclear whether there is a GHT in the human brain. As noted above, there is a large population of NPY neurons in the human pregeniculate nucleus (Fig. 5 ) that should correspond to the rodent IGL. There is no way at this time to determine whether they project to the SCN. The efferent projections of the SCN can probably be assessed by analysis of material prepared with antisera to VIP. For this to be entirely valid, it requires that the SCN be the major locus of VIP neurons in the human hypothalamus, as it is in other animals. This seems likely, but is not yet established. At this time, we have carried out a preliminary analysis of distribution of VIP-immunoreactive axons in the human hypothalamus. Our material only covers the area from the lamina terminalis to the median eminence. Fibers are evident within the SCN from the rostral cells through the caudal border. This plexus is dense and extends beyond the border of the SCN into the adjacent anterior hypothalamic area. It is most dense in the area dorsal to the SCN extending toward the ventral border of the paraventricular nucleus. As in the monkey, there are scattered fibers entering the paraventricular nucleus. The anterior hypothalamic plexus, in contrast, is much more extensive in the human covering much of the rostral anterior hypothalamus. There are few, if any, fibers in the supraoptic nucleus. The dense plexus in the SCN extends caudally into the retrochiasmatic area but appears to become much smaller and less dense as the tuberal area is reached. We do not yet have the material to characterize the caudal extent of the plexus. In summary, we would conclude that the human CTS is similar to that of the rodent and the monkey,

but there are significant differences (Fig. 8). The human RHT is likely to be similar to that in other species, innervating the SCN, lateral hypothalamus, anterior hypothalamus and retrochiasmatic area bilaterally. If a GHT is present, its terminal plexus is obscured by the presence of a unique population of NPY neurons. The human SCN has an organization generally similar to that in other mammals. However, the presence of NPY neurons and a separate, very large population of NT neurons, is distinctly different from other animals and suggests that the human SCN has a functional organization that differs from other mammals. Whether this represents fundamental functional differences, or simply slightly different phenotypic expression of functionally similar neuronal populations, is unclear.

Disorders of circadian timing Disorders of circadian timing have been investigated intensively for only a relatively brief period. The intent of this section is to introduce this subject and relate the disorders to potential pathophysiologic mechanisms, not to review them exhaustively. A list of disorders of circadian timing is presented in Table I. Congenital or acquired blindness, when sufficiently severe, removes direct entraining effects of light. As might be expected, the majority of affected individuals exhibit free-running rhythms (Lewy and

M O W Y BASAL FOREBRAIN EYE - * y N

,-eTHNAMUS THALAMUS PERIAQUEDUCTAL GRAY

HUMAN

.. -. RHT

EYE

BASAL FOREBRAIN /--

4

%

*-\

* HYPOTHALAMUS

/--+

*SCN ,GL/

*%

THALAMUS '2 PERIAOUEDUCTAL GRAY

Fig. 8. Diagrams showing the demonstrated organization of the CTS in the monkey and human. The interrupted lines indicate that the connection has not been demonstrated.

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Newsome, 1983; Sack et al., 1987). Blindness, then, represents a disorder of entrainment. The “hypothalamic” tumors listed in Table I arise from tissue outside the hypothalamus and affect the hypothalamus by compression. These tumors are usually slowly growing and would evolve over a sufficient length of time for disturbances in circadian function to be evident. Further, any alteration in circadian function would likely be masked by other manifestations of the tumor, particularly blindness and endocrine dysfunction. Consequently, there have been few instances of hypothalamic tumors in which circadian disorders have been documented. As an example, we observed a patient with an optic nerve glioma who had symptomatic indications of rhythm disturbance, and evidence of loss of rhythmicity in several functions demonstrated by detailed testing, that correlated with compression of the chiasmal hypothalamus visualized by CT scan (L.P. Morin and R.Y. Moore, unpublished results). These effects are best interpreted by analogy with SCN lesion effects in animals as reflecting a loss of pacemaker function. A similar

TABLE I Disorders of circadian timing Congenital or acquired blindness Hypothalamic tumors Craniopharyngioma Optic nerve glioma Pituitary adenoma Disorders of the sleep-wake schedule Rapid time zone change (jet lag) Work shift disorder Advanced sleep phase syndrome Prolonged sleep phase syndrome Non-25-h sleep-wake syndrome Irregular sleep-wake syndrome Affective disorders Seasonal affective disorder Bipolar affective disorder Major depressive disorder Aging and dementia (Alzheimer’s disease)

case was reported recently by Cohen and Albers (1991). Disorders of the sleep-wake cycle generally reflect abnormalities of entrainment. This is clearly true of the “jet-lag” syndrome, work shift disorder and the sleep phase disorders (cf., Wagner, 1990, for review). The sleep phase disorders are of particular interest because they appear to be effectively treated in many instances by a program of re-entrainment. Non-24-h sleep-wake syndrome is a chronic, reasonably steady pattern of delays in sleep onset and waking. The pattern is similar to that observed in normal individuals in constant conditions, or in the blind (see above), such that the individuals appear to be free-running. This also is an instance of a disorder of entrainment but it is unclear whether the problem is in the entraining pathways or the response of the pacemaker to an entraining input. Irregular sleep-wake syndrome is a disorder in which the circadian pattern is lost and, hence, probably represents a disturbance of pacemaker function. Although it is seen in intact individuals, it is more common in individuals with severe developmental or degenerative brain disorders (Wagner, 1990). Over the past 15 years there have been many studies that report alterations in circadian function in affective disorders (cf., Van Cauter and Turek, 1986; Linkowski et al., 1987, for reviews) but the findings are by no means consistent nor is there complete agreement as to the potential mechanisms (Ehlers et al., 1988). Themost consistent finding has been a phase advance of some rhythms, an abnormality that would suggest either an alteration of entrainment or of pacemaker response to entraining cues. One of the most common complaints of the aged is a disturbance of sleep (Vitiello and Prinz, 1990a). Sleep is typically disrupted more frequently than in the young and there is also often a pattern of an advance of the onset of sleep and waking. Many of the changes are consistent with a dampening of the amplitude of circadian function (cf., Vitiello and Prinz, 1990a, for review). It is unclear whether the alteration in amplitude reflects a change in the strength of the signal from the pacemaker or an in-

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ability of output systems to respond to a normal signal. In Alzheimer’s disease, these changes appear to be exaggerated (Vitiello and Prinz, 1990b; Witting et al., 1990). This isconsistent with the observation that there are fewer SCN neurons in the brains of individuals with Alzheimer’s disease than in agematched controls (Swaab et al., 1985). Summary and conclusions The mammalian circadian timing system has three principal components; (I) photoreceptors and visual pathways mediating entrainment; (2) a pacemaker, the suprachiasmatic nucleus of the hypothalamus; and (3) efferent pathways coupling the suprachiasmatic nucleus to effector systems exhibiting circadian function. In most mammals there are two visual entraining pathways, a direct retinohypothalamic pathway terminating in the suprachiasmatic nucleus, for which the transmitter is unknown, and a secondary visual pathway, the geniculohypothalamic tract, from the intergeniculate leaflet of the lateral geniculate to the suprachiasmatic nucleus that is neuropeptide Y-producing. These pathways end in a distinct subdivision of the suprachiasmatic nucleus characterized by the presence of vasoactive intestinal polypeptide neurons. A second suprachiasmatic nucleus division does not receive visual afferents and is characterized by vasopressin neurons. The efferent projections of the suprachiasmatic nucleus are very restricted, predominantly to the hypothalamus. Although we have much less information on the human circadian timing system than on that of other animals, it seems clear that the human conforms to the general animal pattern in most features. There are, however, two significant differences. First, the largest neural component of the human suprachiasmatic nucleus is a population of neurotensin neurons found throughout the nucleus. Few, if any, neurotensin neurons are found in monkey or other mammals. Second, the human suprachiasmatic nucleus contains a large number of neuropeptide Y neurons located where the plexus arising from geniculate neuropeptide Y neurons is found in other

mammals. This is unique and suggests that the geniculohypothalamic projection may be bypassed in the human. It also may imply that the functional organization of the human SCN is fundamentally different from that of other mammals. The function of the circadian timing system is to coordinate the activities of a series of homeostatic regulatory mechanisms with the control of behavioral state in a temporal pattern that facilitates adaptive behavior, including reproduction (Fig. 9). The function of this system, then, is to provide the ap-

LIGHT

HORMONAL MILIEU (Melatonin)

INTERNAL MILIEU (Hypothalamus)

I AFFECTIVE STATE (Limbic Forebrain)

SCN

I EXTERNAL MILIEU (SENSORY INPUT) BEHAVIORAL STATE (Reticular Formation)

HYPOTHALAMIC HOMEOSTATIC REGULATION BEHAVIORAL STATE REGULATION

Feeding Behavior Sleep-WakeCycles Reinforcement Mechanisms Drinking Behavior Temperature Regulation Anterior Pituitary Regulation Osmoregulalion Reproductive Behavior Autonomic Regulation

ADAPTIVE BEHAVIOR Fig. 9. Diagram illustrating the functional organization of the CTS with respect to the temporal organization of physiological processes and behavioral state. The SCN is the principal component of the CTS. Light is the principal “zeitgeber” for the SCN, acting through the RHT and, probably, the GHT. The other inputs that regulate SCN function are from the hypothalamus, the limbic forebrain, the reticular formation and the hormonal milieu. The SCN provides a temporal organization for the variety of hypothalamic regulatory mechanisms and the neural mechanisms of behavioral state regulation required to provide a necessary substrate for adaptive behavior.

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propriate physiological and behavioral background to facilitate adaptation and survival. Acknowledgements The animal experimental work reported here was supported by NIH Grant NS-16304. The work on the monkey and the human brain was supported by a grant from the United States Air Force Office of Scientific Research (AFOSR Grant 91 -0175). I am particularly indebted to Dr. Nancy Peress, Department of Pathology, State University of New York at Stony Brook, and Dr. John MOOSSY, Department of Pathology, University of Pittsburgh, who made the human material available to me.

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115 amic nucleus receiving retinal input in man: the paraventricular nucleus. Brain Rex, 340: 243 - 250. Schwartz, W. and Gainer, H. (1977) Suprachiasmatic nucleus: use of [‘4C]-labeled deoxyglucose uptake as a functional marker. Science, 197: 1089- 1091. Schwartz, W., Davidsen, L. and Smith, C. (1980) In vivo metabolic activity of a putative circadian oscillator, the rat suprachiasmatic nucleus. J. Cornp. Neurol., 189: 157 - 167. Shibata, S. and Moore, R.Y. (1988) Electrical and metabolic activity of suprachiasmatic nucleus neurons in hamster hypothalamic slices. Bruin Res., 438: 374- 378. Shibata, S., Oomura, Y., Kita, H. and Hattori, K. (1982) Circadian rhythmic changes of neuronal activity in the suprachiasmatic slice. Brain Res., 247: 145- 148. Sofroniew, M.V. and Weindl, A. (1978) Identification of parvicellular vasopressin and neurophysin neurons in the suprachiasmatic nucleus of a variety of mammals including primates. J. Cornp. Neurol., 193: 659- 675. Stephan, F.K. and Zucker, I. (1972) Circadian rhythms in drinking behavior and locomotor activity of rats are eliminated by hypothalamic lesions. Proc. Natl. Acud. Sci. U.S.A., 69: 1583- 1586. Stopa, E.G., King, J.D., Lydic, R. and Schoene, W.C. (1984) Human brain contains vasopressin and vasoactive intestinal polypeptide neuronal subpopulations in the suprachiasmatic region. Bruin Rex, 297: 159- 163. Swaab, D.F., Pool, C.W. and Nijveldt, F. (1975) Immunofluorescence of vasopressin and oxytocin in the rat hypothalamo-neurohypophyseal system. J. Neural Transm., 36: 195-215. Swaab, D.F., Fliers, E. and Partiman, T.S. (1985) The suprachiasmatic nucleus of the human brain in relation to sex, age and senile dementia. Brain Res., 342: 37 -44. Swaab,D.F.,Hofman,M.A.andHonnebier,M.B.O.M. (1990) Development of vasopressin neurons in the human suprachiasmatic nucleus in relation to birth. Dev. Bruin Res., 52: 289-293. Swanson, L.W. and Cowan, W.M. (1975) The efferent connections of the suprachiasmatic nucleus of the hypothalamus. J. Cornp. Neurol., 160: 1 - 12. Swanson, L.W., Cowan, W.M. and Jones, E.G. (1974) An autoradiographic study of the efferent connections of the ventral geniculate nucleus in the albino rat and the cat. J. Comp. Neurol., 156: 143 - 164. Tigges, J. and O’Steen, W.K. (1974) Termination of retinofugal fibers in squirrel monkey: a re-investigation using autoradiographic methods. Brain Rex, 79: 489 - 495. Van Cauter, E. and Turek, F.W. (1986) Depression: a disorder of timekeeping? Perspect. Biol. Med., 29: 510-517. Van den Pol, A.N. (1980) The hypothalamic suprachiasmatic nucleus of the rat: intrinsic anatomy. J. Cornp. Neurol., 191: 661 - 702. Van den Pol, A.N. and Tsujimoto, K.L. (1985) Neurotransmitters of the hypothalamic suprachiasmatic nucleus: im-

munocytochemical analysis of 25 neuronal antigens. Neuroscience, 15: 1049- 1086. Vitiello, M.V. and Prinz, P.N. (1990a) Sleep and sleep disorders in normal aging. In: M.J. Thorpy (Ed.), Handbook of Sleep Disorders, Marcel Dekker, New York, pp. 139- 153. Vitiello, M.V. and Prinz, P.N. (1990b) Sleep/wake patterns and sleep disorders in Alzheimer’s disease. In: M.J. Thorpy (Ed.), Handbookof Sleep Disorders, Marcel Dekker, New York, pp. 703-718. Wagner, D.R. (1990) Circadian rhythm sleep disorders. In: M.J. Thorpy (Ed.), Handbook of Sleep Disorders, Marcel Dekker, New York, pp. 493 - 527. Watts, A.G. and Swanson, L.W. (1987) Efferent projections of the suprachiasmatic nucleus 11. Studies using retrograde transport of fluorescent dyes and simultaneous peptide immunohistochemistry in the rat. J. Cornp. Neurol., 258: 230 - 252. Watts, A.G., Swanson, L.W. and Sunchez-Watts, G . (1987) Efferent projections of the suprachiasmatic nucleus. I. Studies using anterograde transport of phaseolus leucagluttinin in the rat. J. Cornp. Neurol., 258: 204-229. Witting, W., Kawa, I.H., Eikelenboom, P., Mirmiran, M. and Swaab, D.F. (1990) Alteration in the rest-activity rhythm in aging and Alzheimer’s disease. Biol. Psychiatry, 27: 563 - 572.

Discussion G.J. Boer: In all your schemes the SCN is the centre through which retinal information is passed on to other brain structures. However, several studies haveshown that lesion of the SCN does not prevent light information to come through (Dunn et al., 1977; Rusak, 1977; Abe et al., 1979). Do you have an anatomical explanation for such a masking effect or d o you consider the lesions just incomplete? R.Y. Moore: These are retinal projections to the lateral hypothalamus that would not be affected by SCN lesions and could mediate masking effects (Johnson et al., 1988). E. Goudsmit: You showed the presence of four neuropeptides in human SCN neurons. Especially neuropeptide Y (NPY), neurotensin and vasoactive intestinal peptide (VIP) neurons showed a considerable overlap in their distribution within the SCN. Do you know whether there is any coexistence of these peptides in SCN neurons? R.Y. Moore: These all appear to be distinct and non-overlapping populations. I cannot exclude the possibility of coexistence of some peptides in a small subpopulation but I would think this is minor. R. Ravid: How did you check for the fact that the neurotensin staining was independent of age, sex and post-mortem delay? Are there any circadian changes in neurotensin in the human brain? R.Y. Moore: We did not do a systematic study of the effect of post-mortem delay, age or sex on neurotensin staining. However, we have brains with short post-mortem delays and relatively poor

’

116 staining and ones with long delays and superb staining, so I doubt that delay has important consequences in this regard. Similarly, there are no evident differences with age or sex but this really needs to be pursued with appropriate, quantitative studies. I do not know of any circadian changes in neurotensin but the animal studies of circadian changes in peptide content suggest that these are usually so small that they would not be detected by immunocytoc hemist ry . D.W. Pfaff: Would you care to speculateabout the findings that SCN transplants can be effective from quite a variety of sites? R.Y. Moore: We have concluded, as have others, that restoration of circadian function by transplants may occur by two mechanisms, development of critical connections between transplant and host, and by a humoral mechanism. It should be noted that only locomotor activity and related rhythms are known to be restored and it is evident that we need much more information before we can establish the critical mechanisms by which transplants are effective. C.B. Saper: In considering Dr. Pfaff’s question, it occurs to me that there is a set of well-characterized humoral factors that regulate wake-sleep cycles, body temperature and adrenal corticosteroid secretion: prostaglandins (Hayaishi, 1991). Phasic elaboration of prostaglandins by transplanted SCN could potentially allow it to exert circadian control, even in the absence of functional connections with the host brain. Do you know of any evidence for the role played by prostaglandins in the circadian cycle? This is a hypothesis that could easily be tested with inhibitors of prostaglandin synthesis, such as indomethacin. R.Y. Moore: That is an excellent suggestion and we should test the hypothesis. G.J. Boer: With respect to the question of Dr. Pfaff 1might add that results of Harrington et al. (1987) and ourselves (H.A. Griffioen, H. Duindam, G. Rietveld and G.J. Boer, unpublished observations) showed that heterotopic placement of fetal SCN in SCN-lesioned and arrhythmic rat failed to show recovery of drinking, eating and locomotor circadian behavior. Moreover, recovery of rhythms in rat witha homotopic placement has a success rate of only about 40%. Both results, therefore, differ from hamster studies in which recovery in SCN-lesioned hamster is much higher and independent of the site of placement. So species differences might be involved. For rat, I d o not believe in a humoral signal from the SCN, since - among other reasons non-functional grafts seem to have all the immunocytochemical characteristics, as do the functional SCN grafts (Boer and Griffioen, 1990). R.Y. Moore: There may well be differences between the hamster and rat with respect to the success rate for transplants in inducing functional recovery. I would doubt very much, however, that the mechanisms by which transplants work would differ among these relatively closely related species. At this point we obviously need much more information to resolve this issue. J.K. Mai: With reference to the regulation of the extraneuronal milieu of the SCN and the homeostatic function of this nucleus, is there any indication for specific roles of astrocytes (which are

not evenly distributed in the human SCN)? R.Y. Moore: There are not sufficient data to comment on thi, question. D.F. Swaab: Firstly, did you really mean to say that the SCN i. the earliest developing nucleus in the human hypothalamus? Ou vasopressin cell counts show that the SCN contains only 20% o the adult VP expressing cell numbers at term (Swaab et al., 1990) whereas the paraventricular nucleus contains already adul vasopressin and oxytocin neuron numbers at 26 weeks of gesta tion (cf., Goudsmit, this volume). Secondly, did you systematically check for the relationshi1 between the SCN as it appears in Nissl and in vasopressin im munocytochemistry in human fetuses? Is the “SCN” in Nissl ii the fetus really the SCN and not a wider area that includes not on ly the SCN but also other structures? In my experienci vasopressin cells might even be situated in a different area thai one would expect on the basis of conventional stainings (sel Swaab et al., 1990). R.Y. Moore: I did not mean to imply that the SCN was thi earliest nucleus to develop in the human hypothalamus. Rather I meant that we can see the SCN in Nissl-stained material ir fetuses at 35 - 40 weeks of gestation as a much more distinc nucleus than is evident in much of the adult material. We did no do vasopressin immunocytochemistry on the fetal material but would emphasize that the location, size and cellular morpholog: of the SCN as seen in the Nissl-stained fetal material is quit1 typical for the SCN and I doubt that other structures are inch ded. 1 should emphasize that the sections we reviewed are quit, thick, 40-100 gm, which often provides a more distinctiv, cytoarchitectural appearance. H. Braak: You did not mention Alzheimer’s disease in your lis of disorders of circadian rhythms. Can you comment on this? R.Y. Moore: Swaab and his colleagues (1985) have shown tha there is a marked decrease in SCN neuron number in Alzheimer’ disease. This correlates with observations of apparent rhythn alteration in these patients. I did not include it because it remain to be determined that this is a true alteration in circadian functioi rather than a masking effect associated with dementia. F.W. van Leeuwen: Comment: in your introduction you men tioned that in the rat the total number of angiotensin I1 (Ang I1 neurons is less than those of vasopressin (VP). However, with recently raised antibody against Ang I1 (Imboden et al., 1989)w could observe a large number of Ang I1 neurons at exactly th same mediodorsal positions as the VP neurons. Therefore, would not be surprised if Ang I1 and VP are colocalized withi SCN neurons similar to what has been found in the VP neuron of the supraoptic nucleus (Kilcoyne et al., 1980) where they ar coreleased and coacting at synaptic sites. I suppose that this in formation is of particular importance for physiologists interestel in the role of VP in SCN functioning. R.Y. Moore: The statement I made about Ang I1 immunoreac tivity in the rat SCN referred to work by Watts and Swanso (1987). Certainly, any statement about numbers of neuron shown by immunocytochemistry is subject to revision if a ne&

117 more sensitive antiserum becomes available and shows a different picture. I am certainly open to the idea that there are more Ang 11-containingneurons than have been reported. In addition, I would agree that SCN neuron peptides should have a significant role in their function. The problem has been in determining that role. J.D. Mikkelsen: You mentioned that the retino- and geniculohypothalamic pathways may mediate two different signals to the SCN related to light and dark pulses, respectively. I wonder whether there is any evidence that the retino- and/or geniculohypothalamic projections terminating outside the SCN (e.g., in the lateral hypothalamic area 'or the supraventricular area; Johnson et al., 1988; Mikkelsen, 1990a,b) mayreceive similar inputs, and thereby play a role in circadian rhythm regulation? R.Y. Moore: No, at this time we have no evidence that retinal projections other than those to the SCN have any role in circadian rhythm regulation.

References Abe, K., Kroning, J., Greer, M.A. and Critchlow, V. (1979) Effect of destruction of the suprachiasmatic nuclei on the circadian rhythms in plasma corticosterone, body temperature, feeding and plasma thyrotropin. Neuroendocrinology, 29: 119- 131. Boer, G.J. andGriffioen, H.A. (1990) Developmental and functional aspects of grafting of the suprachiasmatic nucleus in the Brattleboro and the arhythmic rat. Eur. J. Morphol., 28: 330 - 345. Dunn, J.D., Castro, A.J. and McNulty, J.A. (1977) Effect of suprachiasmatic ablation on the daily temperature rhythm. Neurosci. Lett., 6: 345 - 348. Harrington, M.E., DeCoursey, P.J., Bruce, D. and Buggy, J. (1987) Circadian pacemaker (SCN) transplants into the lateral ventricles failed to restore locomotor rhythmicity in arhythmic

hamsters. Neurosci. Abstr., 85.9. Hayaishi, 0 .(1991) Molecular mechanisms of sleep-wake regulation: roles of prostaglandins D2 and E2. FASEB J., 5 : 2575 - 2581. Imboden, H., Harding, J.W. andFelix, D. (1989) Hypothalamic angiotensinergic fibre systems terminate in the neurohypophysis. Neurosci. Lett., 96: 42 - 46. Johnson, R.F., Morin, L.P. and Moore, R.Y. (1988) Retinohypothalamic projections in the hamster and rat demonstrated using cholera toxin. Brain Res., 462: 301 -312. Kilcoyne, M.M., Hoffman, D.L. and Zimmerman, E.A. (1980) Immunocytochemical localization of angiotensin I1 and vasopressin in rat hypothalamus: evidence for production in the same neurons. Clin. Sci., 59: 57 - 60. Mikkelsen, J.D. (1990a) A neuronal projection from the lateral geniculate nucleus to the lateral hypothalamus of the rat demonstrated with phaseolus vulgaris leucoagglutinin tracing. Neurosci. Lett., 116: 58 - 63. Mikkelsen, J.D. (1990b) Projections from the lateral geniculate nucleus to the hypothalamus of the Mongolian gerbil (Merionesunguiculatus). An anterograde and retrograde tracing study. J. Comp. Neurol., 299: 493 - 508. Rusak, B. (1977) The role of suprachiasmatic nuclei in the generation of circadian rhythms in the golden hamster, Mesocritus auratus. J. Comp. Physiol., 118: 145 - 164. Swaab, D.F., Fliers, E. and Partiman, T. (1985) The suprachiasmatic nucleus of the human brain in relation to sex, age and dementia. Brain Rex, 342: 37 - 44. Swaab, D.F., Hofman, M.A. andHonnebier, H.B.O.M. (1990) Development of vasopressin neurons in the human suprachiasmatic nucleus in relation to birth. Dev. Brain Rex, 52: 289 - 293. Watts, A.G. and Swanson, L.W. (1987) Efferent projection of the suprachiasmatic nucleus 11. Studies using retrograde transport of fluorescent dyes and simultaneous peptide immunohistochemistry in the rat. J. Comp. Neurol., 258: 230 - 252.

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D.F. Swaab, M.A. Hofman, M. Mirmiran, R. Ravid and F.W. van Leeuwen (Eds.) Progress in Brain Research. Vol. 93

0 1992 Elsevier Science Publishers B.V. All rights reserved.

119

CHAPTER 9

Pre-natal development of a hypothalamic biological clock Steven M. Reppert Laboratory of Developmental Chronobiology, Children’s Service, Massachusetts General Hospital and Harvard Medical School, Boston, MA 02114, U.S.A.

Introduction

Circadian rhytms are ubiquitous biological phenomena. The rhythms are normally synchronized (entrained) to the 24-h period by daily environmental time cues, with the daily light-dark cycle being the most potent entraining stimulus. An entrained biological clock plays a major role in optimizing the efficiency of biological systems and better prepares the body to cope with stress, injury and disease. The system responsible for the generation and regulation of circadian rhythms is referred to as the “circadian timing system”. This timing system is made up of a biological clock (circadian pacemaker), input pathways and output pathways (Fig. 1). The suprachiasmatic nuclei (SCN), small paired structures in the anterior hypothalamus, have been established as the site of a biological clock in mammals (see Klein et al., 1991). Input pathways relay entraining information from the external environment to the biological clock in the SCN. A direct retinohypothalamic tract transmits light information from the eye to the SCN and is both necessary and sufficient for light-dark entrainment (Moore et al., 1989). An indirect retinal pathway to SCN relays in the intergeniculate leaflet and modulates the entrainment process. Output pathways from the SCN drive a diverse array of behavioral and hormonal rhythms.

During fetal life, studies in several mammalian species show that the biological clock in the SCN is oscillating in time (phase) with the environmental light-dark cycle. Entrainment of the fetal SCN involves maternal communication of circadian signals to the fetus. This chapter reviews these studies, discusses the functional implicatiogs of an entrainable biological clock during earl develop,$ ment, and comments on the development of circadian rhythms in humans. Tick tock, it’s a fetal clock

In mammals, circadian rhythms in physiology and behavior are not overtly expressed until post-natal life (for review, see Reppert, 1987). Deguchi (1975) was the first to suggest that before rhythm expression a biological clock might be oscillating in the mammalian fetus. He determined in rats the phase of a rhythm monitored under constant conditions during the post-natal period to estimate the phase of the biological clock at earlier developmental stages. Because circadian rhythms persist (free run) under constant conditions with a cycle length close to 24 h, the phase of a rhythm in the post-natal period can be used to infer the phase of the biological clock at earlier developmental stages (Davis, 1989). Deguchi’s findings suggested that a circadian clock oscillates at or before birth and that its phase is coordinated with the dam.

120 LGN

,/4

GHT

Rhythm

SCN

-+Entrainment Pathwoys

Circadian Pacemaker

output Pathways

Fig. 1. Major components of the mammalian circadian timing system. RHT, Retinohypothalamic tract; LGN, lateral geniculate nucleus; GHT, geniculohypothalamic tract; SCN, suprachiasmatic nucleus.

days 13 and 16 of gestation (Ifft, 1972; Altman and Bayer, 1978). A few synapses first appear in the SCN on day 19 of gestation, with the vast majority of synapses appearing post-natally (Lenn et al., 1977; Moore and Bernstein, 1989). A clear daynight rhythm of SCN action potentials is first apparent in fetal hypothalamic slices on day 21 of gestation (Shibata and Moore, 1987) at a time when the metabolic activity rhythm is quite prominent. Thus, the SCN begin displaying circadian Gestational Age (days) +19-20-21-222-

Post-natal paradigms cannot conclusively show that a biological clock actually functions in utero. It is possible that some rhythmic aspect of the birth process itself could start or set the timing of the developing clock. Demonstrating pre-natal function of the biological clock requires a method that can measure an intrinsic, functionally relevant property of the clock itself. A method proven useful for monitoring the oscillatory activity of the SCN in adult rats is 14C-labeled2-deoxyglucose (2DG) autoradiography which provides a biochemical means of visualizing the metabolic (functional) activity of discrete structures in the central nervous system in vivo (Schwartz, 1991). The 2-DG method was used by Reppert and Schwartz (1983) to delineate a circadian rhythm of metabolic activity in the SCN of fetal rats. This study showed that the fetal SCN exhibit a circadian variation of metabolic activity that is “in time” (coordinated) with the rhythm in the dam and with the external lighting cycle (Fig. 2). Remarkably, this fetal rhythm can be detected as early as day 19 of gestation (2 - 3 days before birth) (Reppert and Schwartz, 1984b; all gestational ages have been standardized to day 0 = day of sperm positivity). Recently, a rhythm of 2-DG in the fetal SCN has also been demonstrated in vitro (Shibata and Moore, 1988). This finding suggests that the metabolic activity rhythm in SCN is endogenously generated in the fetus and not passively driven by the mother. Neurogenesis of the rat SCN occurs between

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Fig. 2. Deoxyglucose experiment showing that the fetal SCN manifest a day-night rhythm in metabolic activity. Four pregnant Sprague-Dawley rats were housed with lights on from 0700 to 1900 h until gestational day 19, when the animals were placed in constant darkness in preparation for the deoxyglucose injection (experimental paradigm depicted in upper panel). Two animals were injected i.v. with 2-deo~y[’~C]glucose during the period when the lights would have normally been off (subjective night) on gestational day 20, and the other two were injected during the period when the lights would have normally been on (subjective day) on gestational day 21. At 45 min after injection, the animals were killed and four fetal brains from each pregnant animal were randomly chosen, sectioned and processed for autoradiography. The optical density (OD) of each fetal suprachiasmatic nucleus was measured and the OD of adjacent hypothalamus was used as an internal reference standard for each fetal brain. The data are thus expressed as relative OD (OD of SCN/OD of adjacent hypothalamus). Each vertical bar in the lower panel gives mean relative OD ( & S.E.M.) for the SCN of eight fetal brains. The fetal SCN exhibit a clear daynight rhythm of metabolic activity (P < 0.01); the nuclei are metabolically active during the mother’s subjective day and inactive during the mother’s subjective night. (Modified from Reppert and Schwartz, 1983).

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oscillations in metabolic and electrical activity when the neurons are virtually devoid of neural connections. This early circadian function in the absence of neural connections has led to the notion that individual SCN neurons may function as individual circadian pacemaking units (Moore and Bernstein, 1989). Vasopressin mRNA levels can also be used as an intrinsic marker of the oscillatory activity in the SCN during fetal life (Reppert and Uhl, 1987). Neurons in the dorsomedial SCN contain the neuropeptide arginine vasopressin (Sofroniew and Weindl, 1980; Swaab et al., 1985), and a circadian rhythm of vasopressin levels in cerebrospinal fluid originates in the SCN (Reppert et al., 1987b). Because SCN vasopressin mRNA levels exhibit a day-night variation in adult rats (Uhl and Reppert, 1986), vasopressin mRNA levels were examined throughout early development. A day-night oscillation in vasopressin mRNA levels was found in the fetal SCN beginning on day 21 of gestation, the earliest time that vasopressin mRNA levels were detected in SCN. The rhythm on fetal day 21 was in phase with the circadian time of the dam (Reppert and Uhl, 1987). Mother communicates circadian information to the fetus Role of the maternal biological clock Entrainment of the fetus is due to maternal communication of circadian signal@) to the fetus. Blind pregnant rats were used to show that environmental lighting acts through the maternal circadian system to entrain the rhythm of fetal SCN metabolic activity (Reppert and Schwartz, 1983); the fetal rhythm was synchronous with the circadian time of the blind mothers and not affected directly by ambient lighting. Investigators have used a variety of pre- and post-natal rhythms to confirm the pre-natal coordination of maternal and fetal rhythms in several species (see Davis, 1989; Reppert et al., 1989, for reviews). The involvement of the maternal SCN in the generation of entraining signals for the fetus has

MOTHER OUTPUT SIGNALS

Fig. 3. Conceptual model of maternal-fetal communication of circadian phase. Light-induced neural signals are conveyed to the SCN by her retinohypothalamic pathway (RHP), entraining her circadian rhythms. Maternal output signals then entrain the fetal clock at a time when the innervation of the fetal SCN by the RHP is incomplete.

been demonstrated in rats and hamsters by destroying the maternal SCN early in gestation (Honma et al., 1984a,b; Reppert and Schwartz, 1986b; Davis and Gorski, 1988; Shibata and Moore, 1988; Weaver and Reppert, 1989). These experiments indicate that the entraining signals depend on the integrity of the maternal SCN (Fig. 3). Reppert and Schwartz (1986b) utilized three markers of the developing circadian system in rats to examine the effects of maternal SCN lesions on fetal circadian phase. Maternal SCN lesions on day 7 of gestation disrupted the normal rhythmic population profile of metabolic activity in the fetal SCN (Fig. 4). There was no day-night rhythm of fetal SCN metabolic activity following maternal SCN lesions in the population. Instead, mean values were similar during subjective day (the period of time when the lights would have been on had the animals remained in a light-dark cycle) and subjective night (the period when the lights would have been out), and the mean values were intermediate between the normally high-day and lownight values. Shibata and Moore (1988) have confirmed the disruptive effect of maternal SCN lesions on fetal SCN metabolic activity by monitoring fetal hypothalamic slices in vitro on day 21 of gestation.

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Fig. 4. Upper panel: day-night profiles of relative optical density (OD) of fetal SCN from sham-operated dams (left) and from dams with histologically complete SCN lesions (right). SCN lesions and the sham surgery were performed on gestational day 7. Two days before birth, the dams were transferred from diurnal lighting into constant darkness. Dams were injected with DG during either subjective night on gestational day 20 or subjective day on gestational day 21. For each injection time in the sham-operated group, all fetuses from each of two pregnant rats were studied. For each injection time in the SCN-lesioned group, all fetuses from each of three pregnant rats were studied. Vertical bars: mean k S.E.M.; number of fetuses in parentheses. A significant day-night variation of metabolic activity was present in fetal SCN from sham-operated dams ( P < 0.01) and absent from those of lesioned dams. Lower panel: the relative OD of each fetal SCN examined from mothers with SCN lesions. At each time point, the open circles represent the values of the first litter, closed circles the values from the second litter, and triangles the values of the third litter. Stippled bar of the lighting cycle represents subjective day and solid black bar represents subjective night. (Modified from Reppert and Schwartz, 1986b).

Maternal SCN lesions on gestational day 7 also disrupted the normal rhythmic population profile of pineal NAT activity for 10-day-old pups born to SCN-lesioned dams and reared in constant darkness (Reppert and Schwartz, 1986b). The population profile of these animals did not exhibit a significant daily rhythm, whereas a normal, rhythmic

profile was exhibited by pups born to sham-operated dams and reared by SCN-lesioned dams. Because both the 2-DG and NAT activity rhythms were monitored in a population of animals, their disruption could indicate either loss of rhythmicity for individual animals or desynchronization of the litter, while the rhythms in each animal persisted. The latter interpretation is probably correct, because individual rat pups born to and reared by SCN-lesioned dams and monitored under constant conditions expressed free-running rhythms of drinking behavior after weaning (Reppert and Scnwartz, 1986b). Furthermore, the phases of drinking offsets at weaning within some litters from SCN-lesioned dams were not different from a random distribution over the 24-h period. The phases within other litters from SCN-lesioned dams did show significant within-litter synchrony, but their mean phases were different from each other. In contrast, phases of drinking offset for litters from sham-operated dams were coordinated with each other and with the dam. As in rats, Davis and Gorski (1988) showed in Syrian (golden) hamsters (Mesocricetus auratus) that complete lesions of the maternal SCN on day 7 of gestation disrupt the timing of the developing circadian system. At weaning, the phases of wheelrunning behavior in pups born to and reared by SCN-lesioned dams under constant conditions were scattered throughout the 24-h day. In contrast, the rhythms from pups of sham-operated dams were coordinated with one another and with their dams. Honma and co-workers (1984a,b) provided evidence in rats that maternal-fetal coordination of circadian phase may occur by day 9 of gestation. However, several of the animals used in their studies had incomplete lesions. Furthermore, their conclusion is based on a moderate 4 h phase difference between groups reared post-natally by SCN-intact foster dams; since the corticosterone rhythms used in these studies can clearly be influenced by non-circadian factors, including feeding and stress, these results should be interpreted cautiously. Furthermore, it seems unlikely

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that maternal-fetal communication of circadian phase occurs by day 9 of gestation, as this would require that progenitor cells of the rat SCN can record phase information before their last mitotic division which occurs on gestational days 13 - 16 in rats (Ifft, 1972; Altman and Bayer, 1978). We recently showed that during the post-natal period, rat pups born to SCN-lesioned dams respond appropriately (entrain) to light-dark cycles despite the absence of pre-natal maternal entrainment (Weaver and Reppert, unpublished data). Furthermore, pups from sham-operated and SCNlesioned dams express free-running rhythms with similar cycle lengths (Reppert and Schwartz, 1986b). Thus, normal circadian function (e.g., light-dark entrainment and free-running cycle length) in adulthood does not appear to require pre-natal maternal entrainment.

Nature of the maternal entraining signal@) Because the in utero environment provides a rich source of rhythmic hormones from the mother (e.g., prolactin, corticosterone and melatonin), studies have focused on the possibility that the maternal signal entraining the fetus is a hormone. Melatonin, the principal hormone of the pineal gland, was a prime candidate for the maternal signal communicating phase information to the fetus (Reppert, 1982). There is a robust rhythm of melatonin in the maternal circulation, and because melatonin can cross the placenta, a melatonin rhythm in the dam is reflected in the fetal circulation (Klein, 1972; Reppert et al., 1979; Yellon and Longo, 1987, 1988; Zemdegs et al., 1988; McMillen and Nowak, 1989). However, maternal pinealectomy (which eliminates measurable levels of melatonin; Lewy et al., 1980) does not abolish maternal coordination of fetal circadian phase in rats (Reppert and Schwartz, 1986a), hamsters (F.C. Davis, personal communication), or mice (Weaver and Reppert, unpublished results). Furthermore, removal of the maternal adrenals, thyroid-parathyroids, pituitary or ovaries (in separate experiments) does not abolish the clear day-night rhythm of metabolic ac-

tivity in the fetal rat SCN when performed on or before day 7 of gestation (the time when maternal SCN lesions are disruptive) (Reppert and Schwartz, 1986a). The maternal eyes, a potential source of both neural and endocrine signals, are also not necessary for the communication, since dams enucleated on day 2 of gestation synchronize the circadian clocks of their fetuses (Reppert and Schwartz, 1983). Another approach to determine which aspect of maternal rhythmicity entrains the fetus is to artificially restore rhythmicity in an SCN-lesioned dam (who has no endogenous rhythmicity). Normally, there is a circadian rhythm in food consumption; this rhythm is disrupted by SCN lesions (Nagai et al., 1978). Restricted access to food in SCN-lesioned rats artificially produces a rhythm in food consumption and can also induce rhythms in locomotor activity and temperature (Kreiger et al., 1977; Stephan 1981; Weaver and Reppert, 1989). Using a food access restriction paradigm (food cue) in SCN-lesioned pregnant rats, we showed that the rhythmic ingestion of food can entrain fetuses of SCN-lesioned dams (Weaver and Reppert, 1989). For this study, dams received SCN lesions on day 7 of gestation, and food access was restricted to 4 h/day from day 8 to day 19 of gestation; the time of food access in these animals was opposite the time when it would normally occur in unoperated animals with food ad libitum. In a control group, SCN-lesioned dams were not foodrestricted, but at the beginning and end of the 4-h period of food restriction the food bins were jostled as they were for the food-restricted animals when food was added and removed. Pups were born and reared in constant darkness. The phases of drinking offsets at weaning within each of the litters from SCN-lesioned dams given food cue showed significant within-litter synchrony (Fig. 5 , middle panel). Furthermore, the average phases for the litters were similar and in each case significantly correlated with the phase predicted, assuming the food cue actually entrained the fetuses. In contrast, the phases of drinking offset at weaning for two of the six jostle-control litters

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were not different from a random distribution (Fig. 5, upper panel). The other four litters did show significant within-litter synchrony, but their phases were different from each other and not correlated with the average phase of the food-restricted group. A final experiment controlled for any potential post-natal influence on pup circadian phase induced by the pre-natal food cue. Pups born to food-restricted, SCN-lesioned dams were reared by SCNlesioned dams that had not been food-restricted during pregnancy; these pups exhibited drinking phases similar to pups born to and reared by food-restricted dams (Fig. 5, lower panel). These results and the results of others (Honma and Honma, 1987) indicate that rhythmic food ingestion during pregnancy can entrain developing circadian phase. Rhythmic food ingestion clearly would cause rhythmic fluctuations in nutrient levels in the blood, and this could in turn induce physiological responses to the presence of nutrients. In SCNlesioned dams, food restriction may cause generation of a nutrient-related signal which entrains the fetuses in a manner parallel to that which occurs in intact dams. Alternatively, restricted feeding of SCN-lesioned dams can lead to the generation of other rhythms (e.g., in temperature or activity) which may be involved in setting the phase of the fetus. Because the fetus is exposed to a multitude of maternal rhythms (behavioral rhythms, as well as hormonal), it is also possible that multiple maternal rhythms act in concert to entrain the fetal biological clock (Fig. 3). Thus, eliminating any one of these rhythms would not be sufficient to disrupt maternal entrainment. This redundancy may explain some recent, seemingly contradictory data. As mentioned above, the maternal pineal gland is not necessary for maternal entrainment of the

fetus. However, Davis and Mannion (1988) have convincingly shown that timed injections of pharmacologic doses of melatonin into SCN-lesioned Syrian hamsters restore fetal synchrony. This finding supports the redundancy hypothesis: while melatonin is not necessary for synchronization, it is one of several circadian signals capable of synchronizing the fetal biological clock. Another line of evidence implicating melatonin as an important mediator of time-of-day information from mother to fetus is our recent discovery of putative melatonin receptors in the fetal SCN of several rodent species (Fig. 6; Weaver et al., 1989). Thus, an anatomic substrate exists for a direct action of maternal melatonin on the fetal biological clock. During the post-natal period, the maternal circadian system of altricial rodents continues to coordinate the timing of the developing circadian system (see Takahashi et al., 1989, for review). It appears that maternal influences during the postnatal period serve to maintain or reinforce the coordination of phase that has been established during the pre-natal period. In rats, the post-natal maternal influence persists until the pup develops the potential for direct light-dark entrainment through its own eyes (via the retinohypothalamic tract) at the end of the first week of life (Stanfield and Cowan, 1976; Terubayashi et al., 1985; Duncan et al., 1986). Potential functions of an entrained fetal clock

The existence of pre-natal communication of circadian phase in mammals suggests that this phenomenon is of adaptive value. Maternal entrainment would presumably coordinate the pups to the dam and to the environment, so that when physiological

Fig. 5 . Phases of drinking offset at weaning for pups of SCN-lesioned dams given food cue (middle panel) or jostle control (upper panel) during pregnancy, or given food cue during pregnancy and fostered on the day of birth to dams not previously cued (lower panel). For each litter, the phases of drinking offset for individual pups are depicted (small circles or triangles) around a large circle that represents 24 h on post-natal day 21. The population profile of pup phases for each group is presented in the right-hand column. For each litter or group that was significantlysynchronized ( P < 0.05 by Rayleigh test), themean phase of drinking offset is depicted by the direction of the arrow; arrow length is arbitrary. (Modified from Weaver and Reppert, 1989).

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Fig. 6 . Localization of specific 1251-labeledmelatonin binding to the SCN of fetal rodents by in vitro autoradiography. Specific '"Imelatonin binding was apparent in the autoradiograph as dark densities over the SCN (left-hand column). The section used to generate each autoradiograph was stained to show the location of SCN (right-hand column). GD, Gestational age; - 4, 4 days before birth. (From Weaver and Reppert, unpublished results.)

and behavioral rhythmicity later develop, the rhythms are expressed in proper relationship to one another and to the 24-h day. This allows the young animal to more easily assume its temporal niche. If the pups of altricial rodents were not coordinated to the environment by the mother, they might exhibit inappropriate timing of emergence from the burrow or disadvantageous behavioral patterns when they emerged (Pratt and Goldman, 1986). Furthermore, if each pup were to develop its own rhythmicity, there would be a period of disorganization of the litter that could be detrimental to some or all members of the litter. For example, coordination of a dam's willingness to nurse and

of all pups in the litter to suckle would clearly be of benefit. Circadian disorganization at the level of the individual or litter could thus threaten survival of individual pups (Hudson and Distel, 1989). Another possible function of the developing circadian clock and its entrainment during fetal life is in the initiation of parturition. This potential role of the entrained fetal SCN may be widely applicable (including primates), because the time of day of birth is gated to the daily light-dark cycle by a circadian mechanism in a number of species (Kaiser and Halberg, 1962; Rossendale and Short, 1967; Jolly, 1972; Lincoln and Porter, 1976). Recent experiments in rats show that the maternal SCN are necessary for the normal circadian gating

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of birth and are consistent with a role for the fetal brain in this timing phenomenon (Reppert et al., 1987a). Development of a biological clock in humans Numerous physiological parameters in the human fetus, including heart rate, respiratory movements and circulating prolactin levels, have been shown to exhibit daily rhythmicity (see Honnebier et al., 1989, for review). It is generally believed that these pre-natal rhythms are passively driven by maternal rhythms rather than by a functional circadian system in the fetus. This belief comes from the general observation that circadian rhythms in these functions are not manifested during the immediate post-natal period, but develop gradually over several weeks to months (see Reppert and Rivkees, 1989, for review). However, the human fetus may indeed be capa-

ble of expressing endogenous circadian rhythmicity. DG studies in squirrel monkeys show that an endogenous pacemaker in the primate SCN is functioning during fetal development and suggest that the timing of this pacemaker is coordinated to ambient lighting conditions (Fig. 7; Reppert and Schwartz, 1984a). It is important to add that the SCN are apparent as discrete nuclei in the human fetus by the 18th week of gestation and already have melatonin receptors at that time (Fig. 8; Reppert et al., 1988). Thus, a functioning circadian clock may be oscillating in the SCN of the human fetus, coordinated to prevailing environmental conditions by the maternal melatonin rhythm. Since the sleep-wake cycle is the rhythm that we are most conscious of in ourselves and in our children, especially in newborns, it is not surprising that this cycle has been the most extensively studied post-natal parameter of human circadian development. Kleitman and Engelman (1953) were

Fig. 7. Autoradiograph of coronal brain sections from mothers (upper row) and fetuses (lower row) following deoxyglucose injection into pregnant squirrel monkeys during subjective day (left-hand column) and subjective night (right-hand column). The metabolically active SCN appear as a pair of small ovoid densities in the mother and fetus during the subjective day (arrows), while the nuclei are no longer visible during the night. (Modified from Reppert and Schwartz, 1984a).

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Fig. 8. Localization of specific 'L'I-labeled melatonin binding to the SCN of a human fetus (18 weeks gestation) by in vitro autoradiography. The section used to generate the autoradiograph was stained to show the location of the SCN (arrows on photomicrograph, upper panel). Specific '251-melatonin binding was apparent in the autoradiograph as dark densities over the SCN (lower panel). Magnification, x 5.5. (Modified from Reppert et al., 1988).

the first to systematically study the development of the 24-h sleep-wake cycle in a large group of individual infants. They used direct observation and automatic recordings from a specially constructed crib to continuously monitor the development of the 24-h sleep-wake pattern in 19 infants. When the data were expressed for the entire group, they found a gradual development of a daily sleep-wake profile that had already made itself apparent at 3 weeks of life (Fig. 9A).

One of the infant records was strikingly different from the records of the other infants (Fig. 9B). The parents of this child were described as sufficiently indulgent to allow the infant to set its own sleep-wake and feeding schedule; in essence this constituted a self-selecting, free-running environment. What emerged in this infant over the first 16 weeks of life was a free-running sleep-wake cycle with a cycle length of about 25 h, the same period that is expressed by the vast majority of adults under free-running conditions. Between the 18th and 21st weeks the infant's rhythm appeared to have entrained to the 24-h period, although there was some phase adjustment in the rhythm through the 24th week. Why the rhythm eventually became entrained to the 24-h period is not certain, but it is possible that light-dark entrainment was occurring by this time (see below). The importance of this infant's record is that it reveals the endogenous nature of the 24-h sleep-wake cycle during early development, and thus suggests that a circadian pacemaker is functioning at an early age. It is possible that, as in rodents, the developing circadian system of humans uses the maternal circadian system to coordinate its timing and that maternal-infant interactions are important for the expression of overt rhythmicity. That this might be the case is supported by the studies of Sander and co-workers (Sander and Julia, 1966; Sander et al., 1970, 1972) who have shown the importance of mother-infant interactions in the temporal development of the human sleep-wake cycle. Their studies suggest that mother-infant interactions augment the development of normal daily rhythmicity of the sleep-wake cycle. In contrast, a nursery environment with fragmented maternal care and discordance between infant activity and caretaking interventions impedes the development of a daily sleep-wake cycle. In addition to maternal interactions, the other major exogenous factor that might influence endogenous rhythmicity in the infant is the daily light-dark cycle. The only data bearing on when the human circadian system is first responsive to alterations in light and darkness are those of Mar-

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Klein, D.C., Reppert, S.M. and Moore, R.Y. (Eds.) (1991) Suprachiasmatic Nucleus: the Mind's Clock, Oxford University Press, New York. Kleitman, N. and Engelman, T.G. (1953) Sleep characteristics in infants. J. Appl. Physiol., 6: 269 - 282. Kreiger, D.T., Hause, L. and Krey, L.C. (1977) Suprachiasmatic nuclear lesions do not abolish food-shifted circadian adrenal and temperature rhythmicity. Science, 197: 398 - 399. Lenn, N.J., Beebe, B. and Moore, R.Y. (1977) Post-natal development of the suprachiasmatic nucleus of the rat. Cell Tissue R e x , 178: 463-475. Lewy, A.J., Tetsuo, M., Markey, S.P., Goodwin, F.K. and Kopin, I.J. (1980) Pinealectomy abolishes plasma melatonin in the rat. J. Clin. Endocrinol. Metab., 50: 204-207. Lincoln, D.W. and Porter, D.G. (1976) Timing of the photoperiod and the hour of birth in rats. Nature, 260: 780 - 78 I . Mann, N.P., Haddow, R., Stokes, L., Goodley, S . and Rutter, N. (1986) Effect of night and day on preterm infants in a newborn nursery: randomized trial. Br. Med. J . , 275: 1265 - 1267. Martin du Pan, R. (1974) Some clinical applications of our knowledge of the evolution of the circadian rhythm in infants. In: L.E. Scheving, F. Halberg and J.E. Pauly (Eds.), Chronobiology, Georg Thieme, Stuttgart, pp. 342 - 347. McMillen, I.C. and Nowak, R. (1989) Maternal pinealectomy abolishes the diurnal rhythm in plasma melatonin concentrations in the fetal sheep and pregnant ewe during late gestation. J. Endocrinol., 120: 459- 464. Moore, R.Y. and Bernstein, M.E. (1989) Synaptogenesis in the rat suprachiasmatic nucleus demonstrated by electron microscopy and synapsin I immunoreactivity. J. Neurosci., 9: 2151 -2162. Moore, R.Y., Shibata, S. and Bernstein, M.E. (1989) Developmental anatomy of the circadian system. In: S.M. Reppert (Ed.), Development of Circadian Rhythmicity and Photoperiodism in Mammals, Perinatology Press, Ithaca, NY, pp. 1-24. Nagai, K., Nishino, T., Nakagawa, S., Nakamura, S. and Fukuda, Y. (1978) Effects of bilateral lesions of the suprachiasmatic nuclei on the circadian rhythm of food intake. Brain Res., 142: 384 - 389. Pratt, B.L. and Goldman, B.D. (1986) Maternal influence on activity rhythms and reproductive development in Djungarian hamster pups. Biol. Reprod., 34: 655-663. Reppert, S.M. (1982) Maternal melatonin: a source of melatonin for the immature mammal. In: D.C. Klein (Ed.), Me/atonin Rhythm Generating System, Karger, Basel, pp. 182- 191. Reppert, S.M. (1987) Circadian rhythms: basic aspects and pediatric implications. In: D.M. Styne (Ed.), Current Concepts in Pediatric Endocrinology, Elsevier, Amsterdam, pp. 91 - 125. Reppert, S.M. and Rivkees, S.A. (1989) Development of

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human circadian rhythms: implications for health and disease. In: S.M. Reppert (Ed.), Development of Circadian Rhythmicity and Photoperiodkm in Mammals, Perinatology Press, Ithaca, NY, pp. 245 - 259. Reppert, S.M. and Schwartz, W.J. (1983) Maternal coordination of the fetal biological clock in utero. Science, 220: 969 - 971. Reppert, S.M. and Schwartz, W.J. (1984a) Functional activity of the suprachiasmatic nuclei in the fetal primate. Neurosci. Lett., 46: 145 - 149. Reppert, S.M. and Schwartz, W.J. (1984b) The suprachiasmatic nuclei of the fetal rat: characterization of a functional circadian clock using ‘‘C-labeled deoxyglucose. J. Neurosci., 4: 1677- 1682. Reppert, S.M. and Schwartz, W.J. (1986a) Maternal endocrine extirpations do not abolish maternal coordination of the fetal circadian clock. Endocrinology, 119: 1763 - 1767. Reppert, S.M. and Schwartz, W.J. (1986b) The maternal suprachiasmatic nuclei are necessary for maternal coordination of the developing circadian system. J. Neurosci., 6: 2724 - 2729. Reppert, S.M. and Uhl, G.R. (1987) Vasopressin messenger ribonucleic acid in the supraoptic and suprachiasmatic nuclei: appearance and circadian regulation during development. Endocrinology, 102: 2483 - 2487. Reppert, S.M., Chez, R.A., Anderson, A. and Klein, D.C. (1979) Maternal-fetal transfer of melatonin in a non-human primate. Pediatr. Res., 13: 788 - 791. Reppert, S.M., Henshaw, D., Schwartz, W.J. and Weaver, D.R. (1987a) The circadian-gated timing of birth in rats: disruption by maternal SCN lesions or by removal of the fetal brain. Brain Rex, 403: 398 - 402. Reppert, S.M., Schwartz, W.J. and Uhl, G.R. (1987b) Arginine vasopressin: a novel peptide rhythm in cerebrospinal fluid. Trends Neurosci., 10: 76 - 80. Reppert, S.M., Weaver, D.R., Rivkees, S.A. and Stopa, E.G. (1988) Putative melatonin receptors in a human biological clock. Science, 242: 78- 81. Reppert, S.M., Weaver, D.R. and Rivkees, S.A. (1989) Prenatal function and entrainment of a circadian clock. In: S.M. Reppert (Ed.), Development of Circadian Rhythmicity and Photoperiodism in Mammals, Perinatology Press, Ithaca, NY, pp. 25-44. Rossendale, P.D. and Short, R.V. (1967) The time of foaling of thoroughbred mares. J. Reprod. Fertil., 13: 341 - 343. Sander, L.W. and Julia, H.L. (1966) Continuous interactional monitoring in the neonate. Psychosom. Med., 28: 822 - 835. Sander, L.W., Stechler, G . , Burns, P. and Julia, H. (1970) Early mother-infant interaction and 24-hour patterns of activity and sleep. 1. Am. Acad. Chifd Psychiatry, 9: 103 - 123. Sander, L.W., Julia, H.L., Stechler, G. and Burns, P. (1972) Continuous 24-hour interactional monitoring in infants reared in two caretaking environments. Psychosom. Med., 34: 270 - 282.

Schwartz, W.J. (1991) SCN metabolic activity in vivo. In: D.C. Klein, R.Y. Moore and S.M. Reppert (Eds.), Suprachiasmatic Nucleus: the Mind’s Clock, Oxford University Press, New York, pp. 144- 156. Shibata, S. and Moore, R.Y. (1987) Development of neuronal activity in the rat suprachiasmatic nucleus. Dev. Brain Res., 34: 311-315. Shibata, S. and Moore, R.Y. (1988) Development of a fetal circadian rhythm after disruption of the maternal circadian system. Dev. Brain Rex, 41: 313-317. Sofroniew, M.V. and Weindl, A. (1980) Identification of parvocellular vasopressin and neurophysin neurons in the suprachiasmatic nucleus of a variety of mammals including primates. J. Comp. Neurol., 193: 659 - 675. Stanfield, B. and Cowan, W.M. (1976) Evidence for a change in the retinohypothalamic projection in the rat following early removal of one eye. Brain Res., 104: 129- 133. Stephan, F.K. (1981) Limits of entrainment of periodic feeding in rats with suprachiasmatic lesions. J. Comp. Physiol., 143: 401 -410. Swaab, D.F., Fliers, E. and Partiman, T.S. (1985) The suprachiasmatic nucleus of the human brain in relation to sex, age and dementia. Brain Res., 342: 37 - 44. Takahashi, K., Ohi, K., Shimoda, K., Yamada, N. and Hayashi, S. (1989) Post-natal maternal entrainment of circadian rhythms. In: S.M. Reppert (Ed.), Development of Circadian Rhythmicity and Photoperiodism in Mammals, Perinatology Press, Ithaca, NY, pp. 67 - 82. Terubayashi, H., Fujisawa, H., Itoi, M. and Ibata, Y. (1985) HRP-Histochemical detection of retinal projections to the hypothalamus in neonatal rats. Acta Histochem. Cytochem., 18: 433-438. Uhl, G.R. and Reppert, S.M. (1986) Suprachiasmatic nucleus vasopressin messenger RNA: circadian variation in normal and Brattleboro rats. Science, 232: 390 - 393. Weaver, D.R. and Reppert, S.M. (1989) Periodic feeding of SCN-lesioned pregnant rats entrains the fetal biological clock. Dev. Brain Res., 46: 291 - 296. Weaver, D.R., Namboodiri, M.A.A. and Reppert, S.M. (1989) Iodinated melatonin mimics melatonin action and reveals discrete binding sites in fetal brain. FEBS Lett., 228: 123 - 127. Yellon, S.M. and Longo, L.D. (1987) Melatonin rhythms in fetal and maternal circulation during pregnancy in sheep. A m . J. Physiol., 252: E799- E802. Yellon, S.M. and Longo, L.D. (1988) Effect of maternal pinealectomy and reverse photoperiod on the circadian melatonin rhythm in the sheep and fetus during the last trimester of pregnancy. Biol. Reprod., 39: 1093 - 1099. Zemdegs, I.Z., McMillen, I.C., Walker, D.W., Thorburn, G.D. and Nowak, R. (1988) Diurnal rhythms in plasma melatonin concentrations in the fetal sheep and pregnant ewe during late gestation. Endocrinology, 123: 284 - 289.

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Discussion D.F. Swaab: (1) Was the pars tuberalis also stained for melatonin receptors in the human fetus? Non-specific hot spots below the optic chiasm could be discerned. (2) How did you confirm in the human fetus that the Nisslstained structure which is indeed the region of the SCN but probably much larger than the SCN itself and stains for melatonin receptors is indeed the SCN? Did you stain the SCN immunocytochemically? We have found consistent discrepancies between the dense Nissl-stained areas and the SCN identified by anti-vasopressin in the human fetus (Swaab et al., 1990). S.M. Reppert: (1) No specific ‘251-melatoninbinding was found in the fetal pars tuberalis. There were areas of increased radioactivity below the optic chiasm that represented non-specific binding of the iodinated melatonin (see Fig. 8, this chapter), i.e., the radioactivity was not displaced by a large excess of unlabeled melatonin. (2) The SCN was easily discernable by Nissl staining in human fetuses at 18 - 20 weeks of gestation. M.A. Corner: In rats not exposed to a circadian rhythm during fetal life (because of SCN lesion of the pregnant dam), is the periodicity of their free-running rhythm later in life any different from that in “normal” rats? S.M. Reppert: Pups born to SCN-lesioned dams (and thus not exposed to circadian rhythms pre-natally) express normal freerunning periods and entrain appropriately to the light-dark cycle (Weaver and Reppert, unpublished data). D.R. Repaske: (1) If the human fetus has a biological clock entrained to the mother’s circadian cycle, why do we not see evidence of biological rhythms in the first weeks of life? (2)What then is the purpose of the pre-natal biological rhythm in humans? S.M. Reppert: (1) It appears that the biological clock is oscillating and entrained to the mother’s circadian cycle before most of the output pathways from the SCN to other structures necessary for overt rhythm expression have matured. It is also possible that there are circadian rhythms manifested by the fetus and newborn human that we have not yet been able to detect. (2) An entrainable biological clock during fetal life “prepares”

the fetus for life outside the womb and would help maximize the infant’s responsiveness to circadian changes in the outside world. We have also speculated that the developing biological clock and its entrainment during fetal life may be involved in the initiation of parturition (Reppert et al., 1987). M. Mirmiran: (1) Since you were able to entrain the fetal biological clock by scheduled maternal feeding in an otherwise SCN-lesioned mother, it seems that in the neurotransmitter/hormonal communication between the mother and the fetus, direct neuronal output of maternal SCN does not play a crucial role. What do you think of this proposition? (2) You have shown (as well as Dr. Moore in his lecture) that the fetal SCN both in Nissl staining and melatonin receptor binding autoradiography is more visible than in the adult human hypothalamus. What is the significance of these observations as far as the “endogenous” generation of fetal circadian rhythm is concerned? S.M. Reppert: (1) Our finding that scheduled maternal feeding can entrain the fetal biological clock in SCN-lesioned mothers suggests that rhythmic food consumption, which is normally under the control of the SCN in intact mothers, can entrain the fetus. Thus a neural/humoral output from the maternal SCN would be required in intact mothers to generate the food intake rhythm that is detected by the fetus. (2) The level of melatonin receptors seems to be higher in the fetuses of rodents and humans when compared to receptor levels in adults. This increased level of receptor expression may correlate functionally with the importance of melatonin as a fetal entraining agent.

References Reppert, S.M., Henshaw, D., Schwartz, W.J. and Weaver, D.R. (1987) The circadian-gated timing of birth in rats: disruption by maternal SCN lesions or by removal of the fetal brain. Brain Res., 403: 398 - 402. Swaab, D.F., Hofman, M.A. and Honnebier, H.B.O.M. (1990) Development of vasopressin neurons in the human suprachiasmatic nucleus in relation to birth. Dev. Brain Res., 52: 289 - 293.

D.F. Swaab, M.A. Hofman, M. Mirmiran, R. Ravid and F.W. van Leeuwen (Eds.) Progress in Brain Research, Vol. 93 0 1992 Elsevier Science Publishers B.V. All rights reserved.

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CHAPTER 10

The human hypothalamus: comparative morphometry and photoperiodic influences Michel A. Hofrnan and Dick F. Swaab Netherlands Institute f o r Brain Research, 1105 A Z Amsterdam, The Netherlands

Introduction The concept of the hypothalamus as a distinct neurological entity concerned with a variety of regulatory processes dates back to the end of the 19th century (for reviews, see Morgane and Panksepp, 1979). In 1893, on the basis of embryological work, the Swiss anatomist Wilhelm His (18631934) proposed a subdivision of the brain as a framework for an anatomical nomenclature of the central nervous system (His, 1893). The point of departure was the five brain-vesicles model described by Von Baer in 1828. His subdivided the second of these vesicles, the Zwischenhirn, or diencephalon, into three regions: epithalamus, thalamus and hypothalamus, which were arranged as longitudinal zones in superposition to one another. In 1895 this nomenclature was accepted by the Anatomische Gesellschaft and was incorporated in the Baseler Nomina Anatomica (His, 1895). Comparative anatomical investigations of this part of the brain in subsequent years, indeed, revealed a general plan of organization among vertebrates (Ariens-Kappers et al., 1936; Le Gros Clark, 1938; see also Simmons, 1988). Despite this basic pattern, which consists of a number of invariants and unchanging spatial relationships between the constituent parts, the morphology of the mammalian diencephalon shows variations in relative position, form and dimensions of nuclei from species to species. Grunthal (1950), when studying the com-

parative anatomy of the hypothalamus in mammals, even declared: “Kein Hypothalamus sieht wie die ander aus”. From what is known about hypothalamic functioning, this brain area may even be expected to show a somewhat varying configuration between individuals of the same species, as a result of external factors. These transformations of the original “Bauplan”, however, only concern the relative change in the geometry of the regional and nuclear subdivisions, while the fundamental topographical pattern of the hypothalamus is preserved (Keyser, 1979). Although the complex cellular arrangement and multitude of afferent and efferent projections have made analysis of hypothalamic organization difficult, the mammalian hypothalamus is generally subdivided into three regions in the anteroposterior direction: (1) chiasmatic (preoptic) region; (2) tuberal region; and (3) mammillary region (see Brockhaus, 1942; Simmons, 1988; Saper, 1990). Each of these regions, in turn, contains groups of nerve cells, many of which are nothing more than diffuse and ill-defined condensations of cells, while others are more circumscribed and consist of cells of rather characteristic types. There is no doubt, however, that within regions of the hypothalamus, describable areas and nuclei with distinctive structural and cytoarchitectonic characteristics exist as distinct entities (see Braak and Braak, this volume). The cytoarchitectural plan of the human hypoT thalamus has been quite well mapped out by now,

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although the exact terminology has often been controversial (Le Gros Clark, 1938; Simmons, 1988; Saper, 1990). One major reason for the differences in the nomenclature is that the cell groups in the human hypothalamus are rarely as well differentiated as in most other mammalian species. In recent years, however, more detailed studies of cellular arrangement employing immunohistochemical methods have outlined various cell groups with considerably more precision (Braak and Braak, 1987; Saper, 1990). This technique has also allowed the exploration of chemically defined neuronal systems in the human brain (Swaab et al., 1985, Mai et al., 1991; Braak and Braak, this volume). It is now possible to draw comparisons with other species that can shed light on the functional organization of the human hypothalamus. Before 1900 there were only vague intimations of the function of the brain surrounding the third ventricle and these were based primarily on various pathological and assorted clinical observations (for a review, see Morgane, 1979). Since then a large body of experimental evidence has been derived implicating that this region of the brain contains the control systems which are critically involved in many homeostatic and rheostatic (Mrosovsky, 1990) regulatory processes. Among these are electrolyte balance, food ingestion and energy metabolism, thermoregulation, reproduction, immune response, and various emotional-affective states in addition to vigilance mechanisms. It is clear that the hypothalamus not only regulates the homeostasis within the “milieu interieur” of the organism, but is also involved in modulating relationships between the individual and its external physical, psychological and social environment. Experimental studies, largely in rodents and primates, have made it clear that the hypothalamus performs these faculties by means of its control of three main types of output systems: endocrine, autonomic and behavioral. The executive authority relating to all these processes has repeatedly been centered in the hypothalamus - thus creating a kind of “subcortical phrenology”. Many of the more behaviorally oriented studies of the past 40 years, in particular,

have placed “the hypothalamus as reigning supreme unto itself and interpreted it as some sort of sovereign center of centers” (Morgane, 1979, p. 7). Most of these studies have, accordingly, failed to place the hypothalamus in its proper relation to the remainder of the nervous system and to develop broader integrative conceptions of hypothalamic organization and function. In this context it is important to point out that the hypothalamus is only one of a series of functional levels in the brain and that, whereas it influences more caudal levels of somatic and autonomic function, the hypothalamus itself is likewise under a direct influence from rostra1 levels of the nervous system, most notably the cerebral cortex (see Morgane, 1979; Palkovits and Zaborsky, 1979). Size and scaling of the hypothalamus As Le Gros Clark (1938) noted more than half a century ago, the human hypothalamus accounts for only 4 cm3, or 0.3% of the normal adult brain volume. It should be kept in mind, however, that the human hypothalamus is still twice the size of a rat brain. A really minuscule hypothalamus is found in the pygmy shrew, Sorex minutus, an insectivore which weighs only 5 g when fully grown, with a brain size of 0.10 cm3. This creature, which is active day or night and at all seasons, has a hypothalamus which measures not more than 0.003 cm3. Yet, despite its modest dimensions, this region contains the integrative systems critical for the animal’s basic life support. In insectivores the volume of the hypothalamus relative to brain volume varies between 2% and 3%, whereas in prosimians the ratio is somewhat lower and accounts for about 1.5%. In anthropoid primates, such as monkeys, chimpanzees and human beings, the ratio is generally a fraction of 1%. A graphic representation of the volume of the hypothalamus as a function of brain volume in insectivores and primates (Fig. 1) reveals the reason for the specific differences between these species. It appears that the volume of the hypothalamus in insectivores increases almost as the first power

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Brain volume ( cm3 ) Fig. 1. Volume of the hypothalamus as a function of brain volume in insectivores and primates. Logarithmic scale. The curve represents the second-degree polynomial equation describing the relationship between the two variables for insectivores and primates together (for details see text). The allometric equations for each individual (sub)order are given by: Znsectivoru: log y = 1.41 + 0.96 log x (r = 0.997; P < 0.001); Prosimii: log y = 1.23 + 0.89 logx (r = 0.990; P < 0.001);Anthropoidea: logy = 1.38 + 0.71 log x (r = 0.992; P < 0.001). Note that the slopes of the standard major axes in primates are different from isometry (geometric similarity).

where y is volume of the hypothalamus (mm3) and x is brain volume (cm3) (R2 = 0.993; n = 34 species). Allometric analysis of the volume of the hypothalamus as a function of body size, on the other hand, shows that the hypothalamus scales in a similar way for insectivores and primates, irrespective of size or evolutionary history (Fig. 2). So, here, the distinction between the orders is not a scaling disparity, as with brain size, but rather reflects a difference between regression constants, as a result of which the hypothalamus in anthropoid primates is larger than in insectivores of comparable body size. This means that if a shrew, such as the Sorex minutus, were scaled up as the 0.6 power to the size of man it would have a hypothalamus of only about 880 mm3. The actual volume of the human hypothalamus, however, is 3600 mm3 (Stephan et al., 1981), which is four times larger than would be predicted from the equation for insectivores. Taken together, these findings indicate that the hypothalamus in mammals, like the cerebral cortex (Hofman, 1985, 1988), is highly correlated with brain size, irrespective of the ecological strategy or evolutionary history of the species considered. 1o5

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prosimians and anthropoid primates the proportion of the hypothalamus relative to brain size decreases in species with larger brains. Consequently, most primates have a relatively small hypothalamus as compared with less encephalized mammals, such as insectivores and rodents. Although not enough data are available at present to consider the allometric relationship between the volume of the hypothalamus and brain volume in all major mammalian taxa, the available data suggest that the general relationship between both structures can be described by a second-degree polynomial equation. For insectivores and primates the relationship is expressed by: logy

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136

Moreover, the human hypothalamus has just the size we may expect of such a large-brained mammal, but it is considerably larger than would be predicted from its body size. A completely different picture emerges from a comparative study of the pineal gland, or epiphysis, a hormone-producing organ located in the midline roof of the third ventricle (McKinley and Oldfield, 1990), which receives photic information from the eye via cell groups in the preoptic region of the hypothalamus (see next section). Allometric analysis of this part of the brain indicates that in primates the pineal volume is basically a linear function of brain volume, but that in insectivores the pineal increases disproportionally with brain size (Fig. 3). In other words, the volumetric relationship between the pineal and the brain in mammals, in contrast to the hypothalamus, depends on the taxonomic group studied. The present analysis further 1o3

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reveals that the relative pineal volume of nocturnal species does not deviate from that of diurnal species. From the allometric analysis of the pineal-body size relationship (Fig. 4) it is clear that the pineal scales in a similar way in insectivores and primates and that the scaling factors in both cases do not significantly deviate from unity. In other words, the epiphysis in insectivores and primates, and presumably in many other mammalian orders as well, is a linear function of body weight. For insectivores and primates the general relationship is given by:

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Brain volume ( cm3 ) Fig. 3. Volume of the pineal gland as a function of brain volume in insectivores and primates. Logarithmic scde. Insectivora: log y = -0.695 + 1.51 log x ( r = 0.993; P < 0.001); and primatexlogy = -1.12 + l.O6logx(r = 0.967;P < 0.001). No significant distinction was found between prosimians and anthropoid primates. Notice the large pineal volume of Tarsius (T) relative to its brain size and the relatively small size of the pineal gland in Gorilla (G). The slope of the standard major axis in insectivores is significantly different from isometry (P < 0.01).

where y is volume of the epiphysis (mm3) and x is body weight (8) (R2 = 0.939; n = 32 species). As the epiphysis in mammals is an endocrine gland, it is logical to consider its interspecific growth being bound to the volume of circulating blood and thus to body weight (see also Legait et al., 1976). The finding that total blood volume is directly proportional to body mass (Prothero, 1980), together with the comparative investigations presented in this

137

essay, support the idea that, across species, the size and synthetic activity of the mammalian pineal varies directly with the animal’s size. However, variations in the morphology and functional activity of certain brain regions, such as the pineal and suprachiasmatic nucleus, may also occur among individuals belonging to the same species, under the influence of environmental stimuli, such as the natural light-dark cycle and seasonal variations in photoperiod. The preoptic region of the hypothalamus Sexually dimorphic nucleus In mammals the preoptic region of the hypothalamus is thought to be implicated in the neural control of endocrine functions (Kelley and Pfaff, 1978). Particularly the medial part of the preoptic area, a region of the hypothalamus which is bordered rostrally by the lamina terminalis and caudally by the posterior edge of theoptic chiasm, is criticallyinvolved in the regulation of sexually differentiated functions under the influence of gonadal steroids (see Anderson et al., 1986; Watson et al., 1986). Recent studies have attempted to identify sex differences in the structure of the preoptic-anterior region that may underlie these functional differences (see De Vries et al., 1984). In 1978, Gorski and his co-workers identified and described an intensely staining cell group within the preoptic area of the rat, the size of which showed a markedly sexual dimorphism (Gorski et al., 1978). This sexually dimorphic nucleus of the preoptic area (SDN-POA) is still the most conspicuous morphological sex difference in the mammalian brain. In mature rats, in which the SDN-POA is 3 - 8 times larger in males than in females, this difference has been shown to be independent of the steroidal environment. Instead, the SDN-POA seems to be profoundly influenced by androgens circulating perinatally (Jacobson et al., 1980; Bloch and Gorski, 1988). Although the SDN-POA has been studied most extensively in the rat (see also Anderson et al., 1985; Robinson et al., 1986), similar sexual differences have been documented in homologous structures in guinea

pigs (Hines et al., 1985; Byne and Bleier, 1987), ferrets (Tobet et al., 1986), gerbils (Commins and Yahr, 1984) and in man (Swaab and Fliers, 1985; Hofman and Swaab, 1989). The human SDN-POA, which corresponds to the intermediate nucleus as described by Braak and Braak (1987), can already be distinguished in the fetal brain around mid-pregnancy. In a developmental study we found that it is only after the age of about 4 years that the human SDN-POA differentiates according to sex, due to a dramatic cell loss in females (Swaab and Hofman, 1988). As a result, the size, shape and cellular morphology of the SDNPOA in adulthood exhibit a striking sexual dimorphism, as well as a sex-dependent pattern of aging (Swaab and Fliers, 1985; Hofman and Swaab, 1989; Swaab et al., this volume). Whereas numerous studies have implicated the medial preoptic area in the mediation and regulation of masculine sexual behavior and reproductive functions (Dohler et al., 1986; Byne and Bleier, 1987) it is not possible at this time to specify the precise role of the SDN-POA within this area. Considering its marked sexual dimorphism and its androgenic sensitivity during development, the SDNPOA might be part of the neural circuitry underlying masculine reproductive processes and scentmarking (Byne and Bleier, 1987; Turkenburg et al., 1988; De Jonge et al., 1990; Yahr and Finn, 1990). Suprachiasmatic nucleus In addition to its involvement in reproductive functions the preoptic region of the mammalian hypothalamus is also considered to be implicated in the temporal organization of biological rhythms (Rusak and Zucker, 1979; Moore-Ede et al., 1982; Meijer and Rietveld, 1989). The suprachiasmatic nucleus (SCN), a collection of parvocellular neurons, located in the basal part of the hypothalamus, just above the optic chiasm, is thought to be the principal component of this central clock mechanism. This bilateral cell group, first described by the Hungarian anatomist Lenhossek in 1887, is less impressive in humans than in many other mammalian species (Hofman et al., 1988) and may be dif-

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Fig. 5 . Vasopressin-stained frontal sections (6pm) of the human hypothalamus. A. The SCN is situated just dorsal to the optic chiasm. The principal mass of VP-immunoreactive cells lies in the dorsal crescent of the SCN, from which strands of loosely packed cells descend on all sides, surrounding a central unlabeled area. Scale bar represents 0.2 mm. B. Higher magnification of the same section. Scale bar is 0.1 mm. OC, Optic chiasm; SCN, suprachiasmatic nucleus; 111, third ventricle.

ficult to recognize in Nissl-stained material (Saper, 1990). The homology of this cell group with the suprachiasmatic nucleus in rodents and non-human primates has only recently been partly established by application of immunohistochemical methods (Lydic et al., 1980; Stopa et al., 1984; Swaab et al., 1985; see also Fig. 5 ) . Morphometric studies have shown that there is no significant sexual dimorphism in the volume and cellular morphometry of the human SCN, with the exception of a difference in shape (Swaab et al., 1985; Hofman et al., 1988). The human SCN has been found to be elongated in females and more spherical in males. Such a sexual dimorphism in shape could conceivably have functional consequences by virtue of differences in contacts between the suprachiasmatic nucleus and structures in its vicinity. Other indications for the assumed func-

tional sex differences of the SCN come from studies in the rat, where it has been found that the volume of the SCN in males is larger than that in females (Gorski et al., 1978; Robinson et al., 1986). It is plausible, therefore, to assume that the SCN of male rats contains more nerve cells than the female SCN, which is relevant in view of the hypothetical relationship between cell number and the pacemaker properties of the SCN (Van den Pol and Powley, 1979; Pickard and Turek, 1985). On the other hand, the reduced size of the neuronal nucleoli in the SCN of male rats (Guldner, 1983) may point to a lower metabolic activity of male SCN neurons (Zambrano and De Robertis, 1968). As these properties may have a compensatory effect, the overall functional capacity of the SCN in male rats does not necessarily have to be different from that of female rats. Whether there is a differential sex effect on the

139

functional activity of the human SCN is not known but seems not very likely in view of the similarities in the SCN cellular morphology of men and women. On the other hand, the human SCN, being only 3.7 times larger than the SCN of the rat, is reduced in size relative to other hypothalamic nuclei, such as the paraventricular nucleus and supraoptic nucleus (Hofman et al., 1988, 1990). Whether this relative reduction in size and neuronal content of the human SCN is a consequence of a diminished significance of the SCN as the central biological clock in humans, in favor of other neuronal oscillators, or is merely an allometric scaling phenomenon, is not known. If, however, the disproportional reduction of the human SCN is due to allometry, it would mean that a relatively small population of neurons in this nucleus is already sufficient to generate and coordinate a wide spectrum of physiological and behavioral rhythms in mammals, irrespective of the size of the organism or its ecological strategy.

and Zucker, 1979; Follett and Follett, 1981; Gwinner, 1986). In recent years it has become clear that in mammals the pineal gland and the suprachiasmatic nucleus are essential for the regulation of these photoperiodic responses (see, e.g., Goldman and Darrow, 1983; Mess and Ruzsas, 1986; Cassone, 1990; Meijer, 1990). Interruption of the retino-hypothalamic pathway, or changes in environmental lighting conditions were found to influence the rhythm of the suprachiasmatic pacemaker, and consequently the synthesizing activity of the pineal gland. In mammals, the circadian synthesis and secretion of the hormone melatonin exhibited by the pineal gland is generated by oscillators in the SCN and is communicated to the pineal via a complex multisynaptic pathway (Moore, 1983; Tamarkin et al., 1985; Binkley, 1988; Krause and Dubocovich, 1990; see also Fig. 6). Disruption of any portion of this pathway from the SCN to the pineal gland abolishes melatonin rhythmicity. These findings strongly suggest that the endocrine

The suprachiasmatic nucleus as neuronal clock The mammalian SCN is generally considered to be the major component of a biological clock, which generates and coordinates a wide spectrum of biochemical, physiological, endocrine and behavioral circadian rhythms (for reviews, see Rusak and Zucker, 1979; Turek, 1985; Meijer and Rietveld, 1989). The SCN receives photic information by a direct neural pathway from the retina, which exists in all mammals studied so far, including man (Sadun et al., 1984). This retinohypothalamic tract appears to be both necessary and sufficient for synchronization of the period and phase of circadian rhythms to the environmental light-dark cycle. Furthermore, indirect retinal projections have been found to enter the SCN via the ventral lateral geniculate nucleus and raphe nuclei (Moore et al., 1978; Harrington et al., 1987; Mikkelsen, 1990). Consistent with its role in the temporal organization of circadian processes, investigations in rodents and non-human primates suggest that the SCN is also involved in the seasonal control of reproductive and metabolic phenomena (for reviews, see Rusak

Fig. 6. Diagram of the human brain (mid-sagittal section) showing the neural pathways (dashed line) by which photoperiodic information reaches the pineal. Abbreviations: SCN, suprachiasmatic nucleus; PVN, paraventricular nucleus; SCG, superior cervical ganglion.

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activity of the mammalian pineal is under neural control, and receives a major input from the SCN. This means that in addition to its role as a circadian pacemaker, the SCN may also be involved in the seasonal timing of a number of physiological and behavioral processes by regulation of the photoperiod-dependent changes in melatonin secretion (Arendt and Broadway, 1987; Cassone, 1990). Seasonal variations in the human SCN Since the environmental light-dark cycle is the main "Zeitgeber" for circadian and seasonal rhythms in most species, including man, photic information could have substantial effects, not only on the neural activity of the suprachiasmatic nucleus but also on its underlying structure. Recently, we conducted a study to determine whether variations in light intensity, diurnal as well as seasonal, affect the morphology of the SCN in human beings (Hofman and Swaab, 1992). A marked seasonal variation was observed in the volume, total cell number and number of vasopressin cells of the human SCN (Table I). The volume of the SCN was, on average, twice as large in the autumn as in the summer and contained almost twice as many cells. Similar seasonal differences were found for the number of vasopressin-contain-

ing neurons. In general, the human SCN is smaller in the summer than in any other season. In contrast, no such seasonal variations could be detected in cell density or in mean cell-nuclear diameter. The sexually dimorphic nucleus of the preoptic area (SDN-POA), located in the immediate vicinity of the SCN, did not show any significant periodic changes over the year in either volume or cell number. Differentiation of the data by gender did not affect the outcomes: no significant seasonal variations in the SDN-POA could be detected for either sex (Fig. 7). In contrast with the annual cycle of the human SCN, no significant diurnal variations in either volume or cell number were observed. The SDNPOA, like the SCN, did not show diurnal variations, irrespective of whether sexes were pooled or analyzed separately. Moreover, no diurnal changes could be detected in cell density or in mean cell-nuclear diameter in either the SCN or the SDN-POA, except for the mean diameter of cell nuclei in the SDN-POA of male subjects, which was significantly larger during the daytime (06.00 - 18.00 h) than during the nighttime (18.00-06.00 h), suggesting a diurnal variation in cell activity in this hypothalamic nucleus in males under the influence of light. Taken together, these results indicate that the morphology of the human SCN is significantly affected by

TABLE I Seasonal variations in the volume, total cell number and vasopressin cell number of the human suprachiasmatic nucleus Season

Winter Spring Summer Autumn

No. of subjects

Period

306"-35" midpoint: 21 December 36" - 125' midpoint: 23 March 126" -215" midpoint: 22 June 216" - 305" midpoint: 21 September

' Values are given as mean

t_

S.E.M.

Suprachiasmatic nucleus' Volume (mm3)

Total cell number ( x I d )

Vasopressin cell number ( x lo3)

17

0.240 k 0.025

48.67 k 3.90

7.00 f 0.75

9

0.278 f 0.031

46.30 f 4.05

7.41 f 0.89

11

0.174 f 0.019

30.28 f 4.19

4.45 f 0.70

11

0.344 f 0.050

58.35 k 5.82

9.95 k 1.38

141

SCN

0

SDN-POA

MALES

40

-8 -

FEMALES

t

-80

80

80

40

40

o

0

-40

-40

t

L

al

n

s

--

a,

-

Winter

Spring

Summer

Autumn

-80

‘

Winter

Spring

Summer

Autumn

Fig. 7. Seasonal variations in the volume and total cell number of the suprachiasmatic nucleus (SCN) and sexually dimorphic nucleus of the preoptic area (SDN-POA) in human subjects, differentiated by gender. Subjects were grouped into four annual periods of equal length based on the time of death (see Table I). Data are expressed as deviations from the annual mean. In general, the human SCN shows a marked seasonai variation with low values in the summer and higher values in other seasons. The observed seasona1fluctuations in the morphology of the SDN-POA are less prominent, and statistically not significant ( P > 0.6).

seasonal variations in photoperiod, but not by the daily light-dark cycle. Although human beings are not considered to be particularly photoperiodic, many of the annual rhythms of human biology are under environmental control, and seem to be driven by an endogenous clock synchronized with the seasons. Several investigations indicate that the SCN and its vasopressin transmitter system are part of the neural substrate that mediates these circannual rhythms (for reviews, see Follett and Follett, 1981; Mess and Rdzsas, 1986; Hermes, 1991). The striking seasonal variation in the total volume and vasopressin cell number of the human SCN is in accordance with this view. The morphology of other parts of the brain that are thought to be critical for the generation of annual cycles, such as the pineal gland, have also been found to undergo seasonal variations. Investigations in rodents showed that both the pineal vo-

lume and nuclear size of pinealocytes are considerably larger in winter than during the summer period, while the number of “synaptic” ribbons in the pineal, associated with the cyclic biosynthesis of melatonin, have also been found to undergo seasonal variations (Peschke et al., 1989; McNulty et al., 1990). Whether there are cyclic variations in the morphology of the human pineal has not been investigated so far. Since the SCN innervation of the paraventricular nucleus (PVN) of the hypothalamus represents the primary, if not the sole, pathway by which photoperiodic information is relayed to the pineal (Pickard and Turek, 1985; Tamarkin et al., 1985; Smale et al., 1989) the neural connections between the SCN and the PVN seem critical for the generation of seasonal cycles. Therefore, we looked for seasonal changes in the structure of the PVN as we observed in the SCN. A preliminary study conduct-

142

0 SCN 0 PVN

I

9

t

-40

-''

SON

1

I

Winter

Spring

Summer

Autumn

Fig. 8. Seasonal variations in the volume of three vasopressincontaining cell groups in the human hypothalamus: SCN, suprachiasmatic nucleus; PVN, paraventricular nucleus; SON, supraoptic nucleus. Significant changes were observed in the volume of the SCN ( - 33%, P < 0.01) during the summer, and in the volume of the PVN ( + 41070, P < 0.02) during the spring. For further details see legend to Fig. 7 .

ed on the human PVN in relation to photoperiod indeed showed a notable seasonal variation in the total volume of the PVN (Fig. 8). The human PVN, however, seems to reach its peak during the spring, whereas the SCN has its maximum size in early autumn. Several brain regions, on the other hand, which are not known to be involved in the temporal organization of biological processes, such as the supraoptic nucleus, do not show significant annual oscillations (Fig. 8). This means that not every cell group in the human hypothalamus is subject to seasonal changes, but probably only those regions which are directly involved in the transmission of photoperiodic information.

form of the SCN annual cycle shows two maxima with a time interval of about 6 months: one peak, with a low amplitude, close to the spring equinox, and a second, more prominent peak, near the vernal equinox, moments of the year when the length of daylight is equal to the length of darkness. Furthermore, the annual minimum was found to coincide with the summer solstice; i.e., the longest photoperiod, whereas a second minimum was observed near the winter solstice. These findings indicate that the bimodal SCN curve reaches its positive amplitude at the same moment when the photoperiod undergoes its largest rate of change (Fig. 10). Sho tening days in autumn, as well as lengthening days early spring, seem to induce morphological changes in the human SCN. These findings indicate that photoperiod may be considered as a potential environmental factor controlling the size of the SCN, and that photoperiodic change rather than day length itself is the essential parameter governing this process. If photoperiod is the effective seasonal Zeitgeber in man, as it is in many vertebrate species (see, e.g., Gwinner, 1986), existence of human beings near the equator, where major seasonal variations in day length do not occur, may, therefore, lead to a desynchronization of the underlying

R

SUPRACHlASMATlC NUCLEUS

Photoperiodic influences on the SCN

U

U ,

At higher latitudes, as in the Netherlands, where changes in day length are pronounced, light-dark information is the main Zeitgeber for the endogenous annual timing system. To investigate the assumed phase relationship between the annual variations in the SCN, the dynamic changes in photoperiod variations in SCN cell number over the year have been correlated with the natural photoperiodic conditions at 52" Northern latitude (Fig. 9). The wave

MAR

JUN

SEP

Time of

DEC

MAR

1

JUN

SEP

DE(

year (months)

Fig. 9. Annual variations in total cell number of the human suprachiasmatic nucleus (SCN) in relation to the photoperiodic cycle of the temperate zone (52" N). In these double-plotted histograms the SCN data are grouped into 1.5 month periods and expressed as deviations from the annual mean, whereas the photoperiodic deviations from the annual mean are given as a cosine curve. Notice the bimodal wave form of the SCN curve with one maximum close to the spring equinox (March) and the other near the vernal equinox (September - October).

143 SUPRACHIASMATIC NUCLEUS

I # # ,, 1 1 1 1 1 1 1 1 , 1 1 , , 1 , 1 1 1 , 1 MAR

JUN

SEP

DEC

MAR

JUN

SEP

OEC

Time of year (months)

Fig. 10. Schematic representation of the latitude-specific influence of photoperiod on the cell number of the human suprachiasmatic nucleus (SCN). The best correlation (Spearman’s e = 0.732, P < 0.05) between the two variables was achieved by correlating the deviations of the total number from the annual mean (upper curve) with the first derivative of the photoperiodic cycle (lower curve). Note that large cell numbers in the SCN coincide with the moment that the photoperiod undergoes its largest rate of change and vice versa.

periodic processes and, consequently, to a subduction of the amplitudes of the SCN cycle. The complexity of the seasonal variations in the SCN, however, suggests that, in addition to the environmental lighting conditions, other non-photic signals are involved in generating this phenomenon. There is now substantial evidence that the SCN and pineal gland are involved in a neuroendocrine “feedback loop’’ (for reviews, see Mess and RUzsAs, 1986; Rosenwasser and Adler, 1986; Cassone, 1990). In mammals, pineal synthesis and secretion of melatonin and possibly other hormonal products follow a diurnal and seasonal rhythm that is imposed on the pineal by the SCN via a hypothalamo-pineal pathway (Fig. 6). Melatonin synthesis shows maximum values during the subjective night in both diurnal and nocturnal species (Klein, 1979; Skene et al., 1990) and the pattern is further influenced by photoperiod (Glass, 1984; VanEEek and Illnerova, 1985; Underwood and Goldman, 1987). On long photoperiods, as in summer, the period of high melatonin synthesis is short, while on short periods, as in winter, it is prolonged. Conversely, the SCN may be an important central target mediating the

physiological effects of circulating melatonin (VanEEek et al., 1987; Reppert et al., 1988). Gonadal hormones probably also mediate the period, phase and coherence of activity rhythms which occur in association with natural reproductive cycles including estrous cycles and photoperiodic responses (Morin and Cummings, 1981; Gwinner, 1986; Rosenwasser and Adler, 1986). Steroid effects on circadian rhythms have been observed in both birds and mammals, which has led several authors to suggest that gonadal secretions may play a role in oscillator coupling in the vertebrate timekeeping system (Rosenwasser and Adler, 1986). Normally, the timing of gonadal steroid hormone secretion is controlled by pituitary gonadotrophins, which are themselves strongly influenced by the SCN (Samson and McCann, 1979; Kawakami et al., 1980). Thus a neuroendocrine feedback loop involving SCN-pituitary-gonadal interactions is a distinct possibility. This hypothesis is further supported by observations that gonadal hormones can influence SCN electrophysiological and neurochemical processes (Kow and Pfaff, 1984; Miller et al., 1984). Taken together, these findings support the hypothesis that the suprachiasmatic nucleus, pineal gland and gonads are critically involved in the generation and control of various circadian and circannual rhythms in mammals. Finally, the present findings indicate that photoperiod is a putative environmental factor controlling the size and functional activity of the human SCN. Therefore, disturbances of the annual cycle of vasopressin synthesis in the SCN may have profound effects on the seasonal timing of a variety of physiological and behavioral processes. It might explain, for example, why light therapy of patients with seasonal affective disorders, a syndrome characterized by regularly recurring depressions in autumdwinter, shows such a spectacular remission of symptoms after a few days of treatment with bright light (Lewy et al., 1982; Arendt and Broadway, 1987; Rosenthal et al., 1988). Although melatonin has been invoked as the critical biochemical mediator of the light-dependent effects in such mood disturbances it does not seem to be implicated

144

in this process (Wehr et al., 1986). In view of the assumed seasonal variations in vasopressin synthesis in the human SCN it would be worthwhile to investigate whether the anti-depressant effect of phototherapy is due to an activation of the vasopressin system - or any of the other immunocytochemically defined neuronal clusters - of this hypothalamic nucleus. Summary and conclusions The concept of the hypothalamus as a distinct neurological entity concerned with a variety of regulatory processes dates back to the end of the 19th century. Before 1900 there were only vague intimations of the function of the brain surrounding the third ventricle and these were based primarily on various pathological and assorted clinical observations. Since then a large body of evidence has been derived implicating that the hypothalamus contains the control systems which are critically involved in many physiological, endocrine and behavioral processes. Among these are feeding and drinking, reproduction and the regulation of the sleep-wake cycle and temperature. Although the human hypothalamus accounts for only 4 cm3, or 0.3% of the adult brain volume, it contains the integrative systems critical for all these processes. A comparative morphometric analysis of the hypothalamus among mammals revealed that the volume of this part of the brain is highly correlated with brain size, irrespective of the ecological strategy or evolutionary history of the species considered. It appears that the human hypothalamus has just the size we may expect of such a largebrained mammal, but it is considerably larger than would be predicted from its body size. In mammals the preoptic region of the hypothalamus is implicated in the neural control of endocrine functions and in the temporal organization of a wide spectrum of biological rhythms. In recent years, the pivotal role of two hypothalamic cell groups have been considered in this context: the sexually dimorphic nucleus (SDN-POA) as part of the neural circuitry underlying masculine sexual behavior and

reproductive functions and the suprachiasmatic nucleus (SCN) as the principal component of the central clock mechanism. Consistent with its role in the temporal organization of circadian processes, investigations in rodents and non-human primates suggest that the SCN is also involved in the seasonal control of reproductive and metabolic phenomena. Since the environmental light-dark cycle is the main Zeitgeber for circadian and seasonal rhythms in most species, including man, photic information could have substantial effects, not only on the neural activity of the biological clock, but also on its underlying structure. Our observations on the human SCN in relation to photoperiod indeed revealed a marked seasonal variation in the morphology of the human SCN. The volume of the SCN was, on average, twice as large in the autumn as in the summer and contained more than twice as many vasopressin immunoreactive neurons. In general the human SCN is smaller in the summer than in any other season. In contrast, no such seasonal variations could be detected in cell density or in mean cellnuclear diameter. The SDN-POA did not show any significant periodic changes over the year in either volume or cell number, indicating the specificity of the SCN oscillations. In contrast with the annual cycle of the SCN, no significant diurnal variations were observed in any of the morphological parameters considered in either the SCN or SDN-POA. Taken together, these results indicate that the human SCN, in contrast with the SDN-POA, is significantly affected by seasonal variations in photoperiod, whereas the daily light-dark cycle per se does not seem to induce comparable modulations in either nucleus. The complexity of the annual SCN cycle, however, suggests that, in addition to the environmental lighting conditions, other non-photic signals (e.g., gonadal hormones) are involved in generating this phenomenon. Acknowledgements The authors are indebted to Mr. B. Fisser for his technical assistance, Mr. H. Stoffels for drawing the

145

figures and Ms. W. Verweij for her secretarial help. Brain material was obtained from the Netherlands Brain Bank (coordinator Dr. R. Ravid).

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Discussion R.Y. Moore: First, how do you determine total cell number in the suprachiasmatic nucleus? Second, how do you account for seasonal changes in total cell number as it seems unlikely that new neurons are being formed? Could this be a function of the season of death rather than a seasonal rhythm? M.A. Hofman: In our study the SCN was defined according to immunocytochemical criteria by using vasopressin as a marker. The number of vasopressin neurons was first counted in this material. Subsequently, the total number of cells (i.e., neurons and glial cells) in this subdivision of the SCN was estimated by counting the profile density per unit area in thionin-stained material (Swaab et al., 1985). I agree that the more than two-fold difference in the number of vasopressin-immunoreactive neurons of the human SCN between late spring and early autumn raises a number of questions about the underlying mechanism. It could be that changes in photoperiod influence the rate of vasopressin synthesis of the SCN and that groups of peptidergic neurons are activated during particular periods of the year, depending on day length, light intensity or other photic parameters. Another, more speculative, explanation for the seasonal variations in SCN morphology is that the volumetric changes reflect the periodic production and/or incorporation of new neurons. Further experimental studies should be performed in non-human mammals to examine the exact nature of the seasonally dependent neural plasticity of the biological clock. H.B.M. Uylings: You showed a clear seasonal variation in the number of visibly stained vasopressin (VP) neurons in the SCN: but, in addition to the comments of Dr. Moore concerning the volume of the SCN and its total cell number, do you agree that other criteria than VP, for example, neurotensin, may be crucial for the determination of the volume of the SCN. M.A. Hofman: Since the cytoarchitectural boundaries of the SCN are difficult to delineate in conventionally stained sections it is indeed important that, in addition to vasopressin, other neuropeptides, such as neurotensin or vasoactive intestinal polypeptide, should be used as neuronal markers. It will provide more insight in the dimensions of the human SCN. H.P.H. Kremer: (1) A similar phenomenon of seasonal variations is described for the projection neurons of the song control system of oscine songbirds. (2) Could you comment upon the exact way of counting the neurons? M.A. Hofman: It is correct that in some avian brains, especially in the high vocal center (HVC) of the neostriatum in canaries and zebrafinches, new neurons continue to be added in adult life (Nottebohm et al., 1986;Alvarez-Buylla et al., 1990). In adult male canaries, for example, new HVC neurons are born in the month of September, and survive for at least 8 months (Kim et al., 1991).The longevity of the HVC neurons suggests that these cells remain part of the vocal control circuit long enough to participate in the yearly renewal of the song repertoire. Whether

such an oscillating pattern of neurogenesis occurs in the mammalian SCN remains to be seen. As to your other question, the number of vasopressin neurons in the SCN of each subject was estimated by counting the number of nuclear profiles per unit area followed by a discrete unfolding procedure and a correction for section thickness (Swaab et al., 1985). J.K. Mai: The glial cells within the human SCN have contact with the organum vasculosum of the lamina terminalis, the ependymal lining, pial surface and blood vessels. It could therefore well be that glial cells of the SCN change their volume (e.g., during different seasons) thereby ensuing technical problems in distinguishing neurons and glial cells. My question is: taken into account this possible effect how did soma size and cross-sectional areas of both cell populations overlap? M.A. Hofman: Differentiation between neurons and glial cells has not been made in the morphometric analysis, except for the group of vasopressin-expressing neurons. Your suggestion, though, that changes in the soma size of glial cells might explain the seasonal variations in the volume of the human SCN seems not very likely, since volume changes in glial cells due to osmotic, potassium, or transmitter induced gradients (Walz, 1989)are far too small to account for the 150% difference in SCN volume between May - June and October - November. Moreover, periodic oscillations in the volume of glial cells would affect the numerical cell density of the SCN over the year, which has not been found (Hofman and Swaab, 1992). W.A. Scherbaum: Do you know if there exist osmoreceptor in. puts to the SCN, and if there exist projections to the PVN: e.g. to vasopressin cells or the CRH cells which might explain pe. riodicity in drinking behavior and control rhythmicity? M.A. Hofman: No osmoreceptor inputs to the SCN have beer described, at least not to my knowledge. R. Ravid: Can you dissociate the effect of light from the effeci of temperature on variations in the SCN? M.A. Hofman: It is not easy to differentiate between the specific influences of these environmental factors upon the morphologj of the human hypothalamic clock, but when the subjects werc grouped into four annual periods based on a thermic division 01 theyear instead of on a photoperiodic division, no significant an nual fluctuations in the volume or cell number of the SCN werc observed. These findings suggest that the human SCN is highl! affected by seasonal variations in photoperiod, but not b! changes in temperature.

References Alvarez-Buylla, A., Kim, J.R. and Nottebohm, F. (1990)Birtf of projection neurons in adult avian brain may be related t( perceptual or motor learning. Science, 249: 1444- 1446. Hofman, M.A., and Swaab, D.F. (1992)Annualvariationsin thi morphology of the human suprachiasmatic nucleus. (Submit ted.) Kirn, J.R., Alvarez-Buylla, A. and Nottebohm, F. (1991)Pro

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ductionand survival of projection neurons ina forebrain vocal center of adult male canaries. J. Neurosci., 11: 1756- 1762. Nottebohm, F., Nottebohm, M.E. and Crane, L. (1986) Developmental and seasonal changes in canary song and their relation to changes in the anatomy of song-control nuclei. Behuv. Neurol. Biol., 46: 445 - 47 1.

Swaab, D.F., Fliers, E. and Partiman, T. (1985) The suprachiasmatic nucleus of the human brain in relation to sex, age and dementia. Bruin Res., 342: 37 - 44. Walz, W. (1989) Role of glial cells in the regulation of the brain ion microenvironment. Prog. Neurobiol., 33: 309 - 333.

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CHAPTER 11

Circadian rhythms and the suprachiasmatic nucleus in perinatal development, aging and Alzheimer’s disease M. Mirmiran, D.F. Swaab, J.H. Kok’, M.A. Hofman, W. Witting and W.A. Van Goo12 Netherlands Institute for Brain Research, 1105 A 2 Amsterdam, The Netherlands; and Departments of I Neonatology and Neurology, Academic Medical Center, University of Amsterdam, Amsterdam, The Netherlands

’

Introduction

Many physiological phenomena, such as rest-activity, sleep-wakefulness, body temperature, plasma levels of different hormones and activity of different neurotransmitters in the brain, exhibit an endogenous “circadian rhythmicity”, i.e., they have a free-running period of about 24 h (Pittendrigh, 1974; Aschoff, 1981; Moore-Ede et al., 1982). Different biological rhythms have a strict phase relation to the environmental (e.g., light/dark) cycles and they are temporally interrelated. These phase relations and internal synchronization are aspects of the circadian rhythm-generating system which appear to be important for the optimal functioning of the organism (Van Goo1 and Mirmiran, 1986). Although different areas of the brain and/or body may be able to generate circadian rhythms, a “bidogical clock” in the anterior hypothalamus, i.e., the suprachiasmatic nucleus (SCN), harmonizes these rhythms to induce a single circadian oscillation in mammals (Rusak and Zucker, 1979; Moore, 1983; Turek, 1985; Rosenwasser and Adler, 1986; Meijer andRietveld, 1989; Rusak, 1989; Ralphet al., 1990; Moore, this volume). Light conveyed to the SCN via the retinohypothalamic pathways is the main factor in the daily entrainment of the endogenous activity of the biological clock to 24-h time cues.

Several studies made an important contribution to explainipg the role of the human hypothalamus in the generation of circadian rhythms. First of all, in addition to the conventional staining methods, the use of immunocytochemical and autoradiographic staining techniques enabled the visualization of the human SCN (Dierickx and Vandersande, 1977; Lydicet al., 1980; Sadunetal., 1984; Stopaet al., 1984; Swaab et al., 1985; Reppert et al., 1988; Moore, 1989; Friedmanetal., 1991; Maiet al., 1991; seealso Moore, this volume); secondly, recently two cases were reported which suggested that, also in the human, a lesion in the suprachiasmatic region of the anterior hypothalamus indeed results in disturbed circadian rhythms (Schwartz et al., 1986; Cohen and Albers, 1991). Moreover, contrary to the conventional belief that the human circadian rhythm system is irresponsive to light, Czeisler et al. (1989, 1990) convincingly showed in a series of experiments that in humans, as in animals, light can reset the endogenous circadian oscillator, which is most probably the SCN of the human hypothalamus (see also Wever, 1989). In the present review we describe human circadian rhythms during early development, in aging and’in Alzheimer’s disease (AD) with particular attention to its relation with the changes that occur in the SCN.

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Circadian rhythms in early human development Earlier studies in which the circadian rhythms of newborn infants were investigated did not give any indication of rhythmicity of rest-activity or sleepwake until at 3 months of age (Kleitman, 1963; Parmalee et al., 1964; Hellbrugge, 1974; Mills, 1975; Davis, 1981; Minors and Waterhouse, 1981; Coons and Guilleminault, 1982; Navelet et al., 1982; Alley and Rogers, 1986). However, these studies were hampered by the fact that: (1) the short-term (i,e., 24 h maximally) recordings were carried out in a highly masked irregular environment; and (2) the data from different individuals were pooled and cross-sectionally studied for age effects. It is of course clear that if any endogenous rhythmicity was present in each individual infant, this would not have been notified in such data analysis. Interestingly, in two cases in which the environmental masking effects were minimized and the infants were fed on demand and recorded longitudinally it became apparent that these infants showed an endogenous free-running rhythm of the sleep-wake cycle until around 15 - 16 weeks of age when the rhythm was entrained to the light-dark cycle (Kleitman and Engelman, 1953; Paupousek and Papousek, 1984; Reppert, this volume). These results indicate perinatal emergence of endogenous circadian rhythms most probably induced in the hypothalamus, which only develops its entrainment to the environmental light-dark cycle post-natally. However, recent studies have indicated a much earlier emergence of diurnal rhythms, i.e. in “newborn” infants, than was originally assumed (Spangler, 1991). Many overt rhythms that are driven by the biological clock in the hypothalamus in adulthood have also been shown to be present during human prenatal life. Rest-activity, breathing movements, heart rate and urine production show a circadian rhythm in the fetus (Patrick et al., 1982;Visser et al., 1982; De Vries et al., 1987; Honnebier et al., 1989; Mirmiranet al., 1989;Rabinowitzet al., 1989;Tuffnell et al., 1990). Circadian rhythms of fetal heart rate variability could be recorded as early as at 22

weeks of gestation (De Vries et al., 1987). To establish the time of circadian rhythm emergence it is important to know whether the fetal rhythms recorded during gestation simply reflect the influence of a maternal circadian system or whether the fetus independently generates a certain amount of circadian rhythmicity by virtue of its own endogenous biological clock activity (Mirmiran et al., 1990). It is for obvious reasons impossible to record fetal circadian rhythms in the absence of maternal influences. We have therefore taken advantage of the unique opportunitiy to explore the possibility of the existence of circadian rhythms in very early human development by recording them in pre-term infants under fairly constant environmental conditions, i.e., in a nursery unit. The rectal or skin QUT 29 WEEKS CONCEPTIONAL 200

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temperature, heart rate and rest-activity cycles were recorded in a group of low-risk pre-term infants (29 - 35 weeks of conceptional age). Throughout the recordings the lights were continuously on, the feeding was done intragastrically every 2 h and the incubator temperature was constant. Under these conditions circadian rhythms (with a periodicity ranging between 24 and 27 h) were found in the body temperature and heart rate of about 50% of the infants (Mirmiran et al., 1990; Mirmiran and Kok, 1991; Fig. 1). For comparison, the circadian rhythm of body temperature of another pre-term infant with a conceptional age of 33 weeks and that of a womam in her 33rd week of pregnancy are shown in Fig. 2. These findings are the first evidence in support of the existence of an endogenous circadian rhythm (possibly generated by the SCN in the fetal hypothalamus) in early human development. However, in contrast to adult rhythms, these early emerging circadian rhythms are more variable and they are not synchronized with the time of day (see also Fig.

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2). FLrthermore, whether the individual differences among pre-term infants (see also Abe and Fukui, 1979) with respect to the presence or absence of circadian rhythms are related to differences in the stage of development of the biological clock in the hypothalamus or pre-natal maternal entrainment requires further study. As far as the entrainment of circadian rhythms to the environmental light-dark cycle is concerned, a recent study has shown that an approximately 4week exposure of infants to the cyclic home environment is, sufficient for the entrainment to develop (McMillen et al., 1991). Pre-term infants as young as 34 weeks of age responded to the cyclic environment and developed fully entrained circadian rhythms of sleep and wakefulness as early as at 45 weeks of conceptional age. The emergence of entrainment in human infants requires that the afferent and efferent neural pathways to and from the hypothalamic circadian pacemaker are functional. The study of McMillen et al. (1991) suggests that

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Fig. 2. Circadian rhythm of body temperature of a woman in the 33rd week of pregnancy (top) and that of a pre-term infant with a conceptional age of 33 weeks. Chi-square periodogram analysis showed a significant circadian rhythm with a maximum period length of 24 h in both subjects. However, note that despite the significant periodicity the rhythm is more variable from day to day in the pre-term baby than in the mother. The latter could partly be the result of constantly being in the incubator under continuous illumination in the neonatal intensive care unit.

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these components of the circadian rhythm generating system are present as early as 35 weeks after conception and that the rate-limiting factor in the emergence of an entrained biological rhythm after this age is the length of exposure to cyclic zeitgebers. McMillen et al. also reported one term infant that never developed an entrained sleep-wake cycle throughout the study. This infant was the only one that was fed at night in full light. Sander et al. (1972) reported that the ability of the infant to differentiate between day and night with regard to the sleep-wake rhythm was already evident by the 4th day of life, if it received individual care from birth onwards. However, such recognition of day and night was not present in infants having multiple care givers. Factors such as the intensity of light (which is higher in the neonatal intensive care than at home both day and night), presence or absence of a light-dark zeitgeber, single vs. multiple care givers and feeding on demand vs. continuous feeding (or at fixed intervals of 2 h) are among those that might influence the developing biological clock of the human infant hypothalamus as early as by 30 - 35 weeks of conceptional age. The early appearance of a functional biological clock in human infants is for instance of particular importance for pre-term infants that are kept in the neonatal intensive care units for several weeks. Early exposure to a cyclical light-dark environment would result in earlier synchronization of the infant’s behavioral and hormonal rhythms with the external environment and subsequently an improvement of their development. Two recent intervention studies in pre-term infants were in accordance with this proposition (Mann et al., 1986; Fajardo, 1990).

metabolic changes are present in the fetal SCN region of the rat and squirrel monkey (Reppert and Schwartz, 1984a,b; Reppert, this volume). It is difficult to convincingly visualize the human SCN by means of conventional histological staining techniques (cf., Swaab et al., 1990). However, immunocytochemical staining of the SCN with antibodies against arginine-vasopressin (AVP) seems to be a good marker of this nucleus in the human brain, enabling morphological and morphometric investigations of the human SCN (Dierickx and Vandersande, 1977; Stopa et al., 1984; Swaab et al., 1985; Hofman et al., 1988; Mai et al., 1991). Positive immunocytochemical staining for AVP of parvocellular neurons and the lack of staining for oxytocin in the suprachiasmatic region of the anterior hypothalamus near the third ventricle differentiate the human SCN from the neurons of, e.g., the supraoptic and paraventricular region that stain for both AVP and oxytocin. In 11 fetal brains (27 - 42 weeks of conceptional age) positive staining for AVP was only found from 31 weeks onwards. Fig. 3 summarizes the development of the human SCN. Although both the number of AVP cells and the total cell number at term (i.e., between 38-42 weeks; n = 7) were respectively 13% and 21% of that found in adulthood, it is interesting to note that there was overlap between the

Human SCN changes during early development The observation that, although generally speaking human newborns lack circadian rhythms, some rhythms might be present in pre-term infants under well-controlled environmental conditions, makes the question when exactly the human SCN becomes functional a particularly interesting one. This point was reinforced by the observation that diurnal

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Fig. 3. Development of vasopressin (VP) cell number in the human suprachiasmatic nucleus (SCN) of the hypothalamus. Log-log scale. The period at term (38 - 42 weeks of gestation) is indicsted by the vertical bar. (From Swaab et al., 1990, with perm,ssio.i .)

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fetal and adult AVP cell numbers (Fig. 3). Other evidence in favor of hypothalamic control of circadian rhythms during early human development comes from a study of Reppert et al. (1988). These investigators showed that using specific melatonin receptor ligand enables the visualization of the fetal human SCN region as early as at the gestational age of 7 months. Animal studies have shown that SCN neurons seem to be able to express circadian rhythmicity well before this nucleus is capable of coordinating overt rhythms; in an altricial mammal such as the rat the rhythm of SCN 2-deoxyglucose uptake appears on embryonic day 19 (Reppert and Schwartz, 1983), a prominent dayhight rhythm of AVP mRNA is evident in the SCN on day 21 of gestation (Reppert and Uhl, 1987) and the spontaneous firing rate of SCN neurons shows a diurnal rhythm, at least on the last day of gestation, i.e., day 22 (Shibata and Moore, 1987). Using 2-deoxyglucose uptake as a measure of the level of neuronal activity within the SCN, Reppert and Schwartz (1983) convincingly showed that non-human primates have a clear rhythm in the activity of their fetal biological clock during gestation that was entrained to a light-dark cycle by the mother. These observations support the hypothesis that SCN neurons express their pacemaker circadian activity long before they are able to communicate this rhythm to the rest of the brain. Still, as was suggested by Moore and Bernstein (1989), it is possible that from the moment they originate SCN neurons are able to generate endogenous circadian activity, an activity genetically coded in these pacemakers cells; furthermore, these cells might be able to induce circadian rhythmicity in certain physiological variables (such as body temperature), but not in others (such as rest-activity). However, these rhythms are not internally synchronized and not entrained to the environmental light-dark cycle in the absence of maternal influences either. It should be noted that at present the data on the development of the human SCN obtained so far are based upon the expression of AVP in the SCN, according to animal studies, whereas VIP expression might appear earlier (Roberts et al., 1987; Hares andFoster, 1988;

Laemle, 1988; Moore and Bernstein, 1989; Davis et al., 1990). Our observation on the early development of body temperature in pre-term infants as young as 29 weeks of conceptional age might thus be related to a development of the SCN neurons in the human hypothalamus which is earlier than that of the AVP neurons. Labeling of the fetal human SCN, using new carbocyanine dye (DiI) tracer in combination with immunocytochemical staining (e.g., for GABA, somatostatin as well as glia cells), is required before one can relate the structural and functional development of the human SCN. Circadian rhythms change in aging and in AD Sleep disturbances are common among elderly people and especially among AD patients (Dement et al., 1985; Prinz et al., 1987, 1990). More than 40% of the hypnotics in the United States are prescribed to elderly people, including patients with AD (Morgan, 1983; Prinzet al., 1990). And about 2 - 10% of the American population over the age of 65 suffers from dementia (Mortimer and Hutton, 1985). AD accounts for more than 50% of these cases (Terry and Katzman, 1983). Sleep-wake rhythm disturbances, insomnia and nocturnal wandering are often important factors in a family’s decision to institutionalize their demented relatives (Sanford, 1975; Prinz et al., 1990). The ineffectiveness of hypnotic therapy draws the attention to the underlying mechanism of such complaints. Furthermore, the residual effects of these drugs may worsen the daytime cognitive performance of the individuals. It has been suggested that circadian rhythms play an important role in sleep regulation (Czeisler et al., 1980; Daan et al., 1984; Strogatz et al., 1986). In fact, disorders of the circadian timing system during aging may first be manifested as sleep-wake pathologies (for a review, see Moore-Ede et al., 1983; Brock, 1985; Dement et al., 1985; Van Goo1 and Mirmiran, 1986; Czeisler et al., 1987; Monk, 1989; Stone, 1989; Van Cauter, 1989; Richardson, 1990). Weitzman et al. (1982), who studied young and elderly subjects under conditions of temporal isolation, reported a reduction in the amplitude and

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period length of the body temperature rhythm because of aging. Reduced amplitude of the body temperature rhythm of elderly subjects was also shown in other studies (Richardson et al., 1982). This reduction seems to exist under home and laboratory conditions. However, the age effect diminishes when the environmental masking effect on the circadian timing system is removed (Monk, 1989). In temporal isolation experiments in which the endogenous activity of the biological clock was studied two interesting observations were done concerning aging (Monk, 1989). One is that there seems to be a negative relationship between the period of the clock (measured by monitoring rectal body temperature) and the age of the individual. Secondly, approximately 80% of the subjects in the 50 - 80 year range showed a spontaneous internal desynchronization, whereas for those in the 20 - 30 year age group the rate was 20%. It has been postulated that desynchronization among internal rhythms could affect not only the sleep pattern, but other aspects of biological aging as well (Czeisler et a1., 1980,1987). In a study on elderly oil-refinery operators, Reinberg and colleagues found that a low amplitude of the circadian temperature rhythm was associated with a poor tolerance of shift-work (Reinberg et al., 1980, 1984). A similar reduction in the amplitude of the rest-activity cycles was found in long-term activity records of healthy elderly vs. young men (Renfrew et al., 1987). This difference was even present during weekends. It seems that the reduction in the amplitude of circadian rhythms, early evening sleep onset, early morning awakening and daytime napping are the result of shortening endogenous circadian rhythms probably due to the loss of SCN neurons (cf, Swaab et al., 1985, 1987). These effects are minimized when the elderly subjects are not forced to follow the strict 24-h entrainment scheme of daily life, but rather sleep or wake on the basis of their own endogenous clock time (Monk and Moline, 1988; Monk, 1989). One of the characteristics of sleep changes in clinically well-defined AD patients is an increase in nocturnal wakefulness after sleep onset indicated by an increased number of awakenings at night (i.e.,

sleep fragmentation) (Prinz et al., 1982, 1987, 1990; Mirmiran et al., 1991). Although spontaneous daytime naps increase in aged individuals, the AD group spent significantly more time napping than the agematched controls. Moreover, it seems that there is a positive relationship between the degree of dementia and that of sleep disturbance (Prinz et al., 1987). There are reasons to suggest that circadian rhythms are disturbed in AD. Continuous 24-h sleep-wake records have shown that AD patients

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Fig. 4. Left panel: raw data of subjects’ rest-activity recorded over several days are double-plotted. Right panel: the x2 periodogram statistical analysis of the data is presented; the straight line indicates the level of statistical significance below which a signal is considered to be random. Top and middle data are from a 41-year-old (D.F.S.) and a 78-year-old (L.I.S.) healthy control; data from an Alzheimer patient (aged 79) are illustrated in the two bottom panels. Note the peak of rest-activity rhythm at 24 h for control subjects, a much smaller nonsignificant peak with a shorter periodicity for the Alzheimer patient. (From Mirmiran et al., 1988, with permission.)

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sleep more during day and wake up more often at night compared to age-matched non-demented or young-adult controls (Prinz et al., 1982). Most of the sleep-wake studies are, however, only carried out for a relatively short period, whereas circadian rhythm studies require several days of continuous recording. Longer sleep-wakefulness studies are not convenient, since the attachment of electrodes and the need to carry the ambulatory EEG recorder interfere considerably with regular daily activities. We have recently completed a series of studies in which the circadian rhythms of rest-activity were recorded in AD patients and controls for a period of 1 week using a small activity monitor (Mirmiran et al., 1988; Witting et al., 1990; see also Figs. 4 and 5 ) . Fourteen patients with clinically well-defined AD, 13 age-matched controls and six young healthy controls were studied. An increased nighttime activity was found both in the elderly and the AD group. The AD group showed a significant reduction of rhythm stability on successive days as well as an increased variability of the rhythm on each individual day

(Fig. 6 ) . This led to a significant reduction in the amplitude of the rest-activity of this group. Furthermore, there was a trend towards positive correlation between circadian rhythm disturbances and the degree of dementia in AD. Prinz et al. (1984) recorded the rectal body temperature of elderly and AD patients for 48 h following one night of adaptation to 1.0

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the clinical research center, but found no effect of AD on the circadian rhythm of body temperature. However, they reported an increased intradaily variability of the rhythm of the AD group. In support of our findings it is interesting to refer to the studies of Campbell et al. (1986, 1988a,b), who recorded AD and elderly controls in their home environment and found both phase advance and reduced amplitude of their body temperature (more so in men than in women). Nevertheless, it should be indicated that there are individual differences in elderly and AD patients with regard to circadian rhythm disturbances. Whether individuals with disturbed circadian rhythms are among those showing loss of SCN neurons (or secondary functional changes in the activity of these neurons via reduced light input; Hinton et al., 1986; Campbell et al., 1988a,b) should be clarified by further research (see also Stone, 1989).

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Human SCN changes during aging and in AD Direct evidence of biological clock dysfunctioning during aging and in AD was demonstrated by Swaab and colleagues (Swaab et al., 1985,1987; Hofman et al., 1988). Compared to younger age groups, a marked decrease in SCN volume, AVP cell number and total number of SCN cells was found in 80100-year-old patients (Fig. 7). Corresponding SCN changes in AD patients were even more pronounced than those observed during normal aging. Neither AVP cell density nor total cell density, as determined by thionin staining, showed any significant changes. In rats using deoxyglucose uptake as a measure of neuronal activity in the SCN, a substantial decrease of SCN activity is shown in aged rats (Wise et al., 1987, 1988). Immunocytochemical staining of arginine vasopressin or vasoactive intestinal polypeptide has also shown loss of these neurons in old rats (Roozendaal et al., 1987; Chee et al., 1988). Partial lesion studies in the animal SCN (including that of non-human primates) made it clear that the size of the SCN is crucial for the expression of its pacemaker properties (Albers et al., 1984; Davis and Gorski, 1984; Rusak, 1989; Woll-

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nick and Turek, 1989; Gerkema et al., 1990; Mirmiran and Bos, 1990). The observed decrease in SCN volume and cell number, particularly in AD, might well be the underlying factor for the circadian rhythm disturbances found in these patients. Summary and conclusions Circadian rhythms are already present in the fetus. At a certain stage of pre-natal hypothalamic development (around 30 weeks of gestation) the fetus becomes responsive to maternal circadian signals. Moreover, recent studies showed that the fetal biological clock is able to generate circadian rhythms, as exemplified by the rhythms of body temperature and heart rate of pre-term babies in the absence of maternal or environmental entrainment factors. Pre-term babies that are deprived of mater-

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nal entrainment and kept under constant environmental conditions (e.g., continuous light) in the neonatal intensive care unit run the risk of developing a biological clock dysfunctioning. However, the fact should be acknowledged that at least in mice the development of the circadian pacemaker (i.e., SCN) does not depend on environmental influences (Davis and Menaker, 1981), although other data suggest that severe disruption of the maternal circadian rhythm indeed abolishes the circadian rhythm of the fetal SCN (Shibata and Moore, 1988). During aging and in particular in AD circadian rhythms are disturbed. These disturbances include phase advance and reduced period and amplitude, as well as an increased intradaily variability and a decreased interdaily stability of the rhythm. Among the factors underlying these changes the loss of SCN neurons seems to play a central role. Other contributory factors may be reduced amount of light, degenerative changes in the visual system and the level of activity and decreased melatonin.

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161 Mortimer, J. and Hutton, J . (1985) Epidemiology and etiology of Alzheimer’s disease. In: J. Hutton and A. Kenny (Eds.), Senile Dementia of the Alzheimer’s Type, Alan R. Liss, New York, pp. 177 - 196. Navelet, Y., Benoit, 0. and Bouard, G. (1982) Nocturnal sleep organization during the first months of life. Electr. Clin. Neurophysiol., 54: 71 - 78. Papousek, H. andpapousek, M. (1984)Qualitativetransitions in integrative processing during the first trimester of human postpartum life. In: H.F.R. Prechtl (Ed.), Continuity of Neural Functions: From Prenatal to Postnatal Life, SIMP, Oxford, pp. 220 - 244. Parmalee Jr., A., Wenner, W. and Schulz, H. (1964) Infant sleep patterns from birth to 16 weeks of age. J. Pediatr., 65: 576 - 582. Patrick, J., Campbell, K., Carmichael, L., Natale, R. and Richardson, B. (1982) Patterns of gross fetal body movements over 24 hour observation during the last 10 weeks of pregnancy. Am. J. Obstet. Gynecol., 142: 363-371. Pittendrigh, C.S. (1974) Circadian oscillations in cells and the circadian organization of multicellular systems. In: F.O. Schmidt and F.G. Worden (Eds.), The Neurosciences Third Study Program, MIT Press, Cambridge, MA, pp. 437 - 458. Prinz, P.N., Peskind, E., Vitaliano, P.P., Raskind, M., Eisdorfer, C., Zemcuznikov, N. and Gerber, C. (1982) Changes in the sleep and waking in non-demented and demented elderly. J. A m . Geriatr. SOC.,30: 86 - 93. Prinz, P.N., Christie, C., Smallwood, R., Vitaliano, P., Bokan, J., Vitiello, M. and Martin, D. (1984) Circadian temperature variation in healthy aged and in Alzheimer’s disease. J. Geront ~ l . 39: , 30 - 35. Prinz, P.N., Vitiello, M.V., Bokan, J., Kukull, W.A., Russo, J. and Vitaliano, P.P. (1987) Sleep in Alzheimer’s dementia. In: H. von Hahn, W . Emser, D. Kurtz and W. Webb (Eds.), Znterdisciplinary Topics in Gerontology, Vol. 22, Karger, Basel, pp. 128- 143. Prinz, P.N., Poceta, J.S. and Vitiello, M.V. (1990) Sleep in the dementing disorders. In: F. Boller and J. Grafman (Eds.), Handbook of Neuropsychology, Vol. 4, Elsevier, Amsterdam, pp. 335 - 347. Rabinowitz, R., Peters, M.T., Vyas, S., Campbell, S. and Nicolaides, K.H. (1989) Measurement of fetal urine production in normal pregnancy by real-time ultrasonography. A m . J. Obstet. Gynecol., 161: 1264- 1266. Ralph, M.R., Foster, R.G., Davis, F.C. and Menaker, M. (1990) Transplanted suprachiasmatic nucleus determines circadian period. Science, 247: 975 - 978. Reinberg, A., Andlauer, P., Guillet, P. and Nicolai, A. (1980) Oral temperature, circadian rhythm amplitude, aging and tolerance to shift-work. Ergometrics, 23: 55 - 64. Reinberg, A., Andlauer, P. and De Prins, J. (1984) Desynchronization of the oral temperature circadian rhythms and intolerance to shift work. Nature, 308: 272 - 274. Renfrew, J.W., Pettigrew, K.D. and Rapoport, S.I. (1987)

Motor activity and sleep duration as a function of age in healthy men. Physiol. Behav., 41: 627 -634. Reppert, S.M. andSchwartz, W.J. (1983) Maternalcoordination of the fetal biological clock in utero. Science, 220: 969- 971. Reppert, S.M. and Schwartz, W.J. (1984a) Thesuprachiasmatic nucleic of the fetal rat: characterization of a functional circadian clock using ‘‘C-labeled deoxyglucose. J. Neurosci., 4: 1677- 1682. Reppert, S.M. andSchwartz, W.J. (1984b)Functionalactivityof the suprachiasmatic nuclei in the fetal primate. Neurosci. Lett., 46: 145 - 149. Reppert, S.M. and Uhl, G.R. (1987) Vasopressin messenger ribonucleic acid in the supraoptic and suprachiasmatic nuclei: appearance and circadian regulation during development. Endocrinology, 102: 2483 - 2487. Reppert, S.M., Weaver, D.R., Rivkees, S.A. and Stopa, E.G. (1988) Putative melatonin receptors in a human biological clock. Science, 242: 78 - 81. Richardson, G.S. (1990) Circadian rhythms and aging. In: C.E. Finch and E.L. Schnieder (Eds.), Handbookof theBiology of Aging, Van Nostrand Reinhold, New York, pp. 275 - 305. Richardson, G.S., Carkadson, M.A., Orav, E.J. and Dement, W.C. (1982) Circadian variation of sleep tendency in elderly and young subjects. Sleep, 5: 82-94. Roberts, M.H., Bernstein, M.F. and Moore, R.Y. (1987) Differentiation of the suprachiasmatic nucleus in fetal rat anterior hypothalamus transplanted in oculo. Brain Res., 32: 59- 66. Roozendaal, B., Van Gool, W.A., Swaab, D.F., Hoogendijk, J.E. and Mirmiran, M. (1987) Changes in vasopressin cells of the rat suprachiasmatic nucleus with aging. Brain Res., 409: 259 - 264. Rosenwasser, A.M. and Adler, N.T. (1986) Structure and function in circadian timing systems: evidence for multiple coupled circadian oscillators. Neurosci. Biobehav. Rev., 10: 431 - 448. Rusak, B. (1989) The mammalian circadian system: models and physiology. J. Biol. Rhythms, 4: 121 - 134. Rusak, B. and Zucker, I . (1979) Neural regulation of circadian rhythms. Physiol. Rev., 59: 449 - 526. Sadun, A.A., Schaechter, J.D. and Smith, L.E.H. (1984) A retinohypothalamic pathway in man: light mediation of circadian rhythms. Brain Res., 302: 371 - 377. Sander, L.W., Julie, H.L., Stechler, G. and Burns, P. (1972) Continuous 24-hour interactional monitoring in infants reared in two caretaking environments. Psychosom. Med., 34: 270 - 282. Sanford, J.R.A. (1975) Tolerance of debility in elderly dependents by supporters at home: its significance for hospital practice. Br. Med. J., 3: 471 -473. Schwartz, W.J., Bosis, N.A. and Hedley-Whyte, E.T. (1986) A discrete lesion of ventral hypothalamus and optic chiasm that disturbed the dailytemperature rhythm. J. Neurol., 233: 1 - 4. Shibata, S. andMoore, R.Y. (1987) Development of neuronal activity in the rat suprachiasmatic nucleus. Bruin Res., 34: 311-315.

162 Shibata, S. and Moore, R.Y. (1988) Development of a fetal circadian rhythm after disruption of the maternal circadian system. Dev. Brain Res., 41: 313-317. Spangler, G. (1991) The emergence of adrenocortical circadian function in newborns and infants and its relationship to sleep, feeding and maternal adrenocortical activity. Early Hum. Dev., 25: 197-208. Stone, W.S. (1989) Sleep and aging in animals. Clin. Geriatr. Med., 5: 363 - 379. Stopa, E.G., King, J.C., Lydic, R. and Schoene, W.C. (1984) Human brain contains vasopressin and vasoactive intestinal polypeptide neuronal subpopulation in the suprachiasmatic region. Brain Res., 297: 159- 163. Strogatz, S.H., Kronauer, R.E. and Czeisler, C.A. (1986) Circadian regulation dominates homeostatic control of sleep length and prior wake length in humans. Sleep, 9: 353 - 364. Swaab, D.F., Fliers, E. and Partiman, T.S. (1985) The suprachiasmatic nucleus of the human brain in relation to sex, age and senile dementia. Brain Res., 342: 37 - 44. Swaab, D.F., Roozendaal, B., Ravid, R., Velis, D.N., Gooren, L. and Williams, R.S. (1987) Prader-Willi syndrome. In: R. De Kloet et al. (Eds.), Neuropeptides and Bruin Function -Progress in Brain Research, Vol. 72, Elsevier, Amsterdam, pp. 301 - 310. Swaab,D.F.,Hofman, M.A. andHonnebier, M.B.O.M. (1990) Development of vasopressin neurons in the human suprachiasmatic nuclues in relation to birth. Dev. Brain Res., 52: 289 - 293. Terry, R. and Katzman, R. (1983) Senile dementia of Alzheimer type. Ann. Neurol., 14: 497-506. Tuffnell, D. J .,Buchan, P.C., Albert, D. and Tyndale-Biscoe, S. (1990) Fetal heart rate responses to maternal exercise, increased maternal temperature and maternal circadian variation. J. Obstet. Gynecol., 1 0 387 - 391. Turek, F.W. (1985) Circadian neural rhythms in mammals. Annu. Rev. Physiol., 47: 49-64. Van Cauter, E. (1989) Physiology and pathology of circadian rhythms. In: C. Edwards and D. Lincoln (Eds.), Recent Advances in Endocrinology and Metabolism, Churchill Livingstone, Edinburgh, pp. 109- 134. Van Gool, W.A. and Mirmiran, M. (1986) Aging and circadian rhythms. In: D.F. Swaab, E. Fliers, M. Mirmiran, W.A. van Gool and F. van Haaren (Eds.), Aging of the Brain and AIzheimerS Disease - Progress in Brain Research, Vol. 70, Elsevier, Amsterdam, pp. 255 - 277. Visser, G.H.A., Goodman, J.D.S., Levine, D.H. and Dawes, G.S. (1982) Diurnal and other cyclic variation in human fetal heart rate near term. Am. J. Obstet. Gynecol., 142: 535 - 544. Weitzman, E.D., Moline, M.L., Czeisler, C.A. and Zimmerman, J.C. (1982) Chronobiology of aging: temperature, sleepwake. rhythms and entrainment. Neurobiol. Aging., 3: 299 - 309. Wever, R.A. (1989) Light effects on human circadian rhythms. J. Biol. Rhythms, 4: 161- 185.

Wise, P.M., Walowitch, R.C., Cohen, I.R., Weiland, N.G. and London, E.D. (1987) Diurnal rhythmicity and hypothalamic deficits in glucose utilization in aged ovariectomized rats. J. Neurosci., 7: 3469 - 3473. Wise, P.M., Cohen, I.R., Weiland, N.G. and London, E.D. (1988) Aging alters the circadian rhythm of glucose utilization in the suprachiasmatic nucleus. Proc. Natl. Acad. Sci. W.S.A., 85: 5305 - 5309. Witting, W., Kwa, I.H., Eikelenboom, P., Mirmiran, M. and Swaab, D.F. (1990) Alterations in the circadian rest-activity rhythm in aging and Alzheimer’s disease. Biol. Psychiatry, 27: 563 - 572. Wollnick, F. and Turek, F.W. (1989) SCN lesions abolish ultradian and circadian components of activity rhythms in LEW/Ztm rats. Am. J. Physiol., 256: R1027- R1039.

Discussion R. Ravid: What is the change in rest-activity rhythm in demented patients who are staying at home instead of a nursing home? M. Mirmiran: In our own studies we have compared recordings of the Alzheimer (AD) group monitored in the hospital with elderlypeople recorded at home (Witting et al., 1990). This factor hampers to some extent the conclusion of the study, viz. disturbed circadian rhythmicity in AD. However, we believe that if there is any effect of staying in a hospital on our recordings this would be in favor of the AD group, since environmental zeitgeber effects are stronger in the hospital than at home. Nevertheless, future home recordings of elderly and AD groups - using our newly developed very small ambulatory activity monitor (Van Someren et al., 1992) - are required before definitive conclusions on disturbed circadian rhythms in AD can be drawn. It is important to indicate that in a study by Campbell et al. (1986, 1988b) a significant reduction of both amplitude and period of circadian rhythms was found in AD patients recorded at home. Yet, Prinz et al. (1984) did not find significant differences in the recordingsof bothelderlyand ADpatientsexcept for anincreased intradaily variability in the AD group. S.M. Reppert: Are the rhythms you monitored in pre-term infants diurnal or circadian? M. Mirmiran: Statistical analysis of our data using x2 periodogram showed significant periodicity with a maximum peak in the power spectrum between 24 and 27 h (Mirmiran and Kok, 1991). However, even in those babies in which “mean” period lengths were 24 h, the rhythm was not diurnal since there were daily changes in the period length (including phase jumps). Although variations in body temperature and heart rate with a periodicity “around 24 h” were present in successive days of recording, no indication of any relationship between these variations and the time of the day were found in pre-term infants, M.L. Summar: What effect would you expect from the pre-natal administration of steroids, which are used to induce pulmonary maturation, in the circadian rhythm of premature infants?

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M. Mirmiran: There is one study in rats in which perinatal exposure to corticosteroids induced significant reduction in the amplitude of circadian rhythms post-natally (Krieger, 1972). Though often used in humans, there are no reports on the effect of corticosteroids on circadian rhythms. The fact that total adrenalectomy or blocking fetal-maternal adrenal activity in pregnant women eliminate circadian rhythm in fetal heart rates, suggests that corticosteroids are important prenatal zeitgebers for circacian rhythms (Arduini et al., 1986a,b, 1987). In future studies it is essential to examine “timed” and daily administration of corticosteroids (or melatonin, see Davis and Mannion, 1988; Cassone, 1990; Yuan et al., 1991) and their effect on the development of human circadian rhythms. W.A. Scherbaum: I was interested in the observation in which you refer to the lack of fetal rhythmicity in the adrenalectomized mother. It has been shown that after experimental adrenalectomy colocalized CRF within AVP cells in the PVN is considerably upregulated so that one might speculate that there is either input from the PVN to the SCN or that AVP cells within the SCN respond directly to cortisol. Do you have data on the cellular expression of CRF in the SCN after experimental adrenolectomy? M. Mirmiran: I do not know of any data on CRF changes in the SCN following adrenalectomy. However, CRF as a maternal signal influencing fetal circadian rhythms is an intriguing hypothesis. There are also indications from the literature that show high levels of corticosteroid receptors in the rat SCN, particularly in early (fetal) development (De Kloet et al., 1988). Certainly it is interesting to hypothesize that CRF directly influences the SCN or indirectly via the PVN. It is also known from rat studies that both hyper- and hypocorticism changes the time course of development of both VIP and somatostatin cells in the SCN (Nobou et al., 1985).

References Arduini, D., Rizzo, G., Parlati, E., Dell-Acqua, S., Mancuso, S. and Romanini, C. (1986a) Are the fetal heart rate patterns related to fetal-maternal-endocrine rhythms at term pregnancy? J . Foetal Med., VI: 53 - 57. Arduini, D., Rizzo, G., Parlati, E., Giorlandino, C., Valensise, H., Dell-Acqua, S. and Romanini, C. (1986b) Modification of ultradiadian and circadian rhythms of fetal heart rate after fetal-maternal adrenal gland suppression: double blind study. Prenatal Diagnosis, 6: 409 - 417.

Arduini, D., Rizzo, G., Parlati, E., Dell-Acqua, S., Romanini, C. and Mancuso, S. (1987) Loss of circadian rhythms of fetal behavior in a totally adrenalectomized pregnant woman. Gynecol. Obstet. Invest., 23: 226 - 229. Campbell, S.S., Kripke, D.F., Gillin, J.C. and Hrubovac, J.C. (1988) Exposure to light in healthy elderly subjects and Alzheimer’s patients. Physiol. Behav., 42: 141 - 144. Cassone, V.M. (1990) Effects of melatonin on vertebrate circadian systems. Trends Neurosci., 13: 457 - 464. Davis, F.C. and Mannion, J. (1988) Entrainment of hamster pup circadian rhythms by prenatal melatonin injections to the mother. Am. J. Physiol., 24: R439- R448. De Kloet, E.R., Rosenfeld, P., Van Eekelen, A.M., Sutanto, W. and Levine, S. (1988) Stress, glucocorticoids and development. In: G.J. Boer, M.G.P. Feenstra, M. Mirmiran, D.F. Swaab and F. van Haaren (Eds.), Biochemical Basis of Functional Neuroteratology: Permanent Effects of Chemicals on the Developing Brain - Progress in Brain Research, Vol. 73, Elsevier, Amsterdam, pp. 101 - 120. Krieger, D.T. (1972) Circadian corticosteriod periodicity: critical period for abolition by neonatal injection of corticosteroids. Science, 178: 1205 - 1207. Mirmiran, M. and Kok, J.H. (1991) Circadian rhythms in early human development. Early Hum. Dev., 26: 121 - 128. Nobou, F., Besson, J., Rostene, W. and Rosselin, G. (1985) Ontogeny of vasoactive intestinal peptide and somatostatin in different structures of the rat brain: effects of hypo- and hypercorticism. Dev. Brain Res., 20: 296 - 301. Prinz, P.N., Christie, C., Smallwood, R., Vitaliano, P., Bokan, J., Vitiello, M. and Martin, D. (1984) Circadian temperature variation in healthy age and in Alzheimer’s disease. J. Gerontol., 39: 30 - 35. Van Someren, E.J.W., Van Cool, W.A., Vonk, B.F.M., Mirmiran, M., Speelman, J.D., Bosch, D.A. and Swaab, D.F. (1992) A new method for the ambulatory registration of tremor and movement. (Submitted.) Witting, W., Kwa, I.H., Eikelenboom, P., Mirmiran, M. and Swaab, D.F. (1990) Alterations in the circadian rest-activity rhythm in aging and Alzheimer’s disease. Biol. Psychiatry, 27: 563 - 572. Yuan, H., Lu, Y. and Pang, S.E. (1991) Binding characteristics and regional distribution of [l]indolmelatonin binding sites in the brain of the human fetus. Neurosci. Lett., 130: 229- 232.

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SECTION V

Development, Aging and Dementia

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D.F. Swaab, M.A. Hofman, M. Mirmiran, R. Ravid and F.W. van Leeuwen (Eds.) Progress in Brain Research, Vol. 93 0 1992 Elsevier Science Publishers B.V. All rights reserved.

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CHAPTER 12

Ontogeny of peptides in human hypothalamus in relation to sudden infant death syndrome (SIDS) N. Kopp', M. Najimi', J . Champierl, F. Chigr', Y. Charnay3, J . Epelbaum2 and D. Jordan' I

Laboratoire d'Anatomie Pathologique, Facultt! de Mddecine Alexis Carrel, 69372 Lyon Cedex 08, France; INSERM U 159, Centre Paul Broca, 75014 Paris, France; and Laboratoire de Neuropathologie, Clinique Be1 Air, Geneva, Switzerland

Introduction The mammalian brain is not mature at birth. This is particularly true for humans. During the end of gestation and in the post-natal period, growth and development of the brain are influenced by the hormonal state. This is well illustrated in the hypothalamus. For instance, it has been shown that the hypothalamo-pituitary adrenal axis is transformed from a state of hyperactivity during early stages of gestation to a state of partial control during late stages (Donovan, 1980). The maturation of the regulation of the hypothalamo-pituitary system by the brain is not'completed at birth and goes on during a long post-natal period (Donovan, 1980). In addition, the maturation of the hormonal pattern, as illustrated physiologically by the chronobiological variations of thyrotropin-releasing hormone (TRH) and thyro-stimulating hormone (TSH) of the hypothalamo-pituitary axis, has been well documented in the neonatal period up to adulthood in rat (Jordan et al., 1989). The authors report'that a hypothalamic TRH circadian rhythm was present at all ages studied (between 15 and 70 days old) whereas the low amplitude of pituitary TSH rhythms detected in the young rat disappeared in the adult. In contrast, the serum TSH rhythm becomes consistent to reach the well-characterized circadian mid-day peak in the adult rat. In fact, during the

post-natal period, circadian variations of the concentration of some pituitary hormones, such as thyroid, growth and gonadotrophic hormones, have been observed in both rats and humans. TSH pituitary concentrations are very low at birth and reach a maximum at the end of the second post-natal week (Winter, 1983). The control of growth hormone (GH) secretion does not reach a complete functional state before the post-natal period. Serum concentrations of GH are more elevated in neonates than in 3-month-old humans (Winter, 1983). This suggests a post-natal set-up of the regulatory system and of the control of GH secretion. Gonadotropic hormones play a very important role during the neonatal period in the development of the hypothalamo-pituitary axis. Correlations have been established between temporary changes of the concentration of sexual hormones and the alteration of the hypothalamo-hypophyso-gonadal system during the first post-natal year of cerebral differentiation (Darner, 1985). Serum concentrations of gonadotropins are elevated between birth and the third post-natal month (Forest, 1983). Furthermore, interrelations were established between hormonal changes and their hypothalamic control factors (TRH, somatostatin (SRIF), luteinizing hormone releasing-hormone (LHRH)). Biochemical and immunohistochemical studies performed in rat and man have revealed important

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differences in the distribution of these hypothalamic factors between neonates and adults. Compared to animal research, human data in the field of the anatomical distribution of hypothalamic factors and their binding sites are very limited. To our knowledge, the only reports on SRIF and LHRH distribution based on immunocytochemistry are those by Bugnon et al. (1977a,b), Bloch (1978) and Fellman (1978) in the fetus and by Barry (1978) and Bouras et al. (1987) in adults. No precise binding site study of SRIF or TRH in the human hypothalamus is available. We therefore performed immunohistochemical mapping of SRIF and LHRH, and binding site studies by quantitative autoradiography of SRIF and TRH in the hypothalamus of normal infants and adults. Some of these markers have been applied to four cases of sudden infant death syndrome (SIDS). In addition, we report here the first preliminary data on the immunohistochemical distribution of delta sleep-inducing peptide (DSIP) in the control infant hypothalamus. Disorders of thermoregulation are hypothesized in SIDS and the peptides and areas studied here might be implicated in this regulatory mechanism. Generally speaking, detailed and precise anatomical normative data will serve as a basis for neuropathological studies and might be useful for neuro-imaging of neurologic, psychiatric and neuroendocrinologic patients. Methodological questions raised by studies of peptides and binding sites in the human hypothalamus We shall review briefly the general pitfalls that arise from human neuropathological studies as well as specific inset of hypothalamus and/or peptides. We shall consider ante- and post-mortem factors. Ante-mortem factors Sex. Many studies have failed to take the factor sex into consideration. However, several macro-

scopic studies indicated the possibility of differences associated with sex in brain morphology (Kopp et al., 1977; De Lacoste-Utamsing and Holloway, 1982). Some differences are described at a microscopic level: number of cells in Onuf‘s nucleus of the spinal cord (Forger and Breedlove, 1986), size of the hypothalamic sexually dimorphic nucleus as well as the number of its cells (Swaab and Fliers, 1985). Laterality. Lateralisations for dopamine, cholineacetyltransferase, noradrenaline and GABA have been reported (Oke et al., 1978; Amaducci et al., 1981; Click et al., 1982). Radioimmunological measurement of LHRH and TRH in twelve microdissected areas of the adult human hypothalamus recently revealed a higher concentration of TRH but not LHRH in three areas corresponding to the paraventricular, dorsal and ventromedial nuclei on the left side (Borson-Chazot et al., 1986). Otherfactors. The influence of nutritional factors or drug treatment on peptidergic systems in humans is not, to our knowledge, well taken into account. Such factors should not be underestimated, especially in the hypothalamus (this part of the brain is highly implicated in the control of satiety; some side effects of psychotropic treatments are of the neuroendocrine type). Quite obviously individual variations raise the problem of “normality”, which is as much a philosophical problem as a scientific one. Age is, of course, crucial in an ontogenic study; it should be stressed here that “normal” autopsy cases of late infancy, adolescence and young adulthood are very rare. This often brings a gap, especially around puberty, in the series aiming at covering all periods of life. Post-mortem factors Post-mortem delay is usually understood as the period of time between death and fixation of tissue or freezing of tissue. Both immunohistochemistry and binding site studies of peptides are influenced by post-mortem delay.

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tions, following a plane parallel to the plane defined by the anterior limit of the optic chiasm and of the anterior white commissure. Finally, the sections were mounted on chromalun-gelatin-coated slides Posr-mortem delay and ~mm~no~istochemistry.and stored at - 20°C. We found that storage up to In our experience of immunohistochemistry of 6 months gave no different results. SRIF, LHRH and DSIP, delays of up to 12 h did not For immunohistochemistry, the peroxidaseantiperoxidase (PAP) technique (Sternberger et al., interfere noticeably with the quality of the results. 1970) was used with minor modifications: (1) preWe tried, as others before, TRH immunohistotreatment of the sections with a 1Yo hydrogen peroxchemistry with several antibodies in the human brain without any success. This may have been due ide for 20 min at room temperature to inhibit the ento the rapid post-mortem diffusion, explainable by dogenous peroxidases; and (2) addition of nickelthe very small size of this peptide, or to degradation. ammoniumsulfate to the final colour developing solution (Kitahama et al., 1985) to increase the senBiochemical studies have shown a good stability of LHRH and SRIF, even in cases with a delay as long sitivity. In the immunohistochemical controls, we as 36 h (Parker and Porter, 1982; Sorensen, 1984). first omitted the specific antiserum or substituted it by non-immune serum (which gave no positive reaction); second, each antiserum used was absorbed Post-mortem delay and binding sites. Most autoonly by its homologous peptide (peptide concentraradiographic studies performed on human brain tion of 20 pM) and produced the same results, irtissue tend to show a good stability up to 24 h or even 36 h for peptide binding sites (Cash et al., respective of its being preabsorbed or not by other 1987). Our own results do corroborate previous data peptides tested (such as neurotensin, vasoactive intestinal peptide, beta-endorphin, cholecystokinin (Manaker et al., 1986; Reubi et al., 1986) for SRIF and substance P). and TRH binding sites. This stability may be a The primary antisera used were: antisomatostatin general property of binding sites since we have also 1-12 (Chayvialle et al., 1978), antisomatostatin 15found it in non-peptidergic ligands such as serotonin 28 (SRIF) (Tohyama, Osaka), antisomatostatin 28 and benzodiazepine, confirming data from the liter(Peninsula Laboratories), anti-LHRH (Bugnon et ature (Palacios et al., 1983; Zezula et al., 1988). al., 1977a) and anti-DSIP (Charnay et al., 1989). Irnmunohistochemistry Quantitative autoradiography Fixation was achieved after autopsy by perfusion After the brain had been removed from the skull, a through the carotis interna. After 5 min of perfusion block of fresh tissue including the hypothalamus with 200 ml saline buffer, 1.5 1 of paraformand adjacent structures was excised. This block had aldehyde 4% (0.1 M phosphate buffer, pH 7.4) was approximately the shape of a parallelepiped with perfused in approximately 30 min at room anterior and posterior aspects parallel to the plane temperature. The leptomeninges were carefully defined by the anterior limit of the chiasm and of the peeled away and the entire brain was immersed in anterior white commissure. The caudal plane was the same fixation medium overnight at 4°C. A block just behind the mammilary complex. Inorder to preof brain including the hypothalamus and adjacent vent anatomical distortions as much as possible, this structures was excised and immersed in a renewed fresh block was supported and surrounded by a sort identical fixation medium for 6 - 10 days at 4°C. of extemporaneously prepared and adapted boxFollowing 1 week of immersion in 20070 sucrose at shaped mould made of strong aluminium foil. The 4"C, the tissue block was frozen in liquid nitrogen mould, with the tissue inside it, was laid on a copper and cut with a cryostat into 20 pm thick coronal secPost-mortem delay and morphology. The number and size of dendritic spines are very sensitive to postmortem delay (De Ruiter, 1983).

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block previously cooled at -80°C. By using this freezing procedure, minimal crystal artefacts were obtained. Quantitative autoradiography (Young and Kuhar, 1979) was preferred to a membrane binding method because of (1) the high degree of anatomical resolution and (2) the possibility of quantification with image analysis. The apparatus used was either an Imstar-Starwise (Paris) station or a Rag 200 Biocom image analysis system (Les Ulis, France). For anatomical localization of the hypothalamic nuclei and areas, adjacent sections to those used for autoradiography were stained with cresyl violet. Nuclei and areas were identified according to an atlas of the human hypothalamus developed in our laboratory and according to the cytoarchitectural reports on the human hypothalamus of Diepen (1962) and Braakand Braak (1987).

SRIF binding sites

Preliminary desaturation by guanosine triphosphate (GTP) appeared to be a necessary condition to obtain a good visualization of binding sites in some areas. In vitro GTP treatment has a dramatic desaturating effect on sites occupied by SRIF (Lerouxet al., 1988). Indeed, it was shown that binding sites to SRIF in the brain are coupled to G proteins (Enjalbert et al., 1983). It has also been reported that GTP increases the dissociation of the radioactive ligand from its receptor (Moyse et al., 1989). Leroux et al. (1988) have used this property of GTP by adding it to the preincubation medium; the result was the detection of somatostatinergic binding sites in several more hypothalamic structures (preoptic area, suprachiasmatic periventricular nuclei and mediolateral region of posterior hypothalamus), whereas adjacent sections not treated with GTP were not labeled. In the present study, M of GTP in the preincubation medium dramatically increased SRIF binding in the preoptic area, diagonal band of Broca, ventromedial and dorsomedial nuclei. Conversely, it had no effect on some structures, such as the infun-

dibular nucleus and tuber nuclei. The effect of GTP was not due to a change in affinity but to a difference in accessibility. This would tend to prove, in an indirect manner, the coupling of SRIF binding sites to G proteins. However, this coupling could be differential. After the addition of GTP in different concentrations (10- loM) during incubation, a decrease of autoradiographic labeling (70% at l o p 7 M) was noticed in both the preoptic area and the infundibular nucleus. This supports the hypothesis that SRIF binding sites couple to G proteins in GTP-sensitive regions (i.e., preoptic area) and GTP-insensitive areas (i.e., infundibular nucleus). Saturation studies made in anterior and mediobasal levels of the hypothalamus show that binding sites to SRIF have a high affinity (Kd in the nanomolar range). It is not different in infants and adults. Furthermore, such affinity is comparable to those usually reported for SRIF binding. It should also be mentioned that displacement experiments with SRIF-14 - as well as withTyr-0-Trp 8 SRIF-14 - show that the specific binding is competitively inhibited.

TRH binding sites [3H]3MeTRH was used as a ligand for TRH binding sites in different animal species. This analogue binds to TRH sites with a high affinity, as has been shown both in human brain homogenates (Parker and Capdevila, 1984)and in sections by quantitative autoradiography (Manaker et al., 1986; Jordan et al., 1989). In the present study, the specific binding represented more than 90% of total binding in the hypothalamic structures. Saturation experiments were performed in two different hypothalamic areas (preoptic area and infundibular nucleus). They demonstrated that [3H]3MeTRH binding is saturable. Furthermore, the ligand bounds with a high affinity (dissociation constant of the nanomolar range) to TRH sites in both the preoptic area and the infundibular nucleus. Inhibition experiments showed that among ana-

171

logues tested only 3MeTRH and TRH are efficient in their inhibiting action. However, 3MeTRH is 10 times as potent as TRH. The characteristics are the same in infants and adults. In contrast to the effect on SRIF, GTP preincubation had no significant effect on the density of TRH binding sites.

Distribution of SRIF immunoreactive neurons Our study is based on four male infant brains (26, 30, 40 and 60 days old, respectively). The infants died of circular of umbilical cord, pulmonary hypoplasia associated with operated diaphragmatic hernia, acute staphylococcus sepsis, therapeutic perfusion error. The presence of several molecular varieties leads to cautiousness in the interpretation of our results. Indeed, besides the SRIF-14 variety, other forms of SRIF, also called prosomatostatins, are present in both the brain and the peripheral nervous system (Benoit et al., 1985). Other studies have shown wide differences in the distribution of SRIF-28 immunoreactive neurons and of SRIF-28 1 - 12 immunoreactive neurons in the monkey (Bakst et al., 1985). A predominance of the SRIF-14 has been reported in human and rat brain whereas the SRIF28 proportion varies widely (Emson et al., 1981;Biggins et al., 1984). Immunohistochemical studies show an exhaustive distribution of SRIF-28 in cell bodies as well as in fibres (Bakst et al., 1985). Binding sites of SRIF-14 and of SRIF-28 have the same general distribution (Srikant and Patel, 1981). Comparison of the labeling obtained with our three different antibodies did not reveal any noticeable difference. Immunohistochemical controls consisted of first, the omission of the specific antiserum or its substitution by non-immune serum; second, each antiserum was blocked only with its homologous peptides (1 pm); and third, the same results were obtained whether antiserum was preabsorbed or not with other peptides. Our results (see Fig. 1) are in general agreement with other data in fetuses and adult humans

(Bugnon et al., 1977b; Bouras et al., 1987). In fetuses, immunoreactive cell bodies are found in paraventricular and suprachiasmatic nuclei as well as in the periventricular area. Immunoreactive fibres are localized in the anterior hypothalamus and in mammillary bodies. However, we find a higher density in the paraventricular nucleus, especially in the parvocellular part. We found a higher density of cell bodies in infundibular and posterior nuclei than has been found in adults (Bouras et al., 1987). In the fetus, no fibres have been reported in the dorsomedial, ventromedial, posterior and mammillary nuclei (Bugnon et al., 1977b). In adults, only a low density of fibres has been observed in the ventromedial nucleus and the median eminence (Bouras et al., 1987). In mammillary bodies, a decrease of fibres has been described in adults, but without specifying the nucleus. These differences, observed mainly between infants and adults, could be due to differences in methods, differences in antibodies or differences between the anatomical atlases used by different authors. However, they could also be due to the fact that this peptidergic system is implicated in maturation and development of neurons. In rats, Inagaki et al. (1982) found SRIF immunoreactive neurons in the granular layer of the cerebellar cortex of neonates but not of adults. In our laboratory, a decrease of another peptide, neurotensin, was found in diencephalic structures, especially in mammilary bodies (Sakamoto et al., 1986). Long and large diameter fibres (“woolly” fibres, described in the rat pallidum by Haber and Nauta, 1983, and in the human septum by Gaspar et al., 1987) were found in the median preoptic area, anterior hypothalamic area and, to a lesser extent, in the lateral preoptic area. In our laboratory, in the same cases and in three additional ones, substance P immunoreactive “woolly” fibres were stable from 26 to 60 days of age. Compared with the distribution in rats, our results show a higher density of SRIF cell bodies in the anterior hypothalamus (including the preoptic

172

H

J

. . . . , . . . . . . ...

3v

L

173

area in infants). In rat, the densest concentration of cell bodies is present in the periventricular nucleus,

whereas in human infants, it is in the paraventricular nucleus. As in previous studies in humans, we did not detect SRIF fibres in the fornix, as has been described in rats by Roberts et al. (1982).

Distribution of SRIF binding sites

Fig. 2. Autoradiography of somatostatin binding sites in the tuber nuclei (TN) of an adult human. Thesenucleiareprominently rich in binding sites and can be easily located between optic tract (OT) and fornix (F).

This study was performed on 16 brains: 7 infants and 9 adults. The general distribution pattern (Fig. 2) shows a rostro-caudal decrease in the hypothalamus and inside some of the hypothalamic nuclei and structures. However, in the mediobasal hypothalamus, the SRIF binding sites increase in the infundibular nucleus in a rostro-caudal direction. Similar observations were made in human fetal spinal cord (Charnay et al., 1988). The same distribution was found in infants and adults. However, some differences should be emphasized: there was a higher density of SRIF binding sites in the posterior part of the infundibular nucleus and in the anterior hypothalamic area in infants. In contrast, the developmental pattern of SRIF binding sites was rather delayed in the tuberal nuclei as compared with other regions. It is interesting to note that these nuclei are well differentiated only in humans and not in lower animals (Grunthal, 1929). Thus, a later ontogenic appearance could reflect the recent phylogenic evolution of these nuclei. These specific differences in the regional distribuOf a peptide between infant and suggest that this peptide could be involved in neuronal maturation. Other studies support the presence of a

Fig. 1. Topographical distribution of somatostatin immunoreactive perikarya fibres in the human infant hypothalamus. Asterisks represent cell bodies; large dots, large fibres; small dots, small fibres. Abbreviations: AA, anterior hypothalamic area; AC, anterior commissure; CP, cerebral peduncle; DA, dorsal area; DBBh or DBH, diagonal band horizontalis; DBBV or DBV, diagonal band verticalis; DM, dorsomedial nucleus; DPC, decussatio pedunculorum cerebellorum superiorum; F, fornix; FF, field of Forel; FLM, fasciculus longitudinalis medialis; I, infundibular nucleus; IC, inferior colliculus; IP, interpeduncular nucleus; LH, lateral hypothalamic area; LM, lateral mammillary nucleus; LP, lateral preoptic area; LT, lamina terminalis; LTu, lateral tuberal nucleus; ME, median eminence; MES, mesencephalon; MM, median mammillary nucleus; MP, medial preoptic area; MT, medial tuberal nucleus; OC, optic chiasma; ON, optic nerve; OT, optic tract; PA, posterior hypothalamic area; PE, periventricular nucleus; PV, paraventricular nucleus; s c , suprachiasmatic nucleus; SO, supraoptic nucleus; SPT, supratrochlearis nucleus; TM, tubero mammillary nucleus; VM, ventromedial nucleus; 21, zona incerta; 3V, third ventricle. (Reproduced from Najimi et al., 1989a, with permission.)

174

...

d

b

.......

f SRIF-LR fibers densities 1251-SRIF binding sites densities ,6000

3

+

I +

moderate

verylow

dpm/mm2

6000-5000 dpm/mm2

0 0

5000-2000 dpm/mm2

~ 2 0 0 0dprn/mrn2

Fig. 3. Comparison of somatostatin immunoreactive densities (left side) and binding site densities (right side). Density scales are in dicated on graph. Abbreviations: see legend of Fig. 1. (Reproduced from Najimi et al., 1991a, with permission.)

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similar action of SRIF on molluscs in vitro (Bulloch, 1987; Grimm-Jorgensen, 1987). The difference (higher number of binding sites in medial preoptic area, diagonal band of Broca, ventromedial and dorsomedial nuclei) in labeling of SRIF binding sites in tuber were found in both sexes: this suggests that gonadotrophins and sex steroids do not influence the expression of these SRIF binding sites. The differences (higher number of binding sites in medial preoptic area, diagonal band of Broca, ventromedial nucleus) between our results and those of Reubi et al. (1986) could be explained, at least partly, by the use of GTP. Indeed, GTP preincubation enhanced the binding in both infants and adults, especially in the diagonal band of Broca. Comparison of the distribution of SRIF and its binding sites (Fig. 3)

sites is relatively lower and increases in the posterior parts. In the posterior hypothalamus, a moderate SRIF binding site density was also demonstrated in the lateral mammillary nucleus, where, at least in the newborn infant, moderate to high densities of SRIF immunoreactive fibres had been reported. However, the posterior hypothalamic area (containing a moderate to high density of SRIF immunoreactive fibres) as well as the medial mammillary nucleus (with no SRIF immunoreactive fibres) display a low SRIF binding site density. It was originally suggested (Reubi et al., 1986) that the mismatch between the peptide and its binding sites could be explained by the fact that several members of the somatostatin family, not detectable by immunocytochemistry, could bind to the binding site. Following desaturation with GTP, the SRIF binding sites were generally in good correlation with immunohistochemical data.

There is an overall good correlation between the distribution of SRIF immunoreactive fibres and SRIF binding sites. The highest densities of SRIF binding sites are located in the preoptic area, corresponding to a high density of fibres in the same area. Furthermore, it should be mentioned that both fibres and binding sites display the same rostrocaudal distribution. There is also a good correlation between the distribution of SRIF fibres and SRIF binding sites in the anterior hypothalamic area, where both exhibit moderate densities. In the mediobasal hypothalamus, the dorsomedial and ventromedial nuclei are homogeneous with respect to densities of SRIF binding sites and SRIF fibres. In contrast, the infant ventromedial nucleus, displaying highly SRIF immunoreactive fibres, contains only a moderate density of SRIF binding sites. This could be due to the fact that a large number of these fibres are fibres “en passage”, with their corresponding binding sites located outside the ventromedial nucleus. In the infundibular nucleus, there is a good correlation between immunoreactive fibres and binding sites. Thus, the density of SRIF immunoreactive fibres is higher in the anterior parts of this nucleus, whereas the density of SRIF binding

Functional implications The presence of SRIF fibres terminating on the third ventricle in infants would favour a possible release of the neuropeptide to the cerebrospinal fluid. It has been suggested that this release would be followed by a transport by cerebrospinal fluid to extra-hypothalamic regions in order to evoke some of its behavioural effects. The very dense innervation of the median eminence, where fibres are concentrated around blood vessels, probably originates from the paraventricular and infundibular nuclei. Neuropeptide release in the portal blood stream is in good agreement with the generally proposed neuroendocrine role of hypothalamic SRIF. The differences found between infants and adults support the hypothesis that SRIF concentrations decrease with age. A decrease of the concentrations of some hypothalamic factors has also been described in rat (Steger et al., 1979; Wise and Ratner, 1980; Rice et al., 1983; McDonald, 1987; Pekary et al., 1987;Morimoto et al., 1988). Thedecreasein somatostatin innervation of the median eminence with age in man and rat strongly suggests a decrease of secretory activity of these hypothalamic neuronal

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systems implicated in the regulation of pituitary functions. The results of immunohistochemical and autoradiographic studies reveal the presence of SRIF cell bodies, SRIF fibres and SRIF binding sites in some hypothalamic nuclei and areas implicated in the control of distinct physiological functions. For instance, the preoptic area contains a dense group of SRIF cell bodies and fibres as well as a high density of SRIF binding sites, which is of interest, since this area seems to be implicated in the control of thermoregulation in rat (Briese, 1989). Moreover, other studies indicate that SRIF may indeed play a role in thermoregulation (Lipton and Glyn, 1980; Brown et al., 1981; Chandara et al., 1981;Wakabayashi et al., 1983). All these data confirm a role of SRIF in the control, from birth onwards, of thermoregulation in man. Distribution of TRH binding sites

Anatomical distribution The distribution of TRH binding sites in the

human hypothalamus was studied in 14 cases (8 adults and 6 infants of both sexes) (see Table I). This distribution is widespread, heterogeneous and predominant in the anterior and mediobasal hypothalamus (Figs. 4 and 5 ) . As for SRIF, the study of TRH binding sites reveals regional differences in the distribution pattern between infants and adults. In infants, the density is higher in the diagonal band of Broca, whereas in adults the density is greater in the tuber nuclei. The decrease with aging in the density of TRH binding sites in the diagonal band of Broca suggests that these sites could be implicated in functions other than synaptic transmission such as cellular differentiation and migration (Zagon and McLaughlin, 1986; Hauser et al., 1987). As in the case of SRIF binding, TRH binding sites appear much later in the tuber nuclei than in other hypothalamic areas. This latency would suggest that even though the systems of binding sites of these two hypophysiotropic factors are already in their definitive location in the post-natal period, they have not yet reached their

TABLE 1 Cases in which TRH autoradiographic binding site studies were performed -

Cases

Sex

Age

Post-mortem delay

Cause of death

Adults A B C D E F G H

M F M F F M M F

22 years 27 years 34 years 42 years 45 years 41 years 67 years 82 years

30 h 25 h 28 h 30 12 h 20 h 12 h l h 2 h 30

Cardiac failure Cardiac failure Sudden death Sudden death Coronary thrombosis Cardiac failure Myocardic infarct Cardiac failure

Neonates I J

F M

2h 35 h

14 h 34 h

K

M

1 day

10 h

L M N

M M F

1 month 1 month 1 year

20 h 5h 7h

Pulmonary hypoplasia Amniotic inhalation with gastric regurgitation Oedematic alveolitis with refractory hypoplasia Liver lesions Enterocolitis Hepatic necrosis

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Comparison between TRH binding sites and the radioimmunological distribution of TRH

Fig. 4. Autoradiography of TRH binding sites in an adult human: diagonai band of Broca.

Fig. 5. Autoradiography of TRH binding sites in an adult human: mammillary nuclei (MN).

complete maturation and adult functional level at that time. It should be noted that, as for SRIF binding sites, the distribution of TRH sites is unaffected by sex in humans. Some differences with the rat should be mentioned: no TRH binding site was found in the infundibular, ventromedial and tuber nuclei in this species (Rostkne et al., 1984; Sharif, 1989). The discrepancy may be explained, for tuber nuclei, by their very small size in the rat.

The study of the distribution of TRH in 10 adult human hypothalami performed in our laboratory (Borson-Chazot et al., 1986), based on “punch” microdissection technique and radioimmunological assays showed a significant predominance of the TRH concentration in three areas (ventromedial, dorsal and paraventricular) on the left side. In our TRH binding site study, we studied the distribution on both sides but found no difference in laterality. To our knowledge, no precise and detailed TRH distribution study is available in man since no immunohistochemical data have been reported. This can probably be explained by rapid diffusion of the peptide TRH in post-mortem tissues. Attempts have been made in our laboratory with different antibodies, different fixation media and different procedures, on relatively short post-mortem cases (as short as 5 h), without any success. However, the comparison of our autoradiographic study with available microbiochemical studies in man (Parker and Porter, 1983; BorsonChazot et al., 1986) tends to reveal a generally good correlation between TRH and its binding sites, especially in the preoptic area, the infundibular and ventromedial nuclei and mammillary bodies.

Functional implications Besides its role in the control of the release of prolactin and TSH, the implication of TRH in thermoregulation is well established. In several species, intracerebral injection of TRH induces an alteration of thermoregulation. In rats that are awake, the direct intra-hypothalamic injection of TRH is followed by an evaluation of rectal temperature (Lin and Yang, 1989). The site of action of TRH seems to be the anterior hypothalamus and, more precisely, the preoptic area, since lesions of the preoptic area induce alterations of thermoregulation. In the preoptic area, TRH would be responsible for the excitation of coolness-sensitiveneurons and of inhibition of neurons sensitive to warmth (Lin and Yang, 1989).

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J

Fig. 6. LHRH immunoreactive neurons in human brain. Large dots symbolize cell bodies; small dots represent fibres. Abbreviation! see legend of Fig. 1 . (Reproduced from Najimi et al., 1990, with permission.)

179

Our findings of a high density of TRH binding sites in the human preoptic area could be considered an anatomical support of such a control, in adults as well as infants. No autoradiographic labeling was observed in the median eminence, so that the occurrence of a local action in this structure could not be confirmed. The terminal TRH fibres seen around vessels in that structure in animals would therefore support the hypothesis of a hypophysiotropic action of this peptide. Immunohistochemical distribution of LHRH neurons

Anatomical distribution This study was carried out on five infant hypothalami (ages of subjects: 26 days, 30 days, 40 days, 60 days and 9 months; three males and two females). The highest density of cell bodies is found in the mediobasal hypothalamus, principally in the infundibular nucleus. The preoptic area has the second highest population. In these two structures, the majority of immunoreactive cell bodies are densely marked, large and bipolar. Concerning fibres, three main structures can be distinguished by their density and the distribution: the lamina terminalis, the periventricular and paraventricular nuclei. The median eminence has the highest density of fibres, with a preferential distribution around vessels. The posterior hypothalamus has the lowest density of fibres (see Fig. 6).

Comparison with human data published Our findings are in general agreement with those reported in the literature (Barry, 1976, 1977, 1978; Bugnon et al., 1977a; King and Anthony, 1984). However, these studies showed high densities in the supraoptic nucleus, in contrast with the low density of LHRH immunoreactive neurons which we found. This discrepancy could be explained by the difference in antibodies and by differences in anatomical definitions. The overall distribution of LHRH immunoreac-

tivity is in good agreement with that described in adults and fetuses. However, King and Anthony (1984) do not report a high density of LHRH innervation in the preoptic region and only a low density of fibres in ,adults. In the median preoptic area, we observed a moderate labeling of cell bodies whereas high concentrations were found in the fetus (Bugnon et al., 1977a). In the anterior part of the hypothalamus, LHRH innervates the following structures: lateral preoptic area, anterior hypothalamic nucleus, dorsal hypothalamic nucleus, supraoptic nucleus and suprachiasmatic nucleus. We have also for the first time observed dense aggregations of LHRH fibres and rare cell bodies in periventricular and paraventricular nuclei.

Functional implications The presence of LHRH immunoreactive fibres around thickenings of third ventricle walls may be of great interest. One possibility is that LHRH is released into the cerebroventricular fluid and transported, probably by specialized ependymocytes, towards the median eminence and portal circulation, thus leading to an elevation of plasma LH levels (Knigge et al., 1978). There are several indications for this: the presence of LHRH in the cerebrospinal fluid (Joseph et al., 1975), the absorption by tanycytes of LHRH injected in ventricles inducing an elevation of plasma LH (Uemura et al., 1975). The discrepancies between our results in infants and those published by others in adults (Barry, 1976; King and Anthony, 1984) could be explained by cell death, which is known as one of the major characteristics of normal development (Parnavelas and Cavanagh, 1988). Immunohistochemical and autoradiographic studies have shown that, in the occipital cortex of rats, there is an overproduction of SRIF neurons with a maximum at 2 weeks after birth; immediately after that, a decrease is observed, suggesting neuronal death (Parnavelas and Cavanagh, 1988). Thus, this peptide could be produced “in surplus” and have a role in the establishment of a given con-

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nection, and later disappear. It should be noticed, however, that arguments in favour of atrophic role of this peptide become more and more obvious (Bulloch, 1987; Palacios et al., 1988; Parnavelas and Cavanagh, 1988). Cell death after the neonatal period might indeed also occur in the human hypothalamus. As already mentioned, we have shown the presence of immunoreactive neurons in the suprachiasmatic nucleus. It is well established that this nucleus plays a major role in the control of circadian rhythms (Brown-Grant and Raisman, 1977; Raisman and Brown-Grant, 1977; Moore, 1979; Hery et al., 1982). Our observations may have important functional implications. Thus, in rat, the electrical stimulation of the suprachiasmatic nucleus increases the release of LHRH and LH (Maxwell and Fink, 1988) in the portal circulation (Chiappa et al., 1977). Immunohistochemical studies have revealed the presence of LHRH neurons in the suprachiasmatic nucleus which project towards the median eminence by pathways running dorsally and ventrally to the optic chiasm (Rethelyi et al., 1981). These observations would be in favour of the implication of the suprachiasmatic nucleus in the “generation” of the circadian rhythm responsible for the release of luteinizing hormone. It would be of interest to follow the fate of LHRH neurons in the suprachiasmatic nucleus in diseases such as Alzheimer’s disease and Prader-Willi syndrome (Swaab et al., 1987). The cell bodies present in the preoptic area may project towards the median eminence via the infundibular nucleus. Thus, tracing studies have revealed direct connections between the preoptic area and the mediobasal hypothalamus (Conrad and Pfaff, 1976; Fink and Jamieson, 1976)on the one hand and the increase of metabolic activity of the infundibular nucleus after electrical stimulation of the preoptic area on the other hand (Maxwell and Fink, 1988). Our observations of the presence of LHRH immunoreactive cell bodies in the preoptic region and of LHRH immunoreactive fibres in the infundibular nucleus and median eminence confirm the existence of such a projection in infants.

Comparison of the distribution of LHRH immunoreactive elements in the left and right hypothalamus did not reveal differences in their nature for their density. These results are in good agreement with radioimmunological microchemical studies of Borson-Chazot et al. (1986), which did not show lateralization on the concentration of LHRH in the human adult hypothalamus. Distribution of delta sleep inducing peptide (DSIP) This unpublished study revealed a great similarity in the patterns of distribution of DSIP immunoreactive neurons and that of LHRH (Fig. 7). It has been shown that, in the rabbit, DSIP and LHRH are colocalized in the same neuronal population (Charnay et al., 1989). Electron microscopic studies in the rat median eminence demonstrated that both DSIP and LHRH immunoreactivities are present within single granules of secretion (Vallet et al., 1991). Studies on sudden infant death syndrome Several convergent data presently support the hypothesis that immaturity of brain areas is responsible for the control of vegetative functions (respiration, heart and blood pressure, swallowing,

Fig. 7. Distribution of delta sleep inducing peptide immunoreac. tive neurons in the human hypothalamus. Abbreviations: set legend of Fig. 1.

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temperature, sleep . . .). The areas regulating these processes are mainly confined to the spinal cord, brain-stem and hypothalamus. Possible thermoregulation and respiratory control disorders in SIDS prompted us to look at the hypothalamus and lower brain-stem; we studied SRIF immunoreactive neurons since SRIF is known to stimulate or inhibit respiration. We therefore investigated the topographical distribution of SRIF immunoreactive neurons as well as the distribution of LHRH immunoreactive neurons in the hypothalamus of four cases of SIDS of 1 month, 1 month, 1.5 months and 3 months of age (with a post-mortem delay of 6, 7, 8 and 10 h, respectively). The controls were 1 month, 1.5 months, 2 months and 5 months (post-mortem delay: 4,6,8 and 10 h, respectively). The causes of death were, respectively: pulmonary hypoplasia in a case of operated diaphragmatic hernia, staphylococcus acute sepsis, perfusion (therapeutic) error, congenital cardiomyopathy. In both series, there are one female and three males. The general autopsy includes gross examination and a protocol of 24 tissue samples from lung, myocardium, heart septum, thymus, oesophagus, stomach, liver, adrenal gland, kidney, pancreas, rib. No significant lesion was found in SIDS cases, which were therefore diagnosed as real unexplained SIDS. We found no difference for the SRIF distribution study between controls and SIDS cases. With LHRH immunohistochemistry, the same general distribution was found in the hypothalamus of controls and SIDS cases. However, in the periventricular and paraventricular nuclei, a dramatic difference appeared in the number of LHRH immunoreactive fibres, which was greatly decreased in the four SIDS cases (Fig. 8). This diminution of the number of fibres of LHRH immunoreactive neurons, in four cases of SIDS as compared with four matched controls, has to be considered with cautiousness until artefacts or other causes unrelated to the syndrome can be definitely discarded. However, this deficiency in LHRH fibres cannot be clearly explained by a post-mortem artefact since

Fig. 8. LHRH immunoreactive fibres in human infant hypothalamic paraventricular nucleus. Upper part: control infant; lower part: case of sudden infant death syndrome. (Reproduced from Najimi et al., 1989b, with permission.)

SIDS cases and controls were well matched for postmortem delay. Sex is probably not responsible for our results either since there were males and females in the two series. This deficiency could thus reflect a real diminution of the number of fibres in periventricular and paraventricular nuclei. However, it could also reflect a depletion of LHRH by which fibres could not be visualized any longer. A nycthemeral factor should also be taken into consideration since in SIDS cases death occurred at night, whereas it occurred during the day for the matched controls. Clearly the present data need to be confirmed by further investigations. It should be mentioned here that previous work from our laboratory has shown changes in SIDS in other neuroregulator systems:

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absence of catecholaminergic neurons in the subnucleus gelatinosus of the nucleus tractus solitarius and increase of the number of neurotensin binding sites in the nucleus tractus solitarius (Denoroy et al., 1987; Chigr et al., 1989b, 1991). If the present finding of a decrease of LHRH immunoreactive fibres in the mediobasal part of the hypothalamus in SIDS is confirmed, the functional significance will justify further investigation and reflection. Presently we can only speculate on a possible implication of a deficiency in LHRH fibres in the anterior part of hypothalamus. The LHRH fibres of the periventricular and paraventricular nuclei are known in animals to originate from cell bodies localized in the anterior hypothalamus, an area known to be implicated in thermoregulation and thermoregulation disorders have been mentioned in infants at risk of SIDS. However, we are fully aware of the fragility of this hypothesis. Since DSIP and LHRH are colocalized in the same neurons, one could take also less DSIP in SIDS as an argument in favour of the reality of our finding since DSIP has been implicated in sleep mechanisms and thermoregulation (as well as in other mechanisms) and sleep anomalies are well documented in infants at risk of SIDS (Challamel et al., 1981; Harper et al., 1983). However, this is pure speculation since DSIP was not studied in our SIDS cases. Furthermore, one must notice here that the anterior hypothalamus is strongly implicated in the control sleep-wakefulness states (Sakai et al., 1990). The present findings are to be considered as preliminary data and should encourage investigators in neuroscience and neuroendocrinology to become involved in SIDS research. Summary and conclusions The brains of mammals are not mature at birth, in particular in humans. Growth and brain development are influenced by the hormonal state in which the hypothalamus plays the major regulatory role. The maturation of the hormonal patterns leads to the physiological establishment of chronological variations as revealed by the circadian variations of

both hypothalamic peptides and pituitary hormones (as illustrated for hypothalamic-pituitary-thyroid axis by the determination of thyro-stimulating hormone (TSH) and thyrotropin-releasing hormone (TRH) circadian rhythms in the rat (Jordan et al., 1989)). It has been established that hypothalamic peptide variations are regulated by hormonal feed-back and amine systems, with the maturation of the latter also being dependent upon the whole functional maturation of the brain. Though these systems have been studied in the rat, very little information is currently available with regard to the human brain. The only biochemical or immunohistochemical information published to date concerns either the fetus or the adult. We have studied four main peptidergic systems (somatostatin-releasing inhibiting factor (SRIF), thyrotropin-releasing hormone (TRH), luteinizing hormone-releasing hormone (LHRH) and delta sleep inducing peptide (DSIP) in post-mortem adults and infants and in sudden infant death syndrome (SIDS) brains either by autoradiography and/or immunochemistry of radioimmunology. From a technical point of view, human brain studies display certain pitfalls not present in animal studies. These may be divided into two subclasses: ante- and post-mortem. Ante-mortem problems concern mainly sex, laterality, nutritional and treatment patterns while post-mortem problems concern post-mortem delay and conditions before autopsy and hypothalamic dissection. This might induce dramatic changes in morphological, immunochemical and autoradiographic evaluations. The matching of pathological subjects with controls is particularly difficult in the case of SIDS because of the rapid changes which take place in physiological regulatory processes during the first year of life. Thus, the treatment of hypothalamic tissue samples both for immunochemistry, radioimmunology and autoradiographic studies required techniques which must be rigorously controlled. For example, SRIF studies were carried out with three different antibodies, which gave similar results. The use of different technical procedures as well as dif-

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ferent antibodies is discussed. These types of differences might explain, at least in part, the discrepancy observed until now. As previously described in the fetus (Bugnon et al., 1977b; Bouras et al., 1987), we confirmed that in the infant hypothalamic SRIF immunoreactive cell bodies are present in the paraventricular and suprachiasmatic nuclei and in the periventricular area. We also showed the presence of a higher density of cell bodies in the infundibular and posterior nuclei as compared with adults. In contrast to results obtained in rat (Roberts et al., 1982), we did not detect SRIF fibres in the fornix of either infants or adults. The distribution of SRIF binding sites was similar in infants and adults. However, two major specific regional differences between infants and adults were evident. A higher density of labeling was found in the posterior infundibular nucleus and the anterior hypothalamus of infant brain whereas the tuber nuclei were much more labeled in adults. It is noteworthy that 2 - 3 times as many SRIF binding sites were revealed by adding guanosine triphosphate (GTP) to the preincubation medium. A high density of immunoreactive cell bodies and binding sites for SRIF was found in the preoptic area. This area is known to be involved in thermoregulation and therefore this result is particularly important when considering SIDS pathology. TRH Previous radioimmunological determinations of the concentration of TRH in hypothalamic structures in our laboratory revealed a left prominence for the ventromedial, dorsal and paraventricular nuclei (Borson-Chazot et al., 1986) in adults. Our present results show no evidence of laterality in hypothalamic TRH binding sites in adults or infants. These comparative measurements in infants and adults give evidence of specific structural differences. First of all, the highest density of binding sites was found in the preoptic area of the infant. This density was similar to that found for SRIF; TRH is also known to be involved in thermoregula-

tion. A high density was also found in the diagonal band of Broca in infants while in contrast in adults, the density was higher in the tuber nuclei. The results that levels of both TRH and SRIF binding sites in the tuber nuclei are significantly lower in infants than in adults suggest that this structure has not achieved functional maturation at this stage in development. LHRH and DSIP Here, we report for the first time the presence of a dense aggregation of LHRH fibres in periventricular and paraventricular nuclei. These observations are of particular interest since similar studies performed in four SIDS pathology cases revealed a dramatic decrease in the number of LHRH immunoreactive fibres in these two hypothalamic nuclei. Confirmation of these results would support the hypothesis of a link between the control of thermoregulation and SIDS pathology since, at least in animals, these fibres originate in the anterior hypothalamus. Furthermore, DSIP and LHRH are colocalized in the same neurons (Charnay et al., 1989) and indeed we found, in the human brain, a similar immunohistochemical distribution for the two peptides. DSIP is known to be implicated in sleep mechanisms. Since sleep abnormalities are well documented in infants at risk of SIDS and since in almost all SIDS cases death occurred during the night, it might be of interest to further investigate the poFsible role of DSIP in this pathology. /

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A “.nowledgements Drs. J.A. Chayvialle, B. Bloch and D. Fellman generously donated antisera. V. Thivolle is thanked for secretarial work. This work was done with support from INSERM (grants nos. 876013 and 884009) from the DCpartement de Biologie Humaine, UniversitC Claude Bernard Lyon I and “FCderation Naitre et Vivre”. Brains were obtained from several French institutes: Laboratoire d’Anatomie Pathologique, Hbpital Edouard Herriot, Lyon; Centre Hospitalier

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Discussion D.F. Swaab: Firstly, we found it extremely difficult to find controls for the age in which SIDS children die. Can you tell us something about the cause of death of the 4 controls? N. Kopp: The cause of death of the 4 controls was: (1) pulmonary hypoplasia and diaphragmatic Bochdalek’s hernia operated. (2) acute sepsis (staphylococcus). (3) a wrong perfusion, an unfortunate therapeutic error. (4) congenital cardiopathy. D.F. Swaab: Secondly, are the fibres that terminate on the ventricular wall axons or dendrites? N. Kopp: I cannot answer definitely. I presume they are axons. J.D. Mikkelsen: There has recently been raised some evidence that the pineal gland is dysfunctioning in patients with SIDS (Wurtman et al., 1991). Further, melatonin which is the active hormone secreted from the pinealocytes, inhibits LHRHinduced LH-release from the anterior pituitary (Martin and Klein, 1976). Do you have any evidence from your material or from experimental studies that changes in the distribution of LHRH-containing neurons in SIDS patients are a result from pineal dysfunction, or any other defect of the circadian timing system? N. Kopp: First a remark: it has been shown by Sparks and Hunsacker (1988) that the pineal gland is too small in infant victims of SIDS. However, to my knowledge, in infants that are alive and at risk of SIDS no data are available allowing to say that pineal gland is dysfunctioning. In answer to your question: no, I do not have such evidence. But it is known that some pineal tumors induce precautious puberty. M. Mirmiran: Although the cause of death in sudden infant death syndrome(S1DS) is not yet well-defined, it is suggested that central cardio-respiratory control failure plays a role. Since you have found a decrease of LHRH fibres in the hypothalamus of SIDS cases, do you know of any evidence indicating an involvement of LHRH in cardio-respiratory function control? N. Kopp: Not at all. R. Ravid: Was there an effect of the respiratory stress in the control cases on the brain as compared to the SIDS cases? Are the agonal states comparable? N. Kopp: This is a very difficult question to answer. The four

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SIDS cases were totally inexplicable. There was no pulmonary and/or hypoxic lesion. Of the four controls two had a hypoxia type of disorder (cases 1 and 4). J.B. Martin: Do you have a link between lateral tuberal nucleus and hyperfecundity of Huntington? N. Kopp: LHRH fibres are indeed present (in infant) in the lateral tuberal nucleus. F.W. Van Leeuwen: First of all, I was wondering what your rationale is for studying the distribution of LHRH/somatostatin and delta sleep inducing peptide? Secondly, I would like to know the follow-up of your studies. Wouldn’t it be better to choose a molecular biological strategy (based upon mRNA comparison) of SIDS and control ones? N. Kopp: To answer your first question: LHRH and DSIP are colocalized. DSIP is implicated in sleep induction and thermogenesis, both of which are implicated in infants at risk of SIDS who die during sleep in 90- 95% of the cases. There seems to be a problem, in fact, in the wakefulness reaction during sleep. Ap-

proximately 10% of infants at risk have thermoregulation problems. With respect to your second question: you are undoubtedly right. Immunocytochemical and binding sites approaches are to be considered as screening approaches. In situ hybridization is the next step.

References Martin, J.E. and Klein, D.C. (1976) Melatonin inhibition of the neonatal pituitary response to luteinizing hormone releasing factor. Science, 191: 301 - 302. Sparks, D.L. and Hunsacker, J.C. (1988) The pineal gland in sudden infant death syndrome: preliminary observations. J. PinealRes., 5: 111-118. Wurtman, R.J., Lynch, H.J. and Sturner, W.Q. (1991) Melatonin in humans: possible involvement in SIDS, and use in contraceptives. Adv. Pineal Res., 5: 319- 327.

D.F. Swaab, M.A. Hofman, M. Mirmiran, R. Ravid and F.W. van Leeuwen (Eds.) Progress in Brain Research, Vol. 93 0 1992 Elsevier Science Publishers B.V. All rights reserved.

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CHAPTER 13

LHRH neurons: functions and development Marlene Schwanzel-Fukuda, Li-Mou Zheng, Hugo Bergen, Gary Weesner and Donald W. Pfaff The Rockefeller University New York, New York 10021, U.S.A.

Introduction Because of the recent discovery that the neurons producing luteinizing hormone-releasing hormone (LHRH, GnRH) are born not in the brain itself, but on the medial side of the olfactory placode (Schwanzel-Fukuda and Pfaff, 1989), the question has naturally arisen “why are LHRH neurons born in the nose?”. An equally valid question is “why must LHRH-producing cells reside as neurons in the brain?”. The first question we can only speculate about at the present time. However, the second question can be answered with confidence in the context of the application of modern molecular techniques to neurobiology. That is, using the techniques of molecular biology, especially with application to neuroendocrine systems, we have shown robust, significant steroid hormone effects on specific mRNA levels in specific regions of the brain (for example, Romano et al., 1988, 1989, 1990; Lauber et al., 1990; Chunget al., 1991). Avariety of findings submit to close reasoning about the mechanisms by which individual reproductive behaviors are organized (Pfaff, 1989). Nevertheless, when comparing brain responses to steroid hormones with responses in other target organs, we quickly ask: what exactly is different about the brain - what features are added by central nervous system mechanisms. Theanswer is clear. What’s new about brain mechanisms, compared to other hormone response systems, is the intricate, highly regulated cell-cell connectivity. We take the view that the wealth of

neuronal connectivity adds necessary regulatory features to neuroendocrine systems which are hormone-responsive. For example, in the case of managing reproduction, not only must hormonal preparations of the reproductive organs be adequate, but also there must be adequate food, adequate water, appropriate environmental temperature, appropriate light rhythms (as might signal the proper mating season), nesting material, an absence of stress and danger, and the presence of an appropriate mating partner. How does the animal in question know that all of these necessary features are in place? For the neurons which control reproduction, LHRH neurons, synaptic inputs influencing neuropeptide synthesis and release must carry the brunt of the load. An extreme view would claim that the combinatorial requirements for transcription factors in the initiation of LHRH mRNA synthesis would comprise part of the mechanism by which these various signals about the opportunities for reproduction could have their effects. The purposes of this chapter are, first, to place in physiological perspective some of the roles of LHRH neurons; second, to review the evidence that they are actually born in the olfactory placode and migrate into the brain; and, third, to show the implication of this LHRH neuronal migration for the genesis of a human disease, Kallmann’s syndrome. Normal controls on adult LHRH neurons, in perspective Participating in neuroendocrine controls, as they

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do, LHRH neurons are expected to be under hormonal control. However, as we view the effects of steroid hormones in the brain, these controls on LHRH neurons are neither (a) direct nor (b) unique. With respect to the first point, the initial claim by Shivers et al. (1983) that the great majority of LHRH neurons in the rat brain lack nuclear estrogen receptors, has been confirmed, and extended as well to the demonstration of the absence of nuclear progesterone receptors in LHRH producing cells (Fox et al., 1990).

Effects of steroid hormones on cellular metabolism of brain cells With respect to the second point, effects of steroid hormones on specific neuropeptides represent only part of a coordinated series of actions by which nervous system physiology is altered according to endocrine state. First, effects of sex hormones on processes involved in general aspects of cellular metabolism, often related to cell growth, seem to set the stage for other more specific actions. For example, there is the effect of estrogen on ribosomal RNA synthesis (Jones et al., 1986, 1990), an increase detected by in situ hybridization using probes directed against rat ribosomal RNA. There are also ultrastructural effects of estradiol on rat ventromedial hypothalamic neurons which look like general, growth-related processes: effects on the nucleolus, on nucleolar-associated DNA, the size of the nucleus, an increase in a rough endoplasmic reticulum, and an increase in the number of densecore vesicles, which in turn appear to show up as synaptic vesicles in the midbrain (Cohen and Pfaff, 1981; Cohen et al., 1984; Chung et al., 1984, 1988; Jones et al., 1985; reviewed in Cohen and Pfaff, 1991). This phase of general, preparative actions may set the stage for the synthesis of specific neuropeptides, such as LHRH. Finally, effects of estradiol on heat shock proteins could have implications for new protein trafficking, as has been proposed for heat shock 70 (Mobbs et al., 1989; Olazabal et al., 1991a,b).

Effects of steroid hormones on LHRH neurons In this context, what are the hormonal effects on LHRH, indirect as they are, and what are the physiological roles of those effects? Four groups have now shown that when estrogens are used in their positive feedback mode, such as would be used to stimulate an ovulatory LH surge, estrogen treatment can actually increase LHRH mRNA (Roberts et al., 1989; Rothfeld et al., 1989; Park et al., 1990; Rosie et al., 1990). Under these conditions, the effects of estrogen on LHRH mRNA also would be consistent with its behavioral action. According to these views, a primary role of hormones affecting LHRH neurons would be to support neural control over the ovulatory LH surge and mating behavior. The latter claim rests on the discovery (Moss and McCann 1973; Pfaff, 1973) that LHRH administration can promote female reproductive behavior. Electrophysiologically, we have little evidence that LHRH has this effect through a classical neurotransmitter-like action, but instead it appears to act in the capacity of a neuromodulator (Pan et al., 1986; Ogawa et al., 1991). Biologically, would the increased synthesis and release of LHRH simply add arithmetically to other behaviorally-relevant neuropeptides or neurotransmitters to make the rest of the reproductive behavior circuit work (Pfaff, 1980), or is some more particular role to be envisioned? We take the view that a specific biological role for LHRH action on reproductive behavior is to synchronize behavioral events with peripheral preparations f o r reproduction. In this role, LHRH, as would be the case with any other neuropeptide, must act at normal release sites, where there are LHRH receptors. Under these circumstances, how can the experimentally applied decapeptide have a behavioral effect not already executed by endogenous LHRH? The answer must be that experimentally-delivered LHRH would have its greatest effects when endogenous release is low - that is, that the exogenous LHRH compensates for low endogenous levels under circumstances where more is required for an optimal behavioral effect. An

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analogy can be constructed to the effects of oxytocin on copulatory and maternal behaviors (McCarthy et al., 1991). With oxytocin, the greatest effects are constrained to specific environmental circumstances which may be characterized by conditions of mild stress. Under these circumstances oxytocin may act as an anti-anxiety compound which allows normal, adaptive behavioral responses that otherwise might be blocked because of stress-induced opioid peptide effects. Likewise, LHRH should not necessarily be considered as a monolithic drive on reproductive behavior, but instead as a specific, biologically adaptive synchronizing signal, neuromodulating specific groups of forebrain and midbrain neurons at specific times. Effectsof transmitter inputs on LHRHmRNA. If a valid reason for LHRH-producing cells to be located in the brain derives from the necessity for them to receive biologically-relevant environmental signals, then neurotransmitter and neuropeptide inputs to LHRH neurons in the preoptic area should be important not only for controlling LHRH release, but perhaps, even, for affecting LHRH gene expression. One of the strongest effects of a neurotransmitter on the LH surge is that of GABA. Bergen et al. (1991)tested the hypothesis that GABA actions in the preoptic area could alter LHRH mRNA levels as determined by quantitative in situ hybridization technique. The pattern of results obtained with GABAA agonists and antagonists acting on LHRH cells indicated that GABA, acting through this receptor subtype, would have to be effective primarily by electrophysiological means. However, this neurotransmitter operating through GABA, receptors might, indeed, reduce LHRH mRNA levels consistent with GABA actions on the ovulatory surge. Alpha adrenergic inputs can, under well chosen hormonal conditions, foster the LH surge. Might some of these actions be through effects on LHRH mRNA? Weesner et al. (1991a) used quantitative in situ hybridization to demonstrate that, while an a-1 agonist was not able to increase LHRH mRNA in the ovariectomized rat, a specific a-1 antagonist was

able to reduce LHRH mRNA. In the same experiment, there was no effect on galanin mRNA. The results were consistent with the view that, in the ovariectomized female rat, LHRH gene transcription is proceeding at a high rate (and the a-1agonist alone cannot further increase it); and this high rate is supported, to a marked extent, by endogenous norepinephrine acting through a-1 receptors. In fact estrogen, used in its positive feedback mode, was not only able to stimulate the production of LHRH mRNA, but also, both the LH surge produced and the increase in LHRH mRNA depended on adrenergic a-1 activity, since prazosin was able to block both events (Weesner et al., 1991b). With these types of experiments on LHRH gene expression, as well as other work on LHRH release, the transduction of physiological signals which modify the central control of reproduction can be analyzed. LHRH neuronal migration during development Origin in the medial olfactory pit Investigation into the origin of LHRH neurons in mice, using immunocytochemical procedures, led to the discovery that LHRH neurons originate in the epithelium of the medial olfactory pit and migrate into the brain along branches of the terminalis and vomeronasal nerves (Schwanzel-Fukuda and Pfaff, 1989). In mice, LHRH-immunoreactive neurons were first detected in the epithelium of the medial olfactory pit at about 11 days of gestation. By days 12 and 13 of embryonic life, cords of LHRHimmunoreactive cells were seen migrating across the nasal septum toward the forebrain. From days 16to 20 of gestation, an increase was seen in the number of LHRH-immunoreactive cells in and around the preoptic area of the mouse. Combined tritiated thymidine autoradiography and immunocytochemistry showed that isotope uptake into the cell nuclei of LHRH-immunoreactive neurons was greatest in mice whose mother was injected with the tritiated thymidine on day 10 of pregnancy, consistent with the report of Wray and co-workers (1989). No evidence of LHRH cell division was seen in the brain (Schwanzel-Fukuda and Pfaff, 1990).

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Ultrastructural observations of migrating LHRH neurons Examination of the ultrastructure of the migrating neurons from the epithelium of the olfactory pit (Zheng et al., 1989, 1992) showed two populations of cells with similar morphology, only one of which contained LHRH immunoreactivity. Before and during migration, no LHRH immunoreactivity was seen in Golgi apparatus or in neurosecretory granules. In fact, in the olfactory placode, a large amount of immunoreactive material was seen surrounding the nuclear envelope. The ultrastructural picture was consistent with the notion that these neurons do not have a secretory function before they attain their target sites. Olfactory pit-derived LHRH neurons in cell culture Cell culture of LHRH neurons, which would greatly facilitate studies of synthesis and secretion, has been hampered by the difficulty of obtaining sufficient numbers of these cells to grow in culture. In our laboratory, we have used the fact that tissues of the embryonic olfactory pit and the adjacent nasal mesenchyme offer a source of LHRH neurons for successful tissue culture (Jorgensen et al., 1988, 1991). The most successful cultures of LHRH neurons were those in which they contacted each other, as well as non-immunoreactive cells. The possibility to obtain enough embryonic LHRH neurons for cell culture opens a new field for experimental manipulation, and further confirms the olfactory pit origin of LHRH-expressing cells. Properties of neuroendocrine cells migrating from olfactory placode to basal forebrain Further experiments about the development of LHRH cells address the following questions: do these immunoreactive neurons actually express the LHRH gene when they are located in and near the olfactory epithelium; and is the migration specific to LHRH neurons, among neuroendocrine cells? Methods Tissue preparation.

Sixteen fetuses were taken

at embryonic ages E l l , E12, E13, and E l 6 from eight pregnant Swiss mice. Frozen, cryostat sections (8 - 12 pm) through the vomeronasal organ, nasal septum, and basal forebrain, were cut and mounted onto polylysine-treated slides. The tissue was fixed by immersion in 4% paraformaldehyde + 0.1 M PBS + 0.02% diethylyprocarbonate (DEP) for 5 min, rinsed with PBS (0.1 M, 3 min), and dehydrated in ethanols. Slides were then immersed in a solution of 0.25 Yo acetic anhydride containing 0.1 M triethanolamine (pH 8.0, 10 min) rinsed with 0.2 x SSC (10 min, 2 x SSC = 0.30 M sodium choride, 0.03 M sodium citrate in autoclaved, nanopure water), dehydrated in ethanols, and dried in a desiccator. Sections were stored at - 70°C foi 1 month before use.

Protocol. The in situ hybridization method usec was essentially the same as that recently described bj Gibbs et al. (1989). Sections were thawed in a desic cator and incubated for 3-4 h with 40 pl of i prehybridization buffer (McCabe et al., 1986). Tht prehybrydization solution was then removed anc 40 p1 of heat-denatured hybridization buffer (Mc Cabe et al., 1986) containing a [3H]-labeledsense 01 antisense ribonucleotide probe (2 x lo5 dpm/sec tion) was pipetted onto each section. The riboprobei were transcribed from a 400 bp RsaA fragment o the mouse LHRH gene (including all of exon 1 clonedintoatranscriptionvector withT7and T3pro moters arranged to produce riboprobes of botl orientations (J.F. Hejtmancik et al., in prepara tion). The sense probe was used as a negative contro since it is chemically similar to, has the same G( content, and can be manufactured to the same spe cific activity as the antisense probe, but does no hybridize to the LHRH message. Some sections re ceived hybridization buffer alone as anothe negative control. Hybridization continued for : days at 50°C in humidified, nalgene boxes. Following hybridization, sections were rinsec with2 X , 1 x ,and0.5 x SSC(lOmineach), treatec with ribonuclease A (2.5 pg/ml in 10 mM Tris, p€ 8.0,0.5 MNaCl, 1 mMEDTA, 37"C, 30min, rinsec with RNAse-free buffer (30 min, 37"C), rinse1

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again with SSC, and then incubated in 0.1 x SSC + 10 mM dithiothreitol at 37°C overnight. The next day, sections were rinsed for 1 min each in 300 mM ammonium acetate: ethanol 1:1, 3:7, 1:9, and finally in absolute ethanol. Sections were then dried in a desiccator, dipped in Kodak NTB3 emulsion (44°C) and stored dry in light-tight boxes at 4°C for 3 months. Autoradiograms were developed with Kodak D-19 developer diluted 1:1 with water (4 min, 15”C), fixed with Kodak fix (6 min) stained with cresyl violet, and examined with a Zeiss photomicroscope.

Immunocytochemistry. Immunocytochemical tests for specificity of the LHRH neuronal migration phenomenon were conducted with tissue from fetal mice, aged between days E l l and E13, with samplingto emphasizedates El2 and El3 because of the robust appearance of LHRH cells in the olfactory apparatus at those times. For tests of the effectiveness of the antiseraused, in tissues other than the olfactory placode, adult mouse brain, and 19-dayold rat brain tissues were employed, as well as, in some cases, newborn mouse brain and brain tissue from mice 5 days after birth. For all the antisera whose data are presented in the Results section, positive results were obtained in adult mouse brain. In addition, for the oxytocin and vasopressin antisera, preadsorption controls were run. For most of the experiments, tissues were fixed with Bouin’s solution and, in addition, for work with cryostat sections, fixation with 4% paraformaldehyde in 0. I M phosphate buffer was employed. The immunocytochemical protocol was the same as that previously reported (Schwanzel-Fukuda and Pfaff, 1989; Schwanzel-Fukuda et al., 1989) and is summarized here briefly: in 0.1 M phosphate buffer, tissue was washed three times, and then was exposed for 30 rnin to 0.5% H,02 in water, to eliminate any effect of endogenous peroxidase. After 30 rnin incubation in 2% normal goat serum to reduce background, the primary antibody was used in 0.01 M phosphate buffered saline containing 1‘Yo normal goat serum, and incubation proceeded for 2 days at 4°C. Tissue was washed three times in 0.1 M phos-

phate buffer, followed by a biotinylated secondary antibody and an avidin-biotin-horseradish peroxidase complex (the “ABC” Elite technique, Vector Laboratories). 3, 3 ’diaminobenzidine tetra hydrochloride (DAB, Sigma Chemical Co.) with 0.001 070 H202 in 0.05 M Tris buffer, was the chromagen. The following antisera were used with sections through the olfactory placode, in experiments for which positive controls staining in adult brain were also obtained: anti-corticotropin releasing hormone (Chemicon, Inc.); anti-somatostatin (Chemicon, Inc.); anti-oxytocin (Chemicon); anti-vasopressin (Chemicon); anti-neuropeptide Y (Cambridge Research Biochemicals). Two other antisera were also used, which did not stain any cells in the fetal mouse olfactory placode, but these are not reported below because we did not see corresponding positive control cells in adult mouse brain: these are anti-growth hormone releasing hormone (Chemicon, Inc.), and anti-thyrotropin releasing hormone (two antibodies, one from Dr. M. Suzuki, University of Gunma, Maebashi, Japan; and one from Arne11 Products, New York). Primary antisera were used in concentrations from 1:200 to 1:1000, except for anti-oxytocin and anti-vasopressin, which were used at concentrations from 1:500 to 1:lOOO.

Controls. In order to test the specificity of the various antisera, adjacent sections.were incubated in primary antiserum that had been absorbed with the specificantigen overnight. We routinely used 1Fg of antigen in 1ml of antiserum at the working dilution. The control sections showed no immunoreactivity. Results In situ hybridization. A view of LHRH neurons migrating from the olfactory pit toward the basal forebrain in the fetal mouse is provided in Fig. 1. Examples of LHRH mRNA-containing cells are shown in Fig. 2. Labeled cells were observed only in sections hybridized with the antisense probe. Thus, three lines of evidence show the specificity of hybridization: absence of well-labeled cells with the control, sense riboprobe; absence of labeled cells

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throughout most of the sections, in areas which are off the migration route; and very low numbers of grains in interstitial tissue between cell bodies. We conclude that these probes and hybridization protocols allow specific detection of LHRH mRNA. LHRH-gene expressing cells were detected as early as day E l l . At this time point, all labeled cells were restricted to the area of the medial olfactory pit. Strongly labeled cells were observed, some of which were located in the medial portion of the

epithelial layer while others were located adjacent to the basal layer of the epithelium. Labeled cells tended to be clustered. By day E12, the number of labeled cells had increased, and many were located bilaterally in the submucosa of the nasal septum. In sagittal sections, lines of labeled cells oriented from the vomeronasal organ toward the forebrain were observed. However, no labeled cells were observed in the forebrain at this age. At day E13, the greatest numbers of labeled cells

Fig. 1. lmmunoreactive LHRH neurons streaming from the olfactory pit across the nasal septum toward the basal forebrain in the fetal mouse. LHRH cells are dark, seen against a light counterstain for the other cells. (Data from Schwanzel-Fukuda and Pfaff, 1989.)

Fig. 2. In situ hybridization demonstration that the LHRH gene is actually expressed in these neurons which migrate from olfactor! pit to the brain. LHRH mRNA was detected using a tritiated probe and a protocol described in the text. Left panel: four clusters o LHRH mRNA expressing cells are seen outside the fetal mouse olfactory placode, as detected by the experimental “antisense” probe Right panel: with the control “sense” probe, no labeled cells were seen. Fig. 3. In the very experiments in which the specificity of LHRH neuronal migration was tested, the LHRH cells themselves were con firmed. Here, the dark brown cells are LHRH-immunoreactive neurons outside the fetal mouse olfactory placode as they head for tht basal forebrain (toward the top of the photograph).

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were observed, many of which were located in the submucosa of the nasal septum, and some of which were found in the forebrain. A large group of labeled cells was located along the medioventral surface of the developing olfactory bulb. Cells located in the nasal septum and on the olfactory bulb were always tightly clustered into groups (Fig. 2).

Zmmunocytochemistry. In this material, as a positive control, the presence of LHRH-expressing immunoreactive neurons in and adjacent to the fetal mouse olfactory pit was confirmed (Fig. 3). Experiments looking for immunoreactivity for other neuroendocrine peptides were run on slides neighboring these positive LHRH slides. None of the antisera used, other than LHRH, revealed immunoreactive neurons in the olfactory pit. For antisera to corticotropin releasing hormone, somatostatin, oxytocin, vasopressin, and neuropeptide Y, positive immunoreactivity in the adult mouse brain was obtained using protocols which failed to show cells in the olfactory placode (as illustrated by Fig. 4 for somatostatin, and Fig. 5 for neuropeptide Y). Thus under the conditions tested, for neuroendocrine peptides of interest, so far, LHRH neurons appear to be specific in making this migration from olfactory pit to brain. Discussion These results contain clear evidence that LHRH immunoreactivity in the epithelium of the olfactory pit is not simply the result of the uptake of immunoreactive material produced in another organ, nor is it due to cross reactivity of our antisera. The

in situ hybridization data demonstrating LHRH messenger RNA are specific, based on four arguments: first, the absence of labeled cells with the control, sense riboprobe; second, the absence of labeled cells in widespread regions of these tissue sections, off the migration route; third, the very small number of grains distributed between cells. Finally, the locations and apparent movement of LHRH gene expressing cells as detected by in situ hybridizationmatch precisely theobservations based on immunocytochemistry (Schwanzel-Fukuda and Pfaff, 1989). At the earliest time points when LHRH mRNA could be detected, cells were in the region of the olfactory placode and, during the next 5 days the numbers of labeled cells in the nasal septum increased and then decreased, consistent with the migration route previously proposed (Schwanzel-Fukuda and Pfaff, 1989). LHRH messenger RNA expressing cells tended to be tightly clustered. The mechanism for this is unknown, but it, also, matches the immunocytochemical data and has also been observed in tissue culture of LHRH cells taken from the olfactory placode and nasal septum (Jorgensen et al., 1988). Perhaps the association of LHRH neurons with nerve cell adhesion molecule (NCAM) during migration is related to the clustering (Schwanzel-Fukuda et al., 1990, 1991a). No other immunoreactive cells expressing neuropeptides of neuroendocrine interest have been seen, so far, migrating from olfactory placode into brain. Under the conditions tested, therefore, LHRH neurons appear to have considerable specificity in demonstrating this particular life history. Why this should be the case is currently an unanswered ques-

Fig. 4. Immunocytochemicalevidence that somatostatin neurons do not migrate from olfactory placode to brain, in the mouse. Left panel: section through the fetal mouse olfactory apparatus does not show somatostatin-immunoreactivecells. Note that small brown cells are nucleated red blood cells characteristicof the fetus. Right panel: positive control for the somatostatin immunocytochemical procedure. The antibody and protocol used for the fetal mouse experiments showed positive immunoreactive cells in adult mouse hypothalamus. Fig. 5. Immunocytochemicalevidence that neuropeptideY neurons do not migrate from olfactory placode to brain, in the mouse. Left panel: section through the fetal mouse olfactory apparatus does not show neuropeptide Y immunoreactive cells. The darker stained cells toward the upper right are nucleated red blood cells which were not washed out during fixation. Right panel: positive control for the neuropeptideY immunocytochemicalprocedure. The antibody and protocol used for the fetal mouse experiments showed positive immunoreactivecells in adult mouse hypothalamus.

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tion. From the position of the olfactory placode in the developing fetal mouse head (Jacobson, 1979), we cannot rule out the hypothesis that LHRH neurons are of neural crest origin. This possibility is currently being addressed experimentally. Biologically, it is clear that chemosensory influences on reproduction of lower vertebrates which are aquatic might have required olfactory involvement in LHRH neuronal control, notably through the nervus terminalis (Demski, 1987, 1991; Demski et al., 1987). Likewise, in such animals, the physical condition of the nasal epithelium may help to signal environmental influences important for reproduction. Not only environmental temperature, but also hydrostatic pressure and salinity, for aquatic animals, could be signaled through nervus terminalis. In evolutionary terms, it might be noteworthy that the hypophyseal placode is near the developing olfactory piacode (Jacobson, 1979). Thus, in very early biologic forms, it may have been that LHRH cells could control developing gonadotropes by a paracrine mechanism. Finally, failure of the apparent migration of LHRH-expressing neurons can explain Kallmann’ssyndrome, an inherited hypogonadal disorder associated with anosmia (Schwanzel-Fukuda et al., 1989). While in normal human fetal tissue LHRH immunoreactive cells and axons were found in their expected distributions in the preoptic area and hypothalamus, the fetal Kallmann’s brain, itself, hadnone - instead, on the proposed migration route from the nasal septum, LHRH immunoreactive neurons had halted in the nose and beneath the bottom of the forebrain. This observation provokes further characterization of migrating LHRH neurons and the mechanisms underlying their movement into brain.

Cell adhesion molecules as part of the migration mechanism. In order to learn more about compounds which might guide the migration of LHRH neurons, we used immunocytochemical procedures with antisera to possible components of the migration route: nerve cell adhesion molecule (NCAM), cytotactin, fibronectin, laminin and CTB proteoglycan (Schwanzel-Fukuda et al., 1990, 1991a).

In the same experiments we did double-immunocytochemistry with antiserum for LHRH, in order to determine the relation between migrating LHRH neurons and these various cell adhesion and substrate molecules. The results showed that only NCAM, among the potential components tested, was routinely present along the migration course of LHRH neurons. NCAM is a cell surface glycoprotein produced by all nerve cells early in development. An integral membrane protein, it mediates cell-to-cell adhesion and is present on the cell body, the neurites and on the tips of growing axons. Soon after the olfactory placode invaginates to form the olfactory pit, we could see that “pioneer” NCAM-immunoreactive cells left theolfactorypit and aggregated near the tip of the developing forebrain. Cords of the NCAMimmunoreactive cells and axons appeared to mark the developing vomeronasal and terminalis nerves. When LHRH neurons began to appear, we noted that the same neuron never appears to express both LHRH and NCAM. However, as LHRH neurons migrate, we always see them associated with NCAM-immunoreactive fascicles. Later, the migration of LHRH neurons into the brain depended absolutely on the formation of a “bridge” of NCAMimmunoreactive cells and fibers which joined the olfactory apparatus to the tip of the forebrain. Dramatically, as LHRH neurons migrate across the nasal septum they can be seen with double immunocytochemistry studies, as passing through a scaffolding of NCAM-immunoreactive processes. This association of LHRH cells and NCAM was specific; we saw it with none of the other antibodies used (Schwanzel-Fukuda et al., 1990, 1991a).

PerturbationofLHRHneuronal migration with antiserum to NCAM. With these observations in hand, we wanted to find out if NCAM plays a causal role in LHRH cell migration. Would anti-NCAM disrupt migration? Swiss mice, on day 10 of pregnancy, were anesthetized and 1 p1 of antiNCAM was injected into the olfactory pit of each embryo. Control animals received 1p1 of rabbit IgG into the olfactory pit. Other animals served as un-

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treated controls. We found that if and only if the fetal mouse showed uptake of anti-NCAM into the epithelium of the olfactory pit, there would be few or no LHRH-immunoreactive cells along the migration route on the nasal septum and no LHRH cells in the brain (Schwanzel-Fukuda et al., 1991b). Untreated control animals, and the various surgical controls all showed anormal distribution of LHRHimmunoreactive migrating cells. We noted that our anti-NCAM manipulation was not brutal enough to destroy the NCAM scaffolding on the nasal septum, and also would not claim that this one-time manipulation would be powerful enough to destroy the passage of all LHRH cells at all times during development. Instead, an apparent retardation of migration was noted (Schwanzel-Fukuda et al., 1991b). There are undoubtedly complex forces and multiple chemical signals involved in the orchestration of LHRH cell migration from the nose into the forebrain. The results stated above indicate that NCAM is one important chemical feature of the migration route. Further, the ability of an olfactory placode manipulation to disturb migration extends our evidence for the initial hypothesis regarding the origin of LHRH neurons.

had the privilege of studying a human fetus with Kallmann’s syndrome (Schwanzel-Fukuda et al., 1989). At 19 weeks of gestation, there were no detectable LHRH-expressing cells in any area of the brain including, notably, the preoptic area and the tuberal hypothalamic region. In contrast, dense clusters of LHRH and thick fascicles of LHRH fibers were seen in the nose. Thus, it appeared that the LHRH gene was being transcribed normally, but the cells expressing LHRH were not migrating from the nasal epithelium into the brain (SchwanzelFukuda et al., 1989). In the controls, normal fetal brains of the same age, LHRH-expressing cells were seen in the basal forebrain, the preoptic area, and hypothalamus as expected. In the Kallmann’s tissue, the blockage of cell movement such that LHRH neurons were stuck in the nose further confirms our hypothesis about the migration of LHRH cells from the olfactory placode into the brain. In addition, the absence of LHRH cells in the brain of the Kallmann fetus provides an explanation for the deficiency of LHRH in patients with Kallmann’s syndrome. Other abnormalities of the Kallmann forebrain are currently under study.

LHRH-expressing cells in Kallmann’s syndrome, a human hypogonadal disease

Summary and conclusions

Kallmann et al. (1944) studied familial patterns of the occurrence of anosmia associated with gonadal failure and ascribed a genetic basis for this disorder, which came to be known as Kallmann’s syndrome. The principal endocrine defect of this syndrome, hypogonadotropic hypogonadism, is the failure of secretion of LHRH, resulting in the underdevelopment of the pituitary gonadotropes and the consequent inability to synthesize and release LH and FSH. Since the anosmia of Kallmann’s syndrome is due to the lack of the development of the olfactory bulbs, which depends in turn on contact between the olfactory nerves and the anlage of the forebrain, we hypothesized that the various deficits of the Kallmann’s patient are related, and that they result from failure of migration from the olfactory placode. We

Examination of the properties of developing LHRH neurons, by in situ hybridization procedures or LHRH immunocytochemistry, showed that these cells (1) are unique among neuroendocrine cells in their origin from the epithelium of the medial olfactory pit, and (2) express LHRH mRNA. LHRH neurons, visualized by either method, tended to be clustered when seen along the migration route in the nasal mesenchyme. Neural cell adhesion molecule (NCAM) is present on the central processes of the olfactory, vomeronasal and terminalis nerves, which form the scaffold along which LHRH neurons migrate into the brain. Injection of a small amount (1 pl) of antiserum to NCAM into the olfactory pits of 10-day-old embryonic mice, while not sufficient to break up the NCAM scaffolding, appeared to decrease the number of LHRH-

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immunoreactive cells in the epithelium of the medial olfactory pit, and retarded their migration in the nasal mesenchyme. This suggest that NCAM is important for LHRH cell migration. Never found actually colocalized with LHRH in the same neurons, NCAM nevertheless may be required for the migration of LHRH-expressing cells.

References Bergen, H.T., Hejtmancik, J.F. and Pfaff, D.W. (1991) Effects of gamma-aminobutyric receptor agonists and antagonists on LHRH-synthesizing neurons as detected by immunocytochemistry and in situ hybridization. Exp. Brain Res., 87: 46-56. Chung, S.K.,Cohen, R.S.andPfaff, D.W. (1984)Ultrastructure and enzyme digestion of nucleoli and associated structures in hypothalamic nerve cells viewed in resinless sections. Biol. Cell., 51: 23 -34. Chung, S.K., Pfaff, D.W. and Cohen, R.S. (1988) Estrogeninduced alterations in synaptic morphology in the midbrain central gray. Exp. Brain Res., 69: 522 - 530. Chung, S.K., McCabe, J.T. andPfaff, D.W. (1991)Estrogeninfluences on oxytocin mRNA expression in preoptic and anterior hypothalamic regions studied by in situ hybridization. J. Comp. Neurol., 307: 281 -295. Cohen, R.S. and Pfaff, D.W. (1981) Ultrastructure of neurons in the ventromedial nucleus of the hypothalamus in ovariectomized rats with or without estrogen treatment. Cell Tissue Res., 217: 451 -470. Cohen, R.S. and Pfaff, D.W. (1991) Synthetic consequences of hormone actions on hypothalamic nerve cells. Prog. Neurobiol., in press. Cohen, R.S., Chung, S.K. and Pfaff, D.W. (1984) Alteration by estrogen of the nucleoli in nerve cells of the rat hypothalamus. Cell Tissue Res., 235: 485 - 489. Demski, L.S. (1987) Phylogeny of luteinizing hormone-releasing hormone systems in protochordates and vertebrates. In: The TerminalNerve (Nervus Terminalis):Structure, Function and Evolution - Ann. N. Y . Acad. Sci., 519: 1 - 14. Demski, L.S. (1991) Pathways for GnRH control of elasmobranch reproductive physiology and behavior. J. Exp. Zool., in press. Demski, L.S., Fields, R.D., Bullock, T.H., Schreibman, M.P. and Margolis-Nunno, H. (1987) The terminal nerve of sharks and rays. In: The TerminalNerve (Nervus Terminalis):Structure, Function and Evolution - Ann. N. Y. Acad. Sci., 519: 15 - 32. Fox, S., Harlan, R., Shivers, B. and Pfaff, D.W. (1990) Chemical characterization of neuroendocrine targets for progesterone in the female rat brain and pituitary. Neuruendocrinology, 5 1: 276 - 283.

Gibbs, R.B., McCabe, J.T., Buck, C.R., Chao, M.V. and Pfaff, D.W. (1989) Expression of NGF receptor in the rat forebrain detected with in situ hybridization and immunohistochemistry. Mol. Brain Res., 6: 275 - 287. Jacobson, M. (1979) DevelopmentalNeurobiology,2nd edition, Plenum, New York, NY. Jones, K. J., Pfaff, D.W. and McEwen, B.S. (1985) Early estrogen-induced nuclear changes in rat hypothalamic ventromedial neurons: an ultrastructural and morphometric analysis. J. Comp. Neurol., 239: 255 - 266. Jones, K. J., Chikaraishi, D.M., Harrington, C.A., McEwen, B.S. and Pfaff, D.W. (1986) Insitu hybridization detection of estradiol-induced changes in ribosomal RNA levels in rat brain. Mol. Brain Res., 1: 145 - 152. Jones, K.J., Harrington, C.A., Chikaraishi, D.M. and Pfaff, D.W. (199O)Steroidhormoneregulationofribosomal RNAin rat hypothalamus: early detection using in situ hybridization and precursor-product ribosomal DNA probes. J. Neurosci, 10 ( 5 ) : 1513-1521. Jorgensen, K.L., MacLeish, P.R. and Pfaff, D.W. (1988) Serumfree cell culture of post-natal rat hypothalamic neurons. Soc. Neurosci. Abstr., 14: 868. Jorgensen, K.L., Schwanzel-Fukuda, M. and Pfaff, D.W. (1991) Endocr. Rev., in press. Kallmann, F.J., Schoenfeld, W.A. and Barrera, S.E. (1944) The genetic aspects of primary eunuchoidism. Am. J. Ment. Defic., 48: 203 - 236. Lauber, A.H., Romano, G.J., Mobbs, C.V., Howells, R.D. and Pfaff, D.W. (1990) Estradiol induction of proenkephalin messenger RNA in hypothalamus: dose-response and relation to reproductive behavior in the female rat. Mol. Brain Res., 8: 47 - 54, McCabe, J.T., Morrell, J.I., hell, R., Schmale, H., Richter, D. and Pfaff, D.W. (1986) In situ hybridization to localize rRNA and mRNA in mammalian neurons. J. Histochem. Cytochem., 34: 45 - 50. McCarthy, M.M., Kow, L.-M. and Pfaff, D.W. (1991) Speculations concerning the physiological roles of central oxytocin in the stimulation of maternal behavior. Ann. N. Y. Acad. Sci., in press. Mobbs, C.V., O’Malley, K.L., Lauber, A. and Pfaff, D.W. (1 989) Constitutive heat-shock-70 mRNA in brain is primarily neuronal and is increased by estrogen in hypothalamus. Soc. Neurosci. Abstr., 15 (2): 1128. Moss, R.L. and McCann, S.M. (1973) Induction of mating behavior in rats by luteinizing hormone-releasing factor. Science, 181: 177- 179. Ogawa, S., Kow, L.-M. and Pfaff, D.W. (1991) In vitro electrophysiologic actions of lordosis-relevant neuropeptides on neurons in the rat midbrain central gray. Soc. Neurosci. Abstr., 17: 1061. Olazabal, U., Pfaff, D.W. and Mobbs, C.V. (1991a) Effects of estradiol on heat shock 90 immunoreactive protein in the brain and uterus of the rat. Mol. Cell. Endocrinol., in press.

20 I Olazabal, U., Kleopoulos, S., Lauber, A., Pfaff, D.W. and Mobbs, C.V. (1991b) Heat shock 70 immunoreactive proteins following estrogen treatment in the rat brain and uterus. (Submitted.) Pan, J.-T., Kow, L.-M., Kendall, D.A., Kaiser, E.T. and Pfaff, D.W. (1986) Electrophysiological test of an amphiphilic Bstructure in LHRH action. Mol. Cell. Endocrinol.. 48: 161 - 166. Park, 0.-K., Gugneja, S. and Mayo, K.E. (1990) Gonadotropinreleasing hormone gene expression during the rat estrous cycle: effects of pentobarbital and ovarian steroids. Endocrinology, 127 (1): 365 - 372. Pfaff, D.W. (1973) Luteinizing hormone-releasing factor potentiates lordosis behavior in hypophysectomized ovariectomized female rats. Science, 182: 1148- 1149. Pfaff, D.W. (1980) Estrogens and Brain Function: Neural Analysis of a Hormone-ControlledMammalian Reproductive Behavior, Springer, New York. Pfaff, D.W. (1989) Patterns of steroid hormone effects on electrical and molecular events in hypothalamic neurons. Mol. Neurobiol., 3: 135 - 154. Roberts, J.L., Dutlow, C.M., Jakubowski, M., Blum, M. and Millar, R.P. (1989) Estradiol stimulates preoptic area-anterior hypothalamic pro-GnRH-GAP gene expression in ovariectomized rats. Mol. Brain Res., 6: 127 - 134. Romano, G.J., Harlan, R.E., Shivers, B.D., Howells, R.D. and Pfaff, D.W. (1988) Estrogen increases proenkephalin messenger ribonucleic acid levels in the ventromedial hypothalamus of the rat. Mol. Endocrinol., 2: 1320- 1328. Romano,G.J.,Krust,A.andPfaff,D.W. (1989)Expressionand estrogen regulation of progesterone receptor mRNA in neurons of the mediobasal hypothalamus: an in situ hybridization study. Mol. Endocrinol., 3: 1295 - 1300. Romano, G.J., Mobbs, C.V., Lauber, A., Howells, R.D. and Pfaff, D.W. (1990) Differential regulation of proenkephalin gene expression by estrogen in the ventromedial hypothalamus of male and female rats: implications for the molecular basis of a sexually differentiated behavior. Brain Res., 536: 63 - 68. Rosie, R., Thomson, E. and Fink, G. (1990) Oestrogen positive feedback stimulates the synthesis of LHRH mRNA in neurones of the rostra1 diencephalon of the rat. J. Endocrinol., 124: 285 - 289. Rothfeld, J., Hejtmancik, J.F., Conn, P.M. and Pfaff, D.W. (1989) In situ hybridization for LHRH mRNA following estrogen treatment. Mol. Brain Res., 6: 121 - 125. Schwanzel-Fukuda, M. and Pfaff, D.W. (1989) Origin of luteirhing hormone-releasing hormone neurons. Nature, 338: 161 - 164. Schwanzel-Fukuda, M. and Pfaff, D.W. (1990) Combination of tritiated thymidine autoradiography and neuropeptide immunocytochemistry to determine birthdates and migration routes of luteinizing hormone-releasing hormone neurons. In: P.M. Conn (Ed.), Methods in Neuroscience, Vol. 3, Quantitative and Qualitative Microscopy, Acadamic Press, New

York. Schwanzel-Fukuda, M., Bick, D. and Pfaff, D.W. (1989) Luteinizing hormone-releasing hormone (LHRH)-expressing cells do not migrate normally in an inherited hypogonadal (Kallmann) syndrome. Mol. Brain Res., 6: 3 11 - 326. Schwanzel-Fukuda, M., Abraham, S., Crossin, K.L., Edelman, G.M. and Pfaff, D.W. (1990) Immunocytochemical demonstration of neural cell adhesion molecule (NCAM) along the migration route of luteinizing hormone-releasing hormone (LHRH) neurons in mice. SOC. Neurosci. Abstr., 16 (1): 398. Schwanzel-Fukuda, M., Abraham, S., Crossin, K.L., Edelman, G.M. andPfaff, D.W. (1991a) Immunocytochemical localization of neural cell adhesion molecule (NCAM) along the migration route of luteinizing hormone-releasing hormone (LHRH) neurons in mice. J. Comp. Neurol., 320: 1 18. Schwanzel-Fukuda, M., Abraham, S., Reinhard, G., Crossin, K.L., Edelman, G.M. and Pfaff, D.W. (1991b) Antibodies to neural cell adhesion molecule (NCAM) disrupt the migration of luteinizing hormone-releasing hormone (LHRH) neurons into the brain in mice. SOC.Neurosci. Abstr., 17: 427. Shivers, B., Harlan, R., Morrell, J. and Pfaff, D.W. (1983) Absence of oestradiol concentration in cell nuclei of LHRHimmunoreactive neurons. Nature, 304: 345 - 347. Weesner, G.D., Bergen, H.T. and Pfaff, D.W. (1991a) Alpha-] adrenergic regulation of luteinizing hormone-releasing hormone (LHRH) gene expression in the rat. Endocr. SOC. Abstr., in press. Weesner, G.D., Krey, L.C. and Pfaff, D.W. (1991b) Alpha-1 adrenergic regulation of estrogen-induced increases in luteinizing hormone-releasing hormone (LHRH) mRNA and release. SOC. Neurosci. Abstr.. 17: 1364. Wray, S., Grant, P. and Gainer, H. (1989) Evidence that cells expressing luteinizing hormone-releasing hormone mRNA in the mouse are derived from progenitor cells in the olfactory placode. Proc. Natl. Acad. Sci. U.S.A., 86: 8132-8136. Zheng, L.-M., Pfaff, D.W. and Schwanzel-Fukuda, M. (1989) Electron microscopic identification of luteinizing hormonereleasing hormone (LHRH) immunoreactivity in olfactory placode-derived neurons. SOC.Neurosci. Abstr., 15 (2): 1015. Zheng, L.-M., Pfaff, D.W. and Schwanzel-Fukuda, M. (1992) Electron microscopic identification of luteinizing hormonereleasing hormone (LHRH)-expressing cells in the medial olfactory placode and basal forebrain of mice during embryonic development. Neuroscience, 46: 407 - 418.

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Discussion E.J. van Zwieten: Have you any data about the luteinizing hormone releasing hormone (LHRH) cells/migration in animal models which show hypogonadism and a human syndrome such as the Prader-Willi syndrome? D.W. Pfaff: The classical hypogonadal mouse has an abnormal LHRH gene but no evidence of a migration failure. Evidently,

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from our immunocytochemical data, the Kallmann’s individual had a normal LHRH gene. J.B. Martin: Would you comment on the neurosensory epithelium in Kallmann’s syndrome? Is it normal or abnormal? As you know, the olfactory deficit in Kallmann’s syndrome patients varies considerably. D.W. Pfaff: We have no evidence that the actual olfactory epithelium, itself, is abnormal in Kallmann’s syndrome. However, we would like to characterize it better (beyond the LHRH finding) as well as to study the forebrain better in the material we have. D.F. Swaab: Did the controls of your Kallmann fetus not have LHRH neurons in the nose? D.W. Pfaff: Some, but not to the extent of the Kallmann’s case. With the “controls”, the impressive result was the appearance of the LHRH neurons in the preoptic area and tuberal hypothalamus. We are studying the nasal epithelium and basal forebrain of the normal tissue vs. Kallmann’s to discern the basis of the GnRH abnormalities in the syndrome. D.F. Swaab: Could the lower number of [3H]-thymidine grains over the nucleus of LHRH cells be due to dilution caused by another cell division after labeling? D.W. Pfaff: Yes. A very rapid final cell division in the LHRH neurons could account for the result. This needs further study as it might indicate an unusual cell cycle in these LHRH cells just before migration. L. Gooren: How is it possible that men with Kallman are born with a penis. For pre-natal male differentiation of the external genitaliaa certain amount of testosterone is required of which the production depends on the boy’s own LHRH-LH production. Pubertal children with Kallmann syndrome sometimes have fairly normal first stages of puberty; it seems that subsequently their LHRH-LH production declines (Spratt et al., 1986). How is your view on this observation? D.W. Pfaff: Both the LHRH neuronal migration problem and the associated endocrine deficits may be partial, yielding clinical symptoms in some situations but not others. J.J. Legros: What do you know about the presence of LHRH neurons in adult olfactory epithelium. Indeed your lecture makes me think about an old (_+40years) treatment of sexual impotency which consisted of electrical stimulation of nasal endothelium (“nasal stress”). D.W. Pfaff: In mice, LHRH-expressing cells are prominent in nervus terminalis but not in the olfactory epithelium itself. We would like to study the question in human material. In fish, Leo Demski (1987) showed that nervus terminalis electrical stimulation can lead to sperm release. Likewise, in fish, we have found LHRH neurons in the olfactory apparatus and nervus terminalis (Ishwar Singh et al., in preparation). J.J. Legros: Could you speculate on the common mechanism leading on the one hand to a defect in LHRH neuron migration and on the other hand to anosmia. D.W. Pfaff: It appears that the same migration failure which

blocks LHRH neurons applies to olfactory neurons, thus to a failure of induction of normal olfactory bulbs, thus to anosmia. F.W. Van Leeuwen: You showed that estrogen receptors are not present in LHRH cells of the rat. I wonder whether this is similar to the situation in the supraoptic nucleus of the rat in which no estrogen receptors are present, whereas in the guinea pig they are (Warembourg and Poulain, 1991). Is it possible that in other species estrogen receptors might be present in LHRH cells? D.W. Pfaff: The predominant dissociation between estrogen receptor (in the cell nucleus) and LHRH (in the cytoplasm) reported by Shivers et al. (1983) has been replicated in the rat and extended to mice, guinea pigs, monkeys and humans (see also the chapter by Rance). Also, progresterone receptor containing neurons do not express LHRH (Fox et al., 1990). F.W. Van Leeuwen: Are estrogen responsive elements present in the upstream region of the LHRH gene? D.W. Pfaff: There is not a consensus ERE and the effectiveness of estradiol in transfection assays remains controversial. Important estrogenic effects on LHRH mRNA are likely to be indirect: involving other cells and, perhaps, consequent to release. F.W. Van Leeuwen: You showed that a GABA, antagonist decreases the number of LHRH mRNA transcripts. Do you think that glia endowed with GABA, receptors can be involved as a chain? D.W. Pfaff: The “glia hypothesis” for the GABA, effect sounds like a good idea and deserves testing. F.W. Van Leeuwen: You showed the olfactory placode LHRH at the electron microscopical level and mentioned that it is restricted to theendoplasmic reticulum. Could you discern stained and unstained parts in this organelle? D.W. Pfaff: The most striking ultrastructural localization (Zheng et al., 1992) was the immunoreactivity right around the nuclear envelope when the LHRH cells were in the olfactory placode. Possible compartments (Van Leeuwen et al., 1991) for LHRH have not been ruled out. C.B. Saper: Did the normal human fetuses have LHRHimmunoreactive cells in the olfactory epithelium, and were these present in the olfactory epithelium in term infants or adults? In other words, do all of the LHRH cells migrate into the brain in normal humans, as in the mouse? D.W. Pfaff: Yes, there were some in the normal 19-week human tissue, but this needs further study in older individuals. C.B.Saper: It seems unlikely that neural cell adhesion molecule (NCAM) could be necessary for LHRH expression, given that you see LHRH cells in low density cell culture. Have you tested this hypothesis by staining your neuronal cultures for NCAM? D.W. Pfaff: We have not tested the LHRH cell cultures for NCAM. However, if it turns out that anti-NCAM antibodies actually affect LHRH gene expression such results would have many precedents. After all, theeffects of cell-cell contacts on differentiation of cell types are likely to include transcriptional mechanisms.

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References Demski, L.S. (1987) Phylogeny of luteinizing hormone-releasing hormone systems in protochordates and vertebrates. In: The Terminal Nerve (Nervus Terminalis): Structure, Function and Evolution - Ann. N . Y. Acad. Sci., 519: 1 - 14. Fox, S., Harlan, R., Shivers, B. and Pfaff, D.W. (1990) Chemical characterization of neuroendocrine targets for progesterone in the female rat brain and pituitary. Neuroendocrinofogy, 5 1: 276 - 283. Shivers, B.C., Harlan, R.E., Morrell, J.I. and Pfaff, D.W. (1983) Absence of oestradiol concentration in cell nuclei of LHRH-immunoreactive neurones. Nature, 304: 345 - 347. Spratt, D.I., Finkelstein, J.S., O’Dea, St.L., Badger, Th.M., Rao, P.N., Campbell, J.D. and Crowley, W.F. (1986) Longterm administration of gonadotropin-releasing hormone in

men with idiopathic hypogonadotropic hypogonadism. Ann. Znt. Med., 105: 848- 855. Van Leeuwen, F.W., Van der Beek, E.M., Van Heerikhuize, J.J., Sluiter, A.A., Felix, D. and Imboden, H. (1991) Vasopressin and angiotensin I1 are absent but spontaneously reappear in solitary hypothalamic neurons of the homozygous Brattleboro rat. Neurosci. Lett., 127: 207 -21 1. Warembourg, M. and Poulain, P. (1991) Presence of estrogen receptor immunoreactivity in the oxytocin-containing magnocellular neurons projecting to the neurohypophysis in the guinea pig. Neuroscience, 40: 41 - 53. Zheng, L.-M., Pfaff, D.W. and Schwanzel-Fukuda, M. (1991) Luteinizing hormone-releasing hormone (LHRH)-expressing cells in the medial olfactory placode and basal forebrain of mice during embryonic development. Neuroscience, 46: 407 - 418.

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0 1992 Elsevier Science Publishers B.V. All rights reserved.

CHAPTER 14

The human hypothalamus in relation to gender and sexual orientation D.F. S w a a b l , L. J.G. Gooren’ and M.A. Hofman’ I

Netherlands Institute for Brain Research, 1105 AZ Amsterdam ZO, The Netherlands; and Free University, 1007 MB Amsterdam, The Netherlands

History Concepts of sex differences in the human soul or brain as reported in the ancient literature, generally reflect the position of women in society, rather than being scientific facts (for references for this paragraph, see Swaab and Hofman, 1984). Aristotle (384 - 322 B.C.), Hippocrates (460 - 377 B.C.) and Thomas Aquinas (1225 - 1274) inferred the moment at which the female fetus becomes “animated” to be at a much later stage than the male fetus. The rationalization for this might be deduced from Aquinas’s opinion that a woman is a “mas occasionatas”, i.e., a man who has not reached his full potential of development. Comparatively minor morphological sex differences in the macroscopy of the human brain were often utilized to “prove” female inferiority. On the basis of the observation that the frontal lobe in males is 1% larger ihan in females, Huske (1854) claimed that “woman is a homo parietalis and interparietalis and man a homo frontalis”. The sex difference in brain weight has not only been frequently used to “prove” woman’s inferiority, but also, by Suffragettes and Women’s Lib activists, to “prove” woman’s superiority. On the basis of equivocal calculations both Maria Montessori (1913) and Germaine Greer (1972) have claimed that, taking total body weight into consideration, women have heavier brains. They concluded, respectively, that ‘‘anthropologically

Department of Endocrinology,

woman has the cranium of an almost superior race” (Montessori, 1913) or that “if the frontal lobes are to be considered as the seat of intelligence, then it must also be pointed out that the frontal area of the brain is more developed in women” (Greer, 1972). Controversy on sex differences in the brain is not only a matter of the past. Even today, discussion of sex differences in the human brain often gives rise to strong emotional reactions, as colleagues from various countries have experienced. This holds even more when a possible relation between sexual orientation and brain structures is discussed. Heated debates on nature-nurture, sickness versus health and destiny or self-realization are elicited. Sexual differentiation of the human hypothalamus In analogy with observations in many mammalian species one is inclined to believe that the human brain undergoes sexual differentiation during its development, due to an organizing effect of sex hormones. However, the stage of development in which sex steroids determine sexual differentiation of the human brain and the exact functional implications of such hormonal actions in relation to gender and sexual orientation are unknown. There are three sex-dimorphic peaks in gonadal hormone levels which thus could be of importance for these pro: cesses, viz. during the first half of gestation when the

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genitalia are formed (Reyes et al., 1974), in the perinatal period and during puberty (Winter, 1978). Total brain weight in adults is sexually dimorphic (Swaab and Hofman , 1984) but the brain area speculated to be the primary substrate of sex differences in reproduction, gender identity and sexual orientation is the hypothalamus (Dorner, 1979, 1986; Gladue et al., 1984). The supposition of Dorner and Staudt (1972) that structural sexual differentiation of the human hypothalamus takes place between 4 and 7 months of gestation was based only on the observation that during this period various hypothalamic cell groups, viz. the supraoptic, ventromedial and paraventricular nuclei, can be distinguished histologically (although sex differences in these nuclei were not reported by these authors), and on the observation that the matrix layer around the third ventricle, in which the cells are formed, has disappeared by 7 months of gestation. Only much later it became clear that not cell division, but rather cell death may be the most important mechanism in sexual differentiation of the nervous system (Nordeen et al., 1985; Swaab and Hofman, 1988).

RAl

MAN

0 males @ females

0,001

PVN

SCN

SDN-POA

PVN

SCN

SDN-POA

Fig. 1. Volumes of hypothalamic regions in young adult rats (2 - 5 . 5 months) and in man (20- 40 years). Values represent the mean volume (f S.E.M.) from one hemisphere. The variance of the SCN volume in female rats could not be determined. The sexual differences in the volume of the SDN-POA were statistically significant, both in man and in the rat. (From Hofman and Swaab, 1989, with permission.)

Sexually dimorphic nucleus (SDN) The sexually dimorphic nucleus (SDN) of the preoptic area of the hypothalamus, as first described in the rat by Gorski et al. (1978), is still the most conspicuous morphological sex difference in the mammalian brain. The cell group, which is 3 - 8 times larger in male rats than in female rats (Fig. l), has such a clear cytoarchitectonic sex difference that it can even be noted with the naked eye in Nisslstained sections. Lesions of the SDN affect masculine components of sexual behavior in rat (Anderson etal., 1986;Turkenburgetal., 1988;De Jongeetal., 1989). On the other hand, the extent of the changes in sexual behavior following SDN lesions is so modest that the major function of the SDN has probably not yet been revealed. Human SDN We have found an SDN in the preoptic area of the human hypothalamus that is - judged by its localization and cytoarchitecture - probably homologous to that in the rat (Swaab and Fliers, 1985), although proof for such a homology on the basis of transmitter content, afferents and efferents is lacking at this moment (Fig. 2). Morphometric analysis of the human SDN revealed that the volume is more than twice as large in adult men as it is in women, and contains about twice as many cells in men (Fig. 3). The human SDN corresponds to the intermediate nucleus as described by Braak and Braak (1987; see also Braak and Braak , this volume) and to the intermediate lateral hypothalamic area in early descriptions by Brockhaus (1942) and Feremutsch (1955). However, it does not correspond with the intermediate nucleus of an earlier paper of Feremutsch (1948) who described the islands of neurosecretory cells between the supraoptic and paraventricular nucleus (SON, PVN) by this name. Because of this confusion we will use the name SDN in the present paper. The sex difference which we observed in the SDN was not present in other hypothalamic nuclei (Fig. 1). The magnitude of the sex difference was found not to remain constant throughout adulthood, but to depend on age (Fig. 3). In males, a major reduction in SDN cell number was observed be-

207

LV

v

-9 0

LV

V LV

0 SDN PVN

-

/l

Fig. 2. Topography of the sexually dimorphic nucleus (SDN) in the preoptic area of the human hypothalamus. Third ventricle, 111; anterior commissure, AC; infundibulum, I; lateral ventricle, LV; optic chiasm, OC; organum vasculosum of the lamina terminalis, OVLT; recessus opticus, RO; septum, S; suprachiasmatic nucleus, SCN; paraventricular nucleus, PVN; supraoptic nucleus, SON; commissural fibers of the suprachiasmatic nucleus, CF. (From Swaab and Fliers, 1985, with permission; copyright 1985 by the AAAS.) 50

--0

Sexually Dimorphic Nucleus

-

40

-

30

-

t 20 d 4

5

L

10

Ln

females

0

20

40

60

80

100

A a.e (.v r s )

Fig. 3. Age-relatedchanges in the total cell number of the sexually dimorphic nucleus (SDN) of the preoptic area in the human hypothalamus. The general trend in the data is enhanced by using smoothed growth curves. Note that in males SDN-POA cell number steeplydeclines between theageof 50 - 60years, whereas in females, from the age of about 50 years, a more gradual cell loss is observed, which continues up to old age. These growth curves demonstrate that the reduction in cell number in the human SDN-POA in senescence is a non-linear, sex-dependent process. (From Hofman and Swaab, 1989, with permission.)

tween the age of 50 and 60 years, so that the sex difference became smaller. In females over 70 years of age cell death was found to be more prominent than in males, dropping to values which were only 1015% of the cell number found in early childhood, so that the sex difference in the SDN increases again in old people (Hofman and Swaab, 1989). This sex difference in the pattern of aging, and the fact that sexual differentiation in the human SDN only occurs after the 4th year of age (Swaab and Hofman, 1988) might explain why Allen et al. (1989), who had a sample of human adults biased for age, did not find a significant sex difference in the size of the SDN, which they called interstitial nucleus of the anterior hypothalamus 1 (INAH-1). In the study of Allen et al., 40% of the adult subjects came from the age group in which the SDN sex difference is minimalj compared to29% in our study (Hofman and Swaab, 1989). Moreover, the age group of elderly subjects

208

(over 70 years of age) was underrepresented in Allen’s study: 20% compared to the 37.5% in case of a proportional distribution of all ages. In our study, 32% of the subjects belonged to this old age group. So it seems likely that Allen et al. (1989) were unable to establish a sex difference in the INAH-1 ( = SDN) because they used a biased sample. A further argument for this assumption is that, if we, in our material, had studied only subjects of the age distribution of Allen’s study, the sex difference in SDN volume would have been reduced from 2 (Hofman and Swaab, 1989) to only 1.4 times, and this difference would not have been statistically significant any longer. Moreover, the sex difference in the SDN emerges only between the ages of 4 and puberty (Swaab and Hofman, 1988); therefore the brains of the 5-year-old boy and 4-year-old girl (she indeed had by far the largest volume of the entire series of female INAH-1) also produced a substantial bias of the Allen et al. (1989) study material. The age distribution, however, does not explain why LeVay (1991) could not find a sex difference in the volume of INAH-1 either. However, cell numbers have not been determined by this author.

Other sexually dimorphic nuclei Allen et al. (1989) described two other cell groups (INAH 2 and 3) in the preoptic-anterior hypothalamic area that were larger in the male brain than in the female brain. LeVay (1991) could not confirm the sex difference in INAH 2 , but did find such a difference in INAH 3. Since immunocytochemistry was not performed it is not clear whether the nuclei have to be considered. as islands of the paraventricular nucleus (PVN) or as separate anatomical entities. In addition, cell counts of the INAHs in the two sexes are lacking. A clear sex difference was described by Allen and Gorski (1990) in what they called the “darkly staining posteromedial component of the bed nucleus of the stria terminalis (BNST-dspm), an area of which the volume was found to be 2.5 times larger in males than in females. However, cell counts were not performed in this study either, and therefore these findings remain preliminary.

The suprachiasmatic nucleus (SCN), stained with vasopressin as a marker, showed a sex difference in shape (Swaab et al., 1985). The shape of the SCN was elongated in women and more spherical in men. However, in this study no significant sex difference was observed in either volume, vasopressin cell number or total cell number of the SCN. It is not

103

1

’

05

f Birth

. . 1

10 Age -postconception

50

5

1

100

( yrs)

Fig. 4. Development and sexual differentiation of the human sexually dimorphic nucleus (SDN) of the preoptic area of the hypothalamus. Log-log scale. Note that at the moment of birth the SDN is equally small in boys (A)and girls (0)and contains only about 20% of the cell number found at 2 -4 years of age. The SDN cell number of a female neonate with a pituitary aplasia (A) is fully within the range of other neonates. Cell numbers reach a peak value around 2 - 4 years post-natally, after which a sexual differentiation occurs in the SDN due to a decrease in cell number in the SDN of women, whereas the cell number in men remains approximatelyunchanged up to theage of 50. In women, cell number decreases for the second time after the age of about 60, after a period of relative stability, dropping to values which are only 10- 15% of the cell number found at 2 years postnatally. Note that in men the reduction in cell number in senescence is less dramatic. The largest discrepancy in cell number between men and women is found around 30 years and in people older than 80, whereas the sexual dimorphism in the SDN cell number is least around the age of 60. The SDN cell number in homosexual men (D) does not differ from that in the male reference group. The cell number of the SDN of a woman with a Prader-Willi syndrome (P)is small. The curves are quintic polynomial functions fitted to theoriginal data for males (drawn line) and females (dashed line). (Adapted from Swaab and Hofman, 1988, with permission.)

209

known whether this sex difference in shape goes together with sex differences in SCN afferent or efferent connections. Since so far only few human brain structures in the two sexes have been investigated morphometrically and a sex difference in relative human brain size exists (Swaab and Hofman, 1984), we expect that many more sex differences in the human brain have yet to be revealed.

Development of the human SDN In mid-pregnancy the SDN can already be distinguished in the human fetal brain (Polzovic et al., 1988; Swaab and Hofman, 1988). Yet the SDN cell number (Fig. 4) and volume at term birth are only 22% and 18070, respectively, of the values found between 2 and 4 years of post-natal age. In the second half of gestation and during the first post-natal years there is no significant sex difference in the size of the SDN. During the first post-natal years the SDN cell number rapidly increases in both boys and girls up to the age of 2 - 4 years post-natally. Only after this age does the human SDN differentiate according to sex, due to a decrease in both SDN volume and cell number in women, whereas in men these parameters remain unaltered up to the age of about 50 (Swaab and Hofman, 1988). Our results do not support the proposition that gonadal hormones stimulate mitotic formation, migration or aggregation of SDN cells during the fetal or perinatal period. In mid-gestation and perinatally the levels of sex hormones are much higher in boys than in girls (Reyes et al., 1974; Winter, 1978). Yet, the SDN size and cell number show the same magnitude in boys and girls up to the age of 2 - 4 years. The surprisingly late post-natal sexual differentiation of the human hypothalamus is in agreement with the observation that neither estrogen, androgen nor progestin receptors were found in the human fetal brain at mid-gestation (Abramovich et al., 1987). The sex difference in the volume of the BNST-dspm seems to occur only in adulthood although there is reason for caution. The sample size of subjects between 10 and 20 years of age was small in the study of Allen

and Gorski (1990). Together, these data support the notion that sexual differentiation of the human hypothalamus takes place after the perinatal period and before adulthood rather than during mid-gestation. This, and our observation that sexual differentiation of the human SDN does not take place earlier than 4 years post-natally, calls for a re-evaluation of the possible relationship between the sex dimorphism of the SDN and the perinatal testosterone peak in boys which lasts only some 90 days (Forest and Cathiard, 1975). It is possible that the perinatal testosterone peak promotes cell survival a few years later by preventing the “programmed” cell death which normally occurs in the female SDN. A similar mechanism is supposed to take place in rat spinal cord neurons of the bulbocavernosus musculature (Nordeen et al., 1985). In addition, one may speculate that not only hormones, but also other chemical compounds and social factors such as stress might be involved in sexual differentiation of the brain in this period (cf., Swaab and Hofman, 1988; Gooren et al., 1990). A case history of a 20.5-year-old man who had suffered a complete loss of testes at birth, showed a lack of sequela in terms of gender identity, gender role and sexual functioning (Gooren and Cohen-Kettenis, 1988). This observation may support the idea that other factors than androgens may be involved in sexual differentiation of the brain, or, alternatively, that the hormonal factors in development of gender and sexual orientation exert their effects before birth. SDN development and sex hormones As regards the possible involvement of sex hormones in SDN development in human, there are only very few data available. Sources of information are the “experiments of nature and medicine”. A child that died, perinatally, of a pituitary aplasia has a normal-sized SDN (Fig. 4; Swaab and Hofman, 1988). It is of interest, though, that one 30-year-old woman with Prader-Willi syndrome, which is characterized by a congenital deficiency in luteinizing hormone-releasing hormone (LHRH) and sex hor-

210

mones (Bray et al., 1983), had a small SDN (Fig. 4; Swaab and Hofman, 1988). A small SDN was also found in a 57-year-old man with Klinefelter’s syndrome, a condition that is characterized by a deficient androgen production (Swaab, unpublished results). It deserves further investigation whether these anomalies invariably lead to a smaller SDN in adulthood. It must be commented that women normally do not have significant elevations of sex steroid levels in early development and that the endocrine differences of Klinefelter patients only become apparent in early puberty (Knorr and Weil, 1984). It is, therefore, also conceivable that the genetic anomalies as such have had an influence on the size of the SDN rather than sex steroid levels in development. However, arguing against this is that in two such different conditions as Klinefelter’s and Prader-Willi’s syndrome the same anomalies were observed. One might also wonder whether these observations have implications for the development of sexual orientation. Vogt (1984) did not find an indication for a higher incidence of homosexuality in his sample of 30 (46, XXY) Klinefelter patients. It must be noted, however, that 15 men in his sample were sexually less active than normal males. This prevents any firm conclusion as to the sexual orientation of these subjects. The human hypothalamus, sexual orientation and gender identity We had the opportunity to study the structure of the anterior hypothalamus in relation to sexual orientation, and investigated 34 subjects. Eighteen male subjects from 22 to 74 years of age, the sexual orientation of whom was generally not known, served as a reference group. The homosexual male group consisted of 10non-demented AIDS subjects, aged 25 43. Six non-demented heterosexuals (4 males, 2 females, aged 21 -73 years) who had also died of AIDS served as a control group. Two areas of the hypothalamus were studied; the SDN (see before) and the suprachiasmatic nucleus (SCN). The SCN is considered to be the principal component of the

biological clock generating and coordinating hormonal, physiological and behavioral circadian rhythms (Moore, 1978; Rusak and Zucker, 1979; Moore-Ede et al., 1982). In addition, the SCN is thought to be involved in reproduction, at least in laboratory animals (Sodersten et al., 1981; Swaab et al., 1987). The main results were as follows: cell numbers in the SDN of the reference group, the male homosexuals and the heterosexual subjects did not differ. However, the SCN volume in homosexual men was 1.7 times as large as that of the reference group of male subjects and contained 2.1 times as many cells (Fig. 5 ; Swaab and Hofman, 1990). The SDN and sexual orientation A prominent theory on sexual orientation is that it develops as a result of an interaction between the developing brain and sex hormones (Gladue et al., 1984; Erhardt et al., 1985; Dorner, 1988; McCormick et al., 1990). A multitude of factors, i.e., maternal stress (Dorner et al., 1980; Anderson et al., 1986; Ellis et al., 1988, but compare Schmidt and Clement, 1990; Bailey et al., 1991) and chemicals (Swaab and Mirmiran, 1986) are thought to influence the process of sexual differentiation of the brain and sexual orientation. According to Dorner’s hypothesis, male homosexuals would have a female differentiation of the hypothalamus. So far, however, this hypothesis was based solely on indirect evidence, i.e., the demonstration of a positive feedback on luteinizing hormone secretion in some homosexual men following injection of estrogens (Gladue et al., 1984; Dorner, 1988). However, according to Gooren (1986a,b), this phenomenon is probably related to changes in testicular function rather than to sexual orientation and in his studies it could be demonstrated as often in homosexual as in heterosexual men. Furthermore, it could be shown that one and the same person was able to produce a negative and a positive estrogen feedback effect, depending on the hormonal milieu. This demonstrated that it is not the organization of the hypothalamic-pituitary unit that determines the occurrence of a positive or negative estrogen feedback. Also, it is not certain whether the rise of luteinizing

211

hormone following estrogen administration that Gladue et al. (1984) and Dorner (1988) observed, meet the stringent endocrine criteria of a positive estrogen feedback effect (Gooren, 1990). Dorner’s hypothesis concerning sexual orientation became testable immediately after we had found that the SDN of the preoptic area of the human hypothalamus contains twice as many cells in men as in women (Swaab and Fliers, 1985; Swaab and Hofman, 1988; Hofman and Swaab, 1989). In contrast to this hypothesis, neither the SDN volume nor the cell number in the hypothalamus of homosexual men (who died of AIDS), however, differed from that of the male reference group in the same age range (Fig. 5 ; Swaab and Hofman, 1988). More recent data (Fig. 5 ) confirmed and extended this observation with a heterosexual control group of subjects also suffering from AIDS (Swaab and Hofman, 1990). The fact that no difference in SDN cell number was observed between homo- and heterosexual men who died of AIDS refutes the global formulation of Dorner’s hypothesis that male homosexuals have “a female hypothalamus”.

The SCN and sexual orientation The observation that the SCN in homosexual men contains 2.1 times as many cells as the SCN of the reference group (Fig. 5; Swaab and Hofman, 1990) implies that the differences in SCN volume cannot be attributed to differences in shrinkage of hypothalamic tissue during the histological procedure. The difference in SCN cell number in relation to sexual orientation can, however, not be directly related to sexual differentiation of the brain since no differences in SCN volume or cell number were found betweenmales and females (Swaab et al., 1985;Hofman et al., 1988). The possibility cannot be excluded, yet, that sex hormone levels during brain development do play some part in this phenomenon (see below). The association between a large SCN (and, in particular, an increase in the number of neurons) and male homosexuality raises a number of questions about the way this difference might have developed. It appears very unlikely that homosexual behavior

as such would increase the neuronal number in any brain structure. The nerve cells of the SCN are postmitotic from a few years of age onwards, if not earlier (Swaab et al., 1990). However, an increase in stainability of vasopressin neurons due to homosexual behavior cannot be totally excluded. Yet the developmental course of SCN cell numbers (Swaab et al., 1990) suggests that the explanation for the large SCN in homosexual men most likely may be found in early processes of brain development. At birth, the SCN contains only 13 - 20% of the adult number of vasopressin and total cells, but in the post-natal period development is rapid. Cell counts reach a peak arond 13 - 16 months after birth (Swaab et al., 1990). The SCN cell numbers found in adult homosexual men were in the same order of magnitude as found around 13-16 months postnatally. The normal pattern is that the vasopressin and total cell numbers subsequently decline to the adult value of about 35% of the peak values. In homosexual men, therefore, this programmed postnatal cell death in the SCN seems to have been reduced. The observation that a similarly enlarged SCN was present in a woman with Prader-Willi syndrome (Swaab et al., 1987), a congenital LHRH deficiency in which sex hormone levels are very low (Bray et al., 1983; Bray, this volume), suggests that the interaction with sex hormones in some stage of development might be relevant for the programmed SCN cell death (however, see the caveat earlier in this text). The possibility of sex hormones playing some role in SCN development is reinforced by an observation of Sodersten et al. (1981). They showed that the amplitude of the circadian rhythm in sexual behavior, of which the SCN is the substrate, is enhanced by anti-estrogen treatment of the neonatal animal. This observation and the large SCN in Prader-Willi syndrome (Swaab et al., 1987) make it more likely that a larger SCN, as reported here for homosexual men, may relate to a difference in the interaction with sex hormones during development. This possibility should be tested in animal experiments and further explored in human material. One might argue that the present finding of an enlarged SCN in male homosexuals who died of

212 (A)

SUPRACHIASMATIC NUCLEUS

0:s

-***

I*

. I

T

05

TE

SEXUALLY DIMORPHIC NUCLEUS

04

- 03 QI

5

0

> 02 0 2 01

. I

Reference group

(8)

Homosexuals (AIDS1

Heterosexuals (AIDS)

Transsexuals

SUPRACHIASMATIC NUCLEUS

Reference group

Homosexuals (AIDS)

Heterosexuals (ALDS)

Transsexuals

SEXUALLY DIMORPHIC NUCLEUS

125 . I

0 -

0 2

100

0

L

75

D

$

T

1

Homosexuals (AIDS)

Heterosexuals (AIDS)

= 50 U rn

c

+

25

Reference group

Homosexuals (AIDS)

Heterosexuals (AIDS)

Transsexuals

Reference group

Transsexuals

For Iegend, see p. 213.

AIDS only holds for a particular subset of homosexual men, i.e., those likely to acquire AIDS through a high number of frequently changing sexual partners with whom anal receptive sexual techniques were performed (Curran et al., 1985; Van Griensven et al., 1987). This possibility, i.e., that an enlarged SCN may be related to, e.g., the level of sexual activity rather than to homosexuality certainly warrants further study. Experiments in rats, however,

have shown a close correlation between sexual activity and SDN size (Andersen et al., 1986). Our observation that the size of the SDN in homosexual men did not differ from that of the male reference group nor from that of the heterosexual men that died of AIDS, does not support this possibility. An alternative explanation for the enlarged SCN found in male homosexuals having died of AIDS is that it might be related to the hypogonadism in

213

(c)

SUPRACHIASMATIC NUCLEUS

Reference group

Homosexuals (AIDS)

Heterosexuals (AIDS)

Transsexuals

Fig. 5 . A . Volume of the human suprachiasmatic nucleus (SCN) and sexually dimorphic nucleus (SDN) as measured in four groups of adult subjects: (1) a male reference group (n = 18); (2) male homosexuals who died of AIDS (n = 10); (3) heterosexuals who died of AIDS (n = 6; 4 males and 2 females); and (4) three male to female transsexuals (numbers refer to TI - T3 in Table I). The values indicate medians and the standard deviation of the median. The differences in the volume of the SCN between homosexuals and the subjects from both other groups, are statistically significant (Kruskal-Wallis multiple comparison test, *P < 0.05; **P < 0.01; ***P < 0.001). Note that none of the parameters measured in the SDN (A$) showed significant differences among the three groups ( P always > 0.4).B. Total number of cells in the human SCN and SDN. The SCN in homosexual men contains 2.1 times as many cells as in the reference group of male subjects and 2.4 times as many cells as the SCN in heterosexual AIDS patients. C. The number of vasopressin neurons in the human SCN (the human SDN does not contain vasopressin-producing cells). The SCN in homosexual men contains, on average, 1.9 times as many vasopressin(VP) producing neurons as the reference group of male subjects and 3.6 times as many VP neurons as the SCN in heterosexual AIDS patients. Notice that the SCN of heterosexual individuals who died of AIDS, contains less vasopressin cells than the subjects from the reference group. In the right hand column of each figure the individual values of three transsexual patients (T1-T3) are given. Note that 1 and 2 have a large SCN and a small SDN and that 3 has a small SCN and a large SDN. For clinical details see Table I. (Adapted from Swaab and Hofman, 1990, with permission.)

adulthood that has been found in AIDS patients (Croxson et al., 1989). Our observation that the SCN in heterosexual male AIDS patients is not enlarged seems to exclude this possible explanation, but homosexual men who had not died of AIDS should certainly be studied in the future. In this respect, it is interesting that we observed an enlarged SCN in two (primary) male-to-female transsexuals who did not suffer from AIDS (Swaab et al., 1987; Fig. 5). The functional implications of the association between sexual orientation in men and SCN size are not clear at this moment. Various observations in animals suggest that the SCN, apart from being the biological clock, may be involved in reproductive processes (Sodersten et al., 1981; Swaab et al., 1987). Judged from its nucleolar size, the SCN is also activated around puberty in rats (Anderson, 1981). In addition, lesions of the SCN area in the female rat attenuated the positive feedback response of gonadotropic hormones to estrogens (Gray et al., 1978; Wiegand et al., 1980). However, recently it was observed that lesions in the adult male rat SCN did not alter sexual orientation (F.H. De Jonge et al., in preparation). This observation argues in favor of the possibility that sexual orientation and the size of the SCN are not causally related but may be subject to the same organizing factor in development. The relationship between a large SCN and homosexuality is unexpected and, for the time being, difficult to interpret. The relationship need not be causal in the sense that it is a necessary and sufficient condition for developing a homosexual orientation. It is imperative to study more material before definitive conclusions can be drawn. We have no information on the size of the SCN in female homosexuals, for instance, or of bisexuals. Until more data have been collected our finding is open to various interpretations. It is particularly pertinent to study the SDN and SCN in subjects whose prenatal/post-natal history has been atypical (an excess of androgens in femalesla deficiency or insensitivity to androgens in males) as has been done in earlier

214 TABLE 1 Clinical data of transsexuals (T) Subjects

T1

T2

T3

Hypothalamus

Large SCN, small SDN

Large SCN, small SDN

Small SCN, large SDN

Male-to-female

Primary

Primary

Secondary

Sexual/social orientation

Androphile, married under social pressure, father, last 4 years of life gynephilic, hostile towards men

Asexual but surrounded herself with women

Gynephilic, father, following operation asexual

Hormone treatment

Cyproterone acetate, ethinylestradiol

Cyproterone acetate, stilbestrol, ethinylestradiol

Cyproterone acetate, ethinylestradiol

Age at death Cause of death

50 Suicide

44 Probably cardiovascular

43 Sarcoma

SCN, Suprachiasmatic nucleus; SDN, sexually dimorphic nucleus; primary, gender dysphoria from early childhood onwards; secondary, gender dysphoria only post-pubertally.

work on sexual orientation (e.g., Money et al., 1984). Transsexuality Gender identity, like sexual orientation, has been proposed to develop as a result of an interaction between the developing brain and sex hormones. Transsexuality is considered to be the result of a disturbance of this interaction (Gladue et al., 1984; Dorner, 1988). In view of the similarity between the hypotheses on the development of gender and sexual orientation, it is of interest that 60% of the male to female transsexuals is androphile and that some 10% is biphile. In no less than 95% of the cases, female to male transsexuals are gynaecophilic (Coleman et al., 1992). These data indicate that indeed similar (but as yet unknown) mechanisms may play a role in the development of both gender and sexual orientation. We were given the opportunity to study the hypothalami of three male to female transsexuals (Fig. 5 andTable I). Two of them (Tl, T2) appeared to have

a large SCN with high cell numbers and a small SDN with low cell numbers. The third transsexual subject (T3), however, revealed exactly the opposite, i.e., a small SCN and a large SDN. These two different patterns could not be related to sexual orientation of these three subjects (see Table I) in any simple way. This table suggests, however, that a relationship might exist between: (1) a large SCN and small SDN and primary transsexuality (i.e., awareness of the gender problem from early childhood onwards) on the one hand; and (2) a small SCN and large SDN and secondary transsexuality (i.e., awareness of transsexuality later in life) on the other. It will be obvious that more data are necessary in order to establish whether such a relationship indeed exists. Conclusions and summary In analogy with observations in many mammalian species the human hypothalamus is believed to undergo sexual differentiation during development due to an organizing effect of sex hormones.

215

We have found a sexually dimorphic nucleus (SDN) in the preoptic area of the human hypothalamus that contains about twice as many cells in young adult men as in women. The magnitude of the sex difference in the SDN depends on age. In the literature two other hypothalamic nuclei (INAH 2 and 3) and a part of the bed nucleus of the stria terminalis have been reported to be sexually dimorphic in human. At term, the SDN contains only some 20% of the cell number found at 2 - 4 years of age. The cell number rapidly increases in boys and girls at the same rate until 2 - 4 years of age, after which the SDPJ differentiates according to sex due to a decrease in cell numbers in girls. This period of sexual differentiation of the human hypothalamus is much later than generally presumed in literature, and opens the possibility of interaction of a multitude of post-natal factors in sexual differentiation of the brain, e.g., hormones, other chemical compounds and psycho-social factors. A few preliminary observations available at present in clinical conditions with deficient hormonal production suggest that the size and cell number of the human SDN in adulthood is influenced by sex hormones in development. No difference in SDN cell number was observed between homo- and heterosexual men. This refutes the most global formulation of Dorner’s hypothesis that male homosexuals would have a “female hypothalamus”. In a sample of brains of homosexual men we found that the suprachiasmatic nucleus (SCN) contained twice as many cells as in the reference group. The observation that a similarly enlarged SCN was present in a woman with Prader-Willi syndrome suggests that sex hormones and SCN development might be interrelated. A recent report claimed INAH 3 to be more than twice as large in heterosexual men as in homosexual men. Preliminary data suggest that the SCN is large and the SDN small in primary male-to-female transsexuals and that the SCN is small and the SDN large in secondary maleto-female transsexuals but more data have to be collected to confirm this observation.

In conclusion: differences in size and cell number have been reported in a number of hypothalamic nuclei in relation to sexual orientation and gender. However, the functional implications of these findings are far from clear as yet. Acknowledgements

Brain material was obtained from the Netherlands Brain Bank (coordinator Dr. R. Ravid) and from Dr. L.J.G. Gooren (Department of Endocrinology, Free University, Amsterdam, The Netherlands), Dr. R.S. Williams (Neuropathology Laboratory, Shriver Center, Walton, U.S.A.), Dr. L. Mrzljak and Dr. I. Kostovic (Department of Anatomy, University of Zagreb, Yugoslavia), Dr. P.G. Barth (Department of Pediatrics and Neurology, AMC, University of Amsterdam, The Netherlands) and Dr. H.P.H. Kremer, Department Neurology, University of Leiden, The Netherlands). General pathology and neuropathology were performed either at the Free University of Amsterdam (Dr. W. Kamphorst) or at the AMC, University of Amsterdam (Dr. D. Troost). We want to thank Mr. B. Fisser for his technical assistance and Ms. W. Verweij for her secretarial help. References Abramovich, D.R., Davidson, LA., Longstaff, A. and Pearson, C.K. (1987) Sexual differentiation of the human midtrimester brain. Eur. J. Obstet. Gynecol. Reprod. Biol., 25: 7 - 14. Allen, L.S. and Gorski, R.A. (1990) Sex difference in the bed nucleus of the stria terminalis of the human brain. J. Comp. Neurol., 302: 691- 706. Allen, L.S., Hines, M., Shryne, J.E. and Gorski, R.A. (1989) Two sexually dimorphic cell groups in the human brain. J. Neurosci., 9: 491 - 506. Anderson, C.H. (1981) Nucleolus: changes at puberty in neurons of the suprachiasmatic nucleus and the preoptic area. Exp. Neurol., 14: 780- 186. Anderson, R.H., Fleming, D.E., Rhees, R.W. andKinghorn, E. (1986) Relationship between sexual activity, plasma testosterone and the volume of the sexually dimorphic nucleus of the preoptic area in pre-natally stressed and non-stressed rats. Brain Rex, 370: 1 - 10.

216 Bailey, J.M., Willerman, L. and Parks, C. (1991) A test of the maternal stress theory of human male homosexuality. Arch. SOC. Behav., 20: 277 - 293. Braak, H. and Braak, E. (1987)The hypothalamus of the human adult: chiasmatic region. Anat. Embryo/., 176: 315 - 330. Bray,G.A., Dahms, W.T., Swerdloff, R.S.,Atkinson, R.L. and Carrel, R.E. (1983) The Prader-Willi syndrome: a study of 40 patients and a review of the literature. Medicine, 62: 59 - 80. Brockhaus, H. (1942) Beitrag zur normalen Anatomie des Hypothalamus und der Zona incerta bei Menschen. J. Psychol. Neurol., 51: 96- 196. Coleman, E., Bockting, W.O. and Gooren, L.J.G. (1992) Homosexual and bisexual identity in sex-reassignedfemale-tomale transsexuals. Arch. Sex. Behav., 21: in press. Croxson, T.S., Chapman, W.E., Miller, L.K., Levit, C.D., Senie, R. and Zumoff, B. (1989) Changes in the hypothalamicpituitary-gonadal axis in human immunodeficiency virusinfected homosexual men. J. Clin. Endocrinol. Metab., 68: 317 - 321. Curran, J.W., Meade Morgan, W., Hardy, A.M., Joffe, H.W., Darrow, W.W. and Dowdle, W.R. (1985) The epidemiology of AIDS: current status and future prospects. Science, 229: 1352- 1357. De Jonge, F.H., Louwerse, A.L., Ooms, M.P., Evers, P. and Van de Poll, N.E. (1989) Lesions of the SDN-POA inhibit sexual behavior of male Wistar rats. Brain Res. Bull., 23: 483 - 492. Dorner, G. (1979) Psychoneuroendocrine aspects of brain development and reproduction. In: L. Zichella and P. Pancheri (Eds.), Psychoneuroendocrinology in Reproduction, an Interdisciplinary Approach, Elsevier, Amsterdam, pp. 43 - 54. Dorner, G. (1986) Hormone-dependent brain development and preventive medicine. Monogr. Neural Sci., 12: 17 - 27. Dorner, G. (1988) Neuroendocrine response to estrogen and brain differentiation in heterosexuals, homosexuals, and transsexuals. Arch. Sex. Behav., 17: 57 - 75. Dorner, G. and Staudt, J. (1972) Vergleichende morphologische Untersuchungen der Hypothalamusdifferenzierung bei Ratte und Mensch. Endokrinofogie, 59: 152- 155. Dorner, G., Geiser, T., Ahrens, L., Krell, L., Mum, G., Sieler, H., Kittner, E. and Muller, H. (1980) Pre-natal stress and possible aetiogenetic factor homosexuality in human males. Endokrinologie, 75: 365 - 368. Ehrhardt, A.A., Meyer-Bahlburg, H.F.L., Rosen, L.R., Feldman, J.F., Veridiano, N.P., Zimmerman, I. and McEwen, B.S. (1985) Sexual orientation after pre-natal exposure to exogenous estrogen. Arch. Sex. Behav., 14: 57-75. Ellis, L., Ames, M.A., Peckham, W. andBurk, D. (1988) Sexual orientation of human offspring may be altered by severe maternal stress during pregnancy. J. Sex. Rex, 25: 152- 157. Feremutsch, K. (1948)Die Variabilitat der cytoarchitektonischen Struktur des menschlichen Hypothalamus. Monatschr. Psychiatr. Neurol., 116: 257 - 283.

Feremutsch, K. (1955) Strukturanalyse des menschlichen Hypothalamus. Monatschr. Psychiatr. Neurol., 121: 87 - 113. Forest, M. and Cathiard, A.M. (1975) Patterns of plasma testosterone and 4-androstenedione in normal newborns: evidence for testicular activity at birth. J. Clin. Endocrinol. Metab., 41: 977-981. Gladue, B.A., Green, R. and Helleman, R.E. (1984) Neuroendocrine response to estrogen and sexual orientation. Science, 225: 1496- 1499. Gooren, L.J.G. (1986a) The neuroendocrine response of luteinizing hormone to estrogen administration in heterosexual, homosexual and transsexual subjects. J. Clin. Endocrinol. Metab., 63: 583 - 588. Gooren, L.J.G. (1986b) The neuroendocrine response of luteinizing hormone to estrogen administration in the human is not sex-specific but dependent on the hormonal environment. J. Clin. Endocrinol. Metab., 63: 589 - 593. Gooren, L.J.G. (1990) The endocrinology of transsexualism: a review and commentary. Psychoneuroendocrinology, 15: 3-14. Gooren, L.J.G. and Cohen-Kettenis, P. (1988) Erotosexuality in a man lacking physiological post-natal androgen exposure. J. Psychol. Hum. Sexuality, 1: 129 - 135. Gooren, L.J.G. and Cohen-Kettenis, P. (1991) Development of a male gender identityhole and a sexual orientation towards women in a 46,xy subject with an incomplete form of the androgen insensitivity syndrome. Arch. Sex. Behav., 20: 459 - 470. Gooren, L.J.G., Fliers, E. and Courtney, K. (1990) Biological determinants of sexual orientation. Annu. Rev. Sex. Res., 1: 175 - 196. Gorski, R.A., Gordon, J.H., Shryne, J.E. and Southam, A.M. (1978) Evidence for a morphological sex difference within the medial preoptic area of the rat brain. Brain Rex, 148: 333 - 346. Gray, G.D., Sodersten, P., Tallentrie, D. and Davidson, J.M. (1978) Effects of lesions in various structures of the suprachiasmatic-preoptic region on LH regulation and sexual behavior in female rats. Neuroendocrinology, 25: 174- 191. Hofman, M.A. and Swaab, D.F. (1989) The sexually dimorphic nucleus of the preoptic area in the human brain: a comparative morphometric study. J. Anat., 164: 55 - 72. Hofman, M.A., Fliers, E., Goudsmit, E. and Swaab, D.F. (1988) Morphometric analysis of the suprachiasmatic and paraventricular nuclei in the human brain. J. Anat., 160: 127 - 143. Knorr, D. and Weil, J. (1984) Klinefelter’s syndrome: diagnostic criteriain childhood. In: H.-J. Bandmann and R. Breit (Eds.), Klinefelter’s Syndrome, Springer, Berlin, pp. 29 - 39. LeVay, S. (1991) A difference in hypothalamic structure between heterosexualand homosexual men. Science, 253: 1034 - 1037. McCormick, C.M., Witelson, S.F. and Kingstone, E. (1990) Left-handedness in homosexual men and women: neuroendocrine implications. Psychoneuroendocrinology, 15: 69 - 76.

217 Money, J., Schwartz, M. andLewis, V.G. (1984)Adulterotosexual status and fetal hormonal masculinization: 46,XX congenital virilizing adrenal hyperplasia and 46,XY androgeninsensitivity syndrome compared. PsychoneuroendocrinolOgy, 9: 405 -414. Moore, R.Y. (1978) Central neural control of circadian rhythms. In: W.F. Ganong and L. Martini (Eds.), Frontiers in Neuroendocrinology, Vol. 5, Raven Press, New York, pp. 185 - 206. Moore-Ede, M.C., Sulzman, F.M. and Fuller, C.A. (1982) The Clocks that Time Us. Physiology of the Circadian Timing System, Harvard University Press, Cambridge, MA. Notdeen, E.J., Nordeen, K.W., Sengelaub, P.R. and Arnold, A.P. (1985) Androgens prevent normally occurring cell death in a sexually dimorphic spinal nucleus. Science, 229: 671 -673. Polzovic, A., Marinkovic, R., Gudovic, R., Mihic, N. and Grkovic, D. (1988) Vascularization of the preoptic area in the human fetus. In: M. Bajic (Ed.), Advancesin the Biosciences, Vol. 70. Neuron, Brain and Behaviour, Pergamon, Oxford, pp. 143 - 146. Reyes, F.I., Boroditsky, R.S., Winter, J.S.D. and Faiman, C. (1974) Studies on human sexual development. 11. Fetal and maternal serum gonadotropin and sex steroid concentrations. J. Clin. Endocrinol. Metab., 38: 612-617. Rusak, B. and Zucker, I. (1979) Neural regulation of circadian rhythms. Physiol. Rev., 59: 449- 526. Schmidt, G. and Clement, U. (1990) Does peace prevent homosexuality? Arch. Sex. Behav., 19: 183 - 187. Sodersten, P., Hansen, S. and Srebro, B. (1981) Suprachiasmatic lesions disrupt the daily rhythmicity in the sexual behaviour of normal male rats and of male rats treated neonatally with antioestrogen. J. Endocrinol., 88: 125. Swaab, D.F. and Fliers, E. (1985) A sexually dimorphic nucleus in the human brain. Science, 228: 1112- 1115. Swaab, D.F. and Hofman, M.A. (1984) Sexual differentiation of the human brain. A historical perspective. In: G.J. De Vries, J.P.C. De Bruin, H.B.M. Uylings and M.A. Corner (Eds.), Sex Differences in the Brain - Progress in Brain Research Vol. 61, Elsevier, Amsterdam, pp. 361 - 374. Swaab, D.F. and Hofman, M.A. (1988) Sexual differentiation of the human hypothalamus: ontogeny of the sexually dimorphic nucleus of the preoptic area. Dev. Brain Rex, 44: 314-318. Swaab, D.F. and Hofman, M.A. (1990) An enlarged suprachiasmatic nucleus in homosexual men. Brain Res., 537: 141 - 148. Swaab, D.F. and Mirmiran, M. (1986) Functional teratogenic effects of chemicals on the developing brain. In: M.M. Cohen (Eds.), Monographs in NeuralSciences, 12, Karger, Basel, pp. 4s- 57. Swaab, D.F., Fliers, E. and Partiman, T.S. (1985) The suprachiasmatic nucleus of the human brain in relation to sex, age and senile dementia. Brain Res., 342: 37 - 44. Swaab, D.F., Roozendaal, B., Ravid, R., Velis, D.N., Gooren, L.J.G. and Williams, R.S. (1987) Suprachiasmatic nucleus in

aging, Alzheimer’s disease, transsexuality and Prader-Willi syndrome. In: E.R. De Kloet, V.M. Wiegant and D. De Wied (Eds.), Neuropeptides and Brain Function - Progress in Brain Research, Vol. 72, Elsevier, Amsterdam, pp. 301 - 31 1. Swaab, D.F., Hofman, M.A. andHonnebier, M.B.O.M. (1990) Development of vasopressin neurons in the human suprachiasmatic nucleus in relation to birth. Dev. Brain Rex, 52: 289293. Turkenburg, J.L., Swaab, D.F., Endert, E., Louwerse, A.L. and Van de Poll, N.E. (1988) Effects of lesions of the sexually dimorphic nucleus on sexual behavior of testosterone-treated female Wistar rats. Brain Res. Bull., 22: 215 - 224. Van Griensven, G. J.P., Tielman, R.A.P., Goudsmit, J .,Van der Noordaa, J., De Wolf, F., De Vroome, E.M.M. and Coutinho, R.A. (1987) Risk factors and prevalence of HIVantibodies in homosexual men in The Netherlands. A m . J. Epidemiol., 125: 1048- 1057. Vogt, H.-J. (1984)Sexual behavior in Klinefelter’s syndrome. In: H.-J. Bandmann and R. Breit (Eds.), Klinefelter’sSyndrome, Springer, Berlin, pp. 163 - 169. Wiegand, S.J., Terasawa, E., Bridson, W.E. and Gray, R.W. (1980) Effects of discrete lesions of preoptic and suprachiasmatic structures in the female rat: alterations in the feedback regulation of gonadotropin secretion. Neuroendocrinology, 31: 147- 157. Winter, J.S.D. (1978) Pre-pubertal and pubertal endocrinology. In: F. Falkner and J.M. Tanner (Eds.), Human Growth. I . Principles and Prenatal Growth, Vol. 2, Bailliere Tindall, London, pp. 183 -213.

Discussion R.M. Sibug: (1) Are there data available on the suprachiasmatic nucleus (SCN) and sexual dimorphic nucleus (SDN) in human exhibiting testicular feminization (Tfm). How would you describe their sexual behavior? (2) The SDN of males of normal and Tfm rats were shown to be similar (Gorski et al., 1981). Yet, behavioral studies have shown that adult Tfm mice exhibit asexual behavior, i.e., they do not exhibit typical male or female sexual behavior (Ohno et al., 1974). How do you account for this assuming it is the SDN which is causing the dimorphism in sexual behavior? D.F. Swaab: (1) No, we do not have any data on the size or cell number of hypothalamic nuclei of Tfm patients. Gooren and Cohen-Kettenis (1991) have recently described one 64.XY subject with an incomplete form of the androgen insensitivity syndrome. In adulthood, the only genital sign of masculinization was a clitoris of 4 cm. The vagina was of normal size. The subject was reared as a girl. At the age of 30 the subject applied for gender reassignment treatment to the male sex. The gender identity was unambiguously male and the sexual orientation was towards women. Of course, one may wonder whether the receptors in the periphery and in the brain are differentially affected.

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(2) In Tfm rats the SDN is indeed of normal size probably indicating that sexual differentiation of the brainin one locus (e.g., the SDN) is determined by estrogens and in another locus (e.g., the spinal nucleus of the bulbocavernosus is completely feminine in these animals) by androgens (cf., Gorski, 1984). In the first place, I do not think that the SDN is absolutely essential for sexual behavior, sincelesions lead to only modest alterations and only under certain particular circumstances (Turkenburg et al., 1988; DeJongeetal., 1989). InfactI havetheideathatatpresent we do not know at all the main function of the SDN. In addition, even if the SDN would be important in sexual behavior, i t q o r ma1 size in Tfm rats would of course not mean that the many other brain structures and systems that are crucial for normal sexual behavior would not be affected in these rats (e.g., the spinal nucleus of the bulbocavernosus). C.B. Saper: A comment on Dr. Gooren’s patient with incomplete testicular feminization and male sexual identity. I agree that it is dangerous to extrapolate from individual clinical cases, where the actual genetic defect is not known. For example, it is possible that your patient was a chimera, with normal testosterone receptors in the brain, but not in the lower body. B.S. McEwen: Question and comment: the DES study of Ehrhardt et al. (1985) also showed that males exposed to DES have no increase in bisexuality even though masculinity appears to be decreased. DES undoubtedly reduced T secretions and thus reduced the major signal for masculinization; however, DES is an estrogen and T is activity-aromatized in the developing hypothalamus. This contrasts to the hypothetical situation of transient lack of androgens during development that might lead to a lack of masculine development as well as a lack of estrogens from aromatization. This situation is the one which might lead to altered sexual orientation and altered size of hypothalamic nuclei, as in the study of LeVay (1991). Might estrogens derived from aromatization not play an important developmental role in the differentiation of the SDN and in the differentiation of sexual orientation? This proposal would also apply to the DES effect to increase bisexuality in women. D.F. Swaab: This is an excellent suggestion. M.A. Hofman: You suggest that the large sizeof the SDN in male homosexuals and the increased number of vasopressin cells in this nucleus are due to a diminished cell death in early post-natal life. Is it not possible that the large vasopressincell number in the SCN of homosexuals is caused by an enhanced vasopressin synthesis in these subjects, as a result of which more vasopressin cells become detectable. D.F. Swaab: This is indeed an alternative possibility. Since the AVP cell density in the SCN did not differ between homosexual, heterosexual and reference group (Swaab and Hofman, 1990), this would mean that in one particular peripheral part of the SCN cells would have to be activated by an as yet unknown factor. H. Braak: Has anyone examined circadian rhythms of homosexuals versus heterosexuals? D.F. Swaab: Vilette et al. (1990) showed altered adrenal hormonal circadian rhythms in asymptomatic HIV-infected male

patients as compared to controls, but this difference was not interpreted by the authors to be associated with sexual orientation but was rather looked upon as an early expression of the effect of the HIV virus. In addition, Gooren et al. (1988) have studied temperature rhythms in transsexuals. A sex difference was observed in these rhythms but no difference was found between transsexual and non-transsexual men. J.J. Legros: (1) Were the controls in the homosexual study male or male and female? (2) Comment: from a general point of view it is interesting to note that the differences observed are not related to an androgen-sensitive nucleus (SDN) but to a nucleus more related to environmental (and social?) factors (SCN). This fits in very well with the hypothesis on the origin of homosexuality, i.e., more due to “social” or “environmental” factors than to hormonal ones. D.F. Swaab: (1) The reference group consisted of males, mainly presumed to be heterosexuals. A heterosexual control group was included which consisted of 6 HIV-infected individuals, 4 males and 2 females (Swaab and Hofman, 1990). (2) Thank you for this remark, but I feel that it might be too early to speculate about the causality of the relationship between a large SCN and homosexuality in these terms. R. Ravid: Why does it take so long for fetal or perinatal sex hormones to have an effect since sexual differentiation occurs only by the age of four? D.F. Swaab: If the sexual differentiation of the hypothalamus is actually due to these sexual dimorphic hormone peaks which is something that still has to be established in the human brain, one might speculate that perhaps the receptors for sexsteroids are not situated in the SDN but in other sexually dimorphic structures that are innervated by the SDN. The SDN fibers in females would only “realize” that there is no target for them when they later innervate that structure and subsequently they would degenerate. Such a process might take a considerable amount of time.

References De Jonge, F.H., Louwerse, A.L., Ooms, M.P., Evers, P. an( Van de Pol, N.E. (1989) Lesions of the SDN-POA inhibit sex ual behavior of male Wistar rats. Brain Res. Bull., 23 483 - 492. Ehrhardt, A.A., Meyer-Bahlburg, H.F.L., Rosen, L.R. Feldman, J.F., Veridiano, N.P., Zimmerman, I. an( McEwen, B.S. (1985) Sexual orientation after pre-natal ex posure to exogenous estrogen. Arch. Sex. Behav., 14(1) 57 - 7s. Gooren, L. and Cohen-Kettenis, P.T. (1991) Development o male gender identityhole and a sexual orientation toward women in a 46,XY subject with an incomplete form of an drogen insensitivity syndrome. Arch. Sex. Behav., 20 459 - 470. Gooren, L., Spijkstra, J.J., Spinder, T., Swaab, D., Mirmiran

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M. and Witting, W. (1988) Circadian rhythms in transsexuals. Neuroendocrinol. Lett., lO(4): 197 - 288. Gorski, R.A. (1984) Critical role for the medial preoptic area in the sexual differentiation of the brain. In: G.J. De Vries, J.P.C. De Bruijn, H.B.M. Uylings and M.A. Corner (Eds.), Sex Differences in rhe Brain - Progress in Brain Research, Vol. 61, Elsevier Science Publishers, Amsterdam, pp. I29 - 147. Gorski, R.A., Csernus, V.J. and Jacobson, C.D. (1981) Sexual dimorphism in the preoptic area. In: B. Flerkb, G. Setalo and L. Tima (Eds.), Advances in Physiological Sciences, Vol. 15, Reproduction and Development, Pergamon, London and Akademiai Kiado, Budapest, pp. 121 - 130. LeVay, S. (1991) A difference in hypothalamic structure between heterosexual and homosexual men. Science, 253: 1034 - 1037. Ohno, S., Geller, L.N. and Young, L. (1974) Tfm mutation and

masculinization versus feminization of the mouse central nervous system. Cell, 235: 242. Swaab, D.F. and Hofman, M.A. (1990) An enlarged suprachiasmatic nucleus in homosexual men. Brain Res., 537: 141 - 148. Turkenburg, J.L., Swaab, D.F., Endert, E., Louwerse, A.L. and Van den Poll, N.E. (1988) Effects of lesions of the sexually dimorphic nucleus on sexual behavior of testosterone-treated female Wistar rats. Brain Res. Bull., 21: 215-224. Vilette, J.M., Bourin, P., Doinel, C., Mansour, I., Fiet, J., Boudou, P., Dreux, C., Roue, R., Debord, M. and Levi, F. (1990) Circadian variations in plasma levels of hypophyseal, adrenocortical and testicular hormones in men infected with human immunodeficiency virus. J. Clin. Endocrinol. Metab., 70: 572 - 577.

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CHAPTER 15

Hormonal influences on morphology and neuropeptide gene expression in the infundibular nucleus of postmenopausal women Naomi E. Rance Departments of Pathology and Neurology, University of Arizona College of Medicine, Tucson, AZ 85724, U.S.A.

Introduction Menopause provides an exceptional opportunity to explore morphological and neurochemical changes in the human hypothalamus relative to changes in the hormonal milieu. Unlike many other aging phenomena, menopause is a consistent, well-defined event which eventually occurs in every woman. In addition, menopause is accompanied by pronounced changes in peripheral plasma hormone levels and endometrial histology. Because menopause occurs in middle age, its onset is long before many other non-specific and confounding neuropathological processes. Finally, extensive research in laboratory animals has characterized the hypothalamic-pituitary-ovarian axis and has provided considerable insight into the contributions of different components of this system to the aging process (Wise, 1983; Finch et al., 1984). Ovarian failure is a major component of the transition to menopause. An irreversible loss of ovarian follicles (Block, 1952) results in a drop of ovarian hormone secretion to castrate levels (Baird and Guevara, 1969). There is a secondary rise in plasma LH and FSH (Chakravarti et al., 1976; Gambacciani et al., 1987; Kazer et al., 1987; Veldhuis et al., 1989) similar to the rise in gonadotropin secretion in young women who have had their ovaries surgically removed (Wallach et al., 1970; Monroe et al., 1972;

Chakravarti et al., 1977). Therefore, the rise in serum gonadotropins in post-menopausal women has been attributed to loss of steroid negative feedback on higher centers. Decreased ovarian inhibin secretion may also contribute to the markedly elevated FSH levels in post-menopausal women (McLachlan.et al., 1986). Indeed, the loss of inhibin has been proposed to occur early in the menopausal transition because plasma FSH increases in perimenopausal women before LH (McLachlan et al., 1988). The most common menopausal symptom is the hot flush, which occurs in approximately 75% of post-menopausal women (McKinlay and Jeffreys, 1974; Hammar et al., 1984). Flushes also occur in hypogonadal men (Heller and Meyers, 1944; Frodin et al., 1985). Flushes are secondary to the loss of ovarian steroid secretion because they can be triggered in young women by ovariectomy and treated by estrogen replacement (Chakravarti et al., 1977; Casper and Yen, 1985). Hot flushes are characterized by facial and upper torso flushing, perspiration, tachycardia and the subjective sensation of heat (Casper and Yen, 1985). There is adramatic increase in finger temperature (Molnar, 1975) secondary to peripheral vasodilation and increased cutaneous blood flow (Ginsburg et al., 1981). Although the core temperature is normal at the onset of the hot flush, it declines during and after the flush episode

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(Molnar, 1975). Because the hot flush appears to be an inappropriate activation of heat loss mechanisms in the presence of normal body temperature, hot flushes have been categorized as a disorder of central thermoregulation (Casper and Yen, 1985). Hypothalamic dysfunction is postulated because the anterior hypothalamus is the central regulatory center for thermoregulation (Kobayashi, 1988). Hypothalamic circuits mediating pulsatile LH release may also be responsible for the initiation of hot flushes. Each flush episode coincides with a pulse of LH, indicating that the flushes are integrated with the central reproductive neuroendocrine control center (Casper et al., 1979; Tataryn et al., 1979). The flushes are not caused by pulsed LH because they occur in hypophysectomized patients (Mulley et al., 1977). In addition, flushes occur after estrogen withdrawal in patients with isolated gonatotropin deficiency and anosmia (Gambone et al., 1984). Because patients with isolated gonadotropin deficiency have a defect of gonadotropin-releasing hormone (GnRH) secretion, increased GnRH secretion is not the causative factor of hot flushes (Gambone et al., 1984). In contrast, flushes are not produced by estrogen withdrawal in patients with hypothalamic amenorrhoea which is thought to arise from dysfunctional input to GnRH neurons. Therefore, altered neurotransmitter input to GnRH neurons has been proposed to be the cause of menopausal flushes (Gambone et al., 1984). Despite the profound alterations in circulating hormone levels and the evidence for hypothalamic dysfunction in the form of hot flushes, advances in studying human reproductive senescence have lagged far behind animal studies. In particular, there have been few attempts to examine morphological changes in the central nervous system of postmenopausal women. In 1966, Sheehan and Kovacs described neuronal hypertrophy in a subdivision of the infundibular (arcuate) nucleus of post-menopausal women. They named the subdivision the subventricular nucleus, in reference to its location below and lateral to the third ventricle (caudal to the tuberoinfundibular

sulcus). Using qualitative cell counting techniques, they described neuronal enlargement in the subventricular nucleus of post-menopausal females compared to pre-menopausal females and men. Neuronal hypertrophy was also identified in young women suffering from post-partum hypopituitarism with complete loss of ovarian function. However, enlargement of neurons did not occur in these patients if residual pituitary function resulted in sufficient ovarian estrogen secretion to prevent uterine atrophy (Sheehan 1967). These findings suggested that ovarian failure causes infundibular neuronal hypertrophy. Interestingly, neuronal hypertrophy was also described in patients at the end of pregnancy, although this was characterized as having a different morphological appearance than post-menopausal hypertrophy (Sheehan, 1967). The finding of neuronal enlargement in the hypothalami of post-menopausal women was subsequently confirmed by measurements of nuclear diameters in the infundibular nucleus of premenopausal and post-menopausal women (De Rooij and Hommes, 1974). However, the size of infundibular nuclei in older men was the same as in older women (De Rooij and Hommes, 1974). This discrepancy with the previous studies may be due to differences in methodology. De Rooij and Hommes measured diameters of 50 nuclei, while Sheehan and Kovacs classified and counted cells according to morphologic appearance and size without actual measurements. In addition, inclusion of chronically ill older men may have inadvertently affected the results of both studies. Indeed, infundibular neuronal hypertrophy has been described in chronically ill, hypogonadal men, and in patients suffering from starvation and gonadal atrophy (Hart, 1971; Ule et al., 1983). Neuronal hypertrophy occurs in a subpopulation of neurons in the infundibular nucleus of postmenopausal women Our initial observation of post-menopausal neuronal hypertrophy was made in a 67-year-old female who had died secondary to acute thoracic trauma.

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Fig. 1 . Representative photomicrographsof cresyl violet-stained sections of the infundibular nucleus of premenopausal ( A )and postmenopausal( E )women. The hypertrophied neuronsare distinguishednot only by increased soma size, but also by larger nuclei, nucleoli and increased Nissl substance. Bar, 20 pm for both photomicrographs. (From Rance et al., 1990.)

The hypertrophied infundibular neurons were distinguished not only by enlarged somata but also by increased Nissl substance, and enlarged nuclei and nucleoli (Fig. 1 , see also Sheehan and Kovacs, 1966). The phenomenon of post-menopausal neuronal hypertrophy was intriguing because of its association with estrogen withdrawal and its location within the hypothalamic control center for reproduction (Krey et al., 1975; Knobil, 1980). Therefore, wedecided to study this phenomenon with the hope of prdviding new information on the control of reproduction in the human. Several modifications were made from the previous studies (Sheehan and Kovacs, 1966; De Rooij and Hommes, 1974; Ule et al., 1983). Most important was the sole use of subjects dying from sudden, unexpected causes such as acute myocardial infarcts or trauma. Complete autopsies were used to confirm the lack of systemic or neuropathologic illnesses. In addition, histologic examination of the ovaries and endometria was performed. Because en-

dometria remains responsive to ovarian steroids post-menopausally (King et al., 1979; Demopoulos, 1982), the endometrial biopsy was used as an internal bioassay to confirm post-menopausal status and to determine if hormonal replacement therapy had been given. We also determined that the ovaries of the pre-menopausal group had an appropriate number of follicles. The endometria of the pre-menopausal subjects were in various stages of the menstrual cycle. Our first goal was to quantitate changes in neuronal size in the infundibular nucleus of post-menopausal women (Rance et al., 1990). We compared formalin-fixed, paraffin-embedded, cresyl violetstained sections from hypothalami of pre-menopausal and post-menopausal women. Sagittal sections were used to standardize the plane of sectioning (parallel to the third ventricle), and to examine the infundibular nucleus in its largest dimension. We found that sections of 12 pm were thin enough to appreciate neuronal morphology, but thick

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Fig. 2. Projection drawing of a sagittal section through the human hypothalamus. The dotted lines delineate the dorsal and ventral boundaries of thecaudal infundibular nucleus. The black area indicates the location of the boundary where Nissl-stained neurons were studied (Rance et al., 1990). In subsequent studies of neurons labeled by in situ hybridization, theanterior-posterior dimension of the boundary was doubled. (cf., Nauta and Haymaker, 1969, p. 142, fig. 4-4.) AC, Anterior commissure; F, fornix; IF, interventricular foramina; MB, mammillary body; OC, optic chiasm; PS, pituitary stalk; VM, ventromedial nucleus; POA, preoptic area. (From Rance et al., 1990.) Nissl Stain

-

!OO

~ - P r e r n e n o p . u r a l 100 ~-Poatmenopausal

a, 80 u

80

m

,

Estrogen Receptor mRNA

I

%

o

60

60

40

40

20

20

r )

5 0

b

a

"

" 50-200

>ZOO

50-200

>ZOO

Cross Sectional Area (prn')

Fig. 3. Percentages of neurons less than and exceeding 200 pm2 cross-sectional area in the infundibular nucleus of premenopausal and post-menopausal women. Left: histogram of all neurons stained with cresyl violet; right: histogram of neurons expressing estrogen receptor gene transcripts. (From Rance et al., 1990.)

enough to distinguish nuclear groups. The hypothalami were serially sectioned and every fifth section was stained with cresyl violet. After the slides were coded, a representative section was chosen from each subject and a 0.5 mm2 boundary drawn

within the infundibular nucleus (Fig. 2). Soma areas of more than 3500 neurons were measured using an image-combining computer microscope. The image-combining computer microscope greatly facilitates accurate cell counting and area measurements by the superimposition of the digitized image over the microscopic field (Glaser et al., 1983). This study clearly demonstrated that neurons in the infundibular nucleus of the post-menopausal females were hypertrophied (Figs. 1 and 3). The mean cross-sectional area ( + S.E.M.) of infundibular neurons in the post-menopausal women (190.4 2.1 pm2) was elevated nearly 30% relative to those in the pre-menopausal group (147.0 + 2.2 pm2). There was a distinct increase in neurons exceeding 200 pm2 in area, indicating that the hypertrophy occurred in a subpopulation of neurons (Fig. 3). In contrast, neurons in the medial mammillary nucleus were the same size in pre-menopausal and post-menopausal women. Therefore, the infundibular neuronal hypertrophy was not an artifact of tissue processing or a non-specific generalized effect of aging. Densities of neurons and glia were comparable in both groups. The lack of gliosis was confirmed by immunoperoxidase staining for glial fibrillary acidic protein of adjacent sections. These data suggest that post-menopausal neuronal hypertrophy is not a compensatory response to local cell death. Hypertrophied neurons in the infundibular nucleus of post-menopausal women express estrogen receptor gene transcripts If post-menopausal neuronal hypertrophy was secondary to the loss of ovarian steroids, it seemed likely that the hypertrophied cells were target tissues for estrogen and would contain the estrogen receptor. Because mRNAs are stable for significant postmortem intervals (Johnson et al., 1986), we used in situ hybridization to detect estrogen receptor gene transcripts (Rance et al., 1990). Hypothalami were collected from pre-menopausal and post-menopausal women, dissected into sagittal blocks, snap frozen, and serially sectioned

225

at 20 pm in a cryostat. Every tenth slide was processed for in situ hybridization using a synthetic [35S]-labeled 48-base oligonucleotide probe targeted to bases 1681 - 1728 of human estrogen receptor cDNA (Greene et al., 1986). Adjacent sections were hybridized with a probe against bases 1128 1176 of GnRH cDNA (Seeburg and Adelman, 1984). Northern blot analysis of human breast carcinoma, endometrium and pituitary was performed as a control for specificity for the estrogen receptor probe. An image-analysiscomputer microscope system was used to determine the grain density and measure cross-sectional areas of labeled neurons. The estrogen receptor probe clearly labeled the hypertrophied neurons (Fig. 4). In contrast to our previous Nissl study, where only 36% of the neurons were larger than 200 pm2, nearly all the neurons containing estrogen receptor mRNA were larger than 200 pm2 (Fig. 3). The neurons expressing the estrogen receptor gene in the post-menopausal women were twice as large as those from the pre-meno-

pausal group (Rance et al., 1990). These data provided strong support for the hypothesis that infundibular neuronal hypertrophy is secondary to the loss of ovarian steroids. Additional support for this hypothesis arose from the examination of the hypothalamus of a young, ovariectomized female who did not have estrogen replacement therapy. The infundibular neurons in this subject were enlarged to post-menopausal size and also contained estrogen receptor gene transcripts (Rance et al., 1990). Hybridization of adjacent sections with the GnRH probe labeled small, oval neurons distributed widely in the septa1 region, bed nucleus of the stria terminalis, diagonal band of Broca, medial preoptic area and infundibular nucleus. However, it was very clear that the hypertrophied neurons did not contain GnRH mRNA. This finding was consistent with previous studies in rats showing that the estrogen receptor and GnRH are not colocalized in the same neurons (Shivers et al., 1983). Thus, if estrogen is acting via receptor-mediated mechanisms, estrogen regulation of GnRH secretion may be mediated via interneurons containing estrogen receptors. Hypertrophied neurons in the infundibular nucleus of post-menopausal women contain substance P and neurokinin B messenger RNAs

Fig. 4. Photomicrograph of neurons in the infundibular nucleus of a post-menopausal woman showing hypertrophied neurons labeled by cDNA probes complementary to estrogen receptor mRNA. Bar, 20 pm. (From Rance and Young, 1991.)

A great advantage of studying the hypothalamicpituitary-gonadal axis is the tremendous amount of basic research which has characterized this system. In particular, there is extensive information on neurotransmitters and neuropeptides within arcuatehfundibular neurons of laboratory animals that are colocalized with the estrogen receptor. Nuclear concentration of [3H]-estradiol has been visualized in rat arcuate neurons containing dopamine (Sar, 1984), P-endorphin (Morrell et al., 1985; Jirikowski et al., 1986), dynorphin (Morrell et al., 1985), substance P (SP) (Akesson and Micevych, 1988) and growth hormone-releasing hormone (GHRH) (Shirasu et al., 1990). In addition, there is an extensive literature on classical neurotransmitters and neuropeptides which have been shown to influence gonadotropin release and

226

Fig. 5 . Photomicrographs of neurons in the infundibular nucleus of pre-menopausal ( A and C) and post-menopausal women (B and

D).In the pre-menopausal woman small neurons are labeled with cDNA probes complementary to SP(A) and NKB ( C ) mRNAs. Note the hypertrophied neurons in the infundibular nucleus of a post-menopausal woman containing SP (B)and NKB (0)mRNAs. SP and NKB neurons in the post-menopausal woman also are more numerous and more densely labeled than those in the pre-menopausal woman. Bar, 20 pm on all micrographs. (From Rance and Young, 1991.)

227

are modulated by estrogen (Fuxe et al., 1969; Rance et al., 1981; Kalra and Kalra, 1986; Aronin et al., 1986; Rasmussen et al., 1986; Merchenthaler et al., 1990). We used these data to provide clues to search for the peptide content of the hypertrophied neurons (Rance and Young, 1991). Because we had previously established that estrogen receptor mRNA is a marker for the hypertrophied population (Rance et al., 1990), we compared the estrogen receptor mRNA containing neurons in the infundibular nucleus of post-menopausal women to adjacent sections incubated with various cDNA probes. Frozen hypothalami from three postmenopausal women were serially sectioned and every tenth slide was stained with cresylviolet. These slides were used to locate the level of the infundibular nucleus which contained the hypertrophied neurons. Adjacent unstained slides were chosen for in situ hybridization using synthetic [35S]labeled48base oligodeoxyribonucleotide probes complementary to estrogen receptor, corticotropin releasing factor (CRF), dynorphin, neuropeptide Y,GHRH, cholecystokinin, enkephalin, proopiomelanocortin (POMC), SP, neurokinin B (NKB), tyrosine hydroxylase and galanin cDNAs (Rance and Young, 1991). To verify probe specificity, Northern analyses at the same conditions of stringency were performed on total RNAs (Chirgwin et al., 1979) from human hypothalamus, hippocampus, caudate, pituitary, endometrium and breast carcinoma using the same probes. A second indicator of specificity was the differential distribution of labeled cells using the various probes, many of which had similar percentages of guanine-cytosine base pairs. These studies revealed that the hypertrophied neurons in the infundibular nucleus of postmenopausal women contained SP and NKB mRNAs (Fig. 5B,D). This finding was readily apparent by light microscopic examination and was confirmed by measuring the cross-sectional areas of labeled neurons. The hypertrophied neurons containing NKB and SP gene transcripts were comparable in size to the hypertrophied cells containing estrogen receptor mRNA (Table I). In contrast, neurons containing GHRH, POMC,

TABLE I Mean cross-sectional area of neurons in the infundibular nucleus of post-menopausal women labeled by in situ hybridization with various cDNA probes (from Rance and Young, 1991) Probe

Cell area (cm2 + s.E.M.)

Estrogen receptor Neurokinin B Substance P GHRH POMC Galanin Enkephalin CCK Neuropeptide Y

284.0 275.0

&

5.0

5.7* 5.S 194.4 * 6.1 179.7 f 4.2 f

263.7 r

173.9 f 4.2 153.7 & 3.6 153.7 f 7.8 101.1 k 1.8

Values represent mean & S.E.M. from three subjects except for neuropeptide Y and enkephalin (two subjects). * Mean cross-sectional area not significantly different from cells labeled with estrogen receptor probe.

galanin, enkephalin, cholecystokinin and neuropeptide Y mRNAs were much smaller than those containing estrogen receptor gene transcripts (Table I). Although CRF and dynorphin neurons were large (mean areas of 259.7 k 18.5 and 235.5 f 12.7 Fm2, respectively), there were few of these cells within the infundibular nucleus. Cells labeled with the tyrosine hydroxylase probe were numerous surrounding the infundibulum, but were not identified in the caudal infundibular nucleus where the hypertrophied neurons were the most prominent. Menopause is associated with marked increases in tachykinin gene expression The above procedure indicated that the hypertrophied neurons in the infundibular nucleus of post-menopausal women contained SP and NKB gene transcripts. We next compared the crosssectional areas and gene expression of SP and NKB neurons in the infundibular nucleus of premenopausal and post-menopausal women (Rance and Young, 1991). We predicted that tachykinin gene expression would be increased because hyper-

228

trophy is a morphologic marker of increased activity. Every tenth section from the hypothalami of three pre-menopausal and three post-menopausal women was incubated with [35S]-labeled cDNA probes complementary to SP or NKB mRNAs. After coding the slides, a representative section was chosen and a 2 mm2 boundary drawn within the infundibular nucleus. We determined the density of labeled neurons (defined as > 5 x background) and also measured the size and grain density of labeled neurons using an image-analysis computer microscope system. The cell areas of infundibular NKB and SP neurons in the post-menopausal group were elevated 194% (NKB) and 176% (SP) relative to the premenopausal neurons (Figs. 5 and 6). In addition, there was a striking increase in the total number of 3007 1'

Are:

Premenopausal postmenopausal

200 N

E, 100

cells expressing SP and NKB mRNAs in the postmenopausal infundibular nucleus. This observation was confirmed by our cell density measurements which showed a six-fold (SP) and 15-fold (NKB) increase in the density of labeled neurons in the postmenopausal infundibular nucleus (Fig. 6). The increased gene expression was also evident by the significant increase in grain density (Fig. 6). Although most of the labeled neurons in the post-menopausal women were hypertrophied, there was also an increase in the density of smaller SP and NKB neurons. The finding of increased SP gene expression in the hypothalami of post-menopausal women is consistent with the hypothesis that infundibular neuronal hypertrophy is secondary to ovarian steroid withdrawal. Studies in experimental animals show thai gonadal steroids modulate SP in the hypothalamus. Hypothalamic SP content (Antonowicz et al., 1982; Kerdelhuk et al., 1982; Micevych et al., 1988) and the number and size of immunoreactive SP neuron5 in the arcuate nucleus (Tsuruo et al., 1984)vary with the rat estrous cycle. Moreover, mean SP release rates in preoptic push-pull perfusates art significantly higher in ovariectomized rats than in ovariectomized estrogen-treated rats (Jarry et al. 1988), consistent with the idea that estrogen downregulates SP release. In another study, 10 days oj estrogen replacement in ovariectomized rats in. creased total hypothalamic SP mRNAs. This finding is at variance with what we would predict from our studies (Brown et al., 1990). However, it is we1 established that estrogen feedback may be eithei positive or negative depending on numerous factor: including time and dose (Barraclough, 1973 Karsch, 1987). !

0 Grain Density

60

1

300

1

Cell Density

+

#

T

General discussion "

Substance P Neurokinin B

Fig. 6. Cross-sectional area, grain density and cell density (mean + S.E.M.) of neurons containing SP and NKB mRNAs in the infundibular nucleus of pre-menopausal and post-menopausal women ('P < 0.05; * P < 0.01; * P < 0.001, compared to premenopausal). (From Rance and Young, 1991.)

The regulation of gonadotropin secretion vk negative feedback of ovarian steroids has been ar enduring concept since its description by Moore anc Price in 1932. Although originally hypothesized a: a reciprocal relationship between ovary and pitui. tary, the discovery of the directional flow of thc

229

hypothalamic portal system (Wislocki and King, 1936; Green and Harris, 1949) and the characterization of hypothalamic peptides with gonadotropinreleasing activity (Amoss et al., 1971; Matsuo et al., I97 1) firmly established the hypothalamus as the central neuroendocrine control center. Steroid hormones could act on this control center to regulate GnRH secretion via hormone specific receptors or epigenetic mechanisms (Pfaff and McEwen, 1983). Presumably, removal of steroid negative feedback results in increased GnRH secretion into the portal circulation. Although ovarian steroid withdrawal results in increased gonadotropin secretion in post-menopausal women, it is not known if this is caused by increased GnRH stimulation of the anterior pituitary gland. The hypothalamic content of GnRH decreases in both ovariectomized and post-menopausal women (Parker and Porter, 1984). However, a decline in hormone content may be due to decreased biosynthesis, increased degradation in the hypothalamus, or increased secretion of GnRH into the portal capillary circulation. Measurement of GnRH in peripheral plasma has been uninformative because of the relative confinement of the hormone in portal blood and its short half-life (Arimura et al., 1974; Pimstone et al., 1977; Barron et al., 1982; Crowley et al., 1985). Our studies indicate a hypothalamic site for steroid negative feedback in the human. The following data summarize the current support for the hypothesis that post-menopausal neuronal hypertrophy is due to the loss of ovarian steroids: (1) neuronal hypertrophy occurs in young women suffering from post-partum hypopituitarism with complete gonadal atrophy (Sheehan, 1967) and in a young ovariectomized woman (Rance et al., 1990); (2) neuronal hypertrophy occurs in men who have chronic illness resulting in gonadal atrophy (Ule et al., 1983); (3) the hypertrophied cells are located within the putative regulatory center for reproduction in the primate (Krey et al., 1975) and within the region of the “GnRH pulse generator” (Silverman et al., 1986); (4) the hypertrophied neurons contain estrogen receptor mRNA indicating they are target

tissues for estrogen action (Rance et al., 1990); (5) the hypertrophied neurons contain SP mRNA (Rance and Young, 1991) which is modulated by ovarian steroids (Antonowicz et al., 1982; Kerdelhue et al., 1982; Tsuruo et al., 1984; Jarry et al., 1988; Micevychet al., 1988; Brownet al., 1990); and (6) steroid withdrawal produces proliferative ultrastructural changes in the rat arcuate nucleus (Brawer, 1971; King et al., 1974; Price et al., 1976; Leranth et al., 1985) and conversely, estradiol implantation results in smaller neuronal nucleoli (Lisk and Newlon, 1963). Estrogens modify brain function, in part, by altering gene expression (Beato et al., 1987; Romano et al., 1988, 1989; Lauber et al., 1990). The estrogen receptor belongs to a large superfamily of regulatory molecules which act as ligand-dependent transcription factors (Yamamoto, 1985; Beato et al., 1987; Evans, 1988). Depending on the cell type, a particular hormone can either induce or repress different sets of genes (Beato et al., 1987; Adler et al., 1988). Thus, gene induction or repression provides a molecular mechanism whereby estrogen may have either positive or negative feedback effects. The technique of in situ hybridization allows quantitation of relative changes in mRNAs at the cellular level, while preserving anatomical localization. An elevation in mRNA is generally interpreted as reflecting changes in gene transcription, although changes in mRNA stability cannot be ruled out (Brock and Shapiro, 1983; Shapiro et al., 1987). Regardless of the mechanism that elevates gene transcripts, there is evidence that changes in the activity of peptidergic neurons is correlated to changes in prepropeptide mRNAs (Comb et al., 1987; Young and Zoeller, 1987; Selmanoff et al., 1991). The present data lend support to this hypothesis because elevations in prepropeptide mRNAs are found in cells exhibiting larger somata, nuclei, nucleoli and increased rough endoplasmic reticulum (Nissl substance). These findings indicate that the increased gene transcripts in the hypertrophied neurons reflect increased activity. What are the consequences of the increased activity of the tachykinin neurons? The most logical

230

scenario is that activation of SP and NKB neurons ultimately results in increased GnRH secretion. In the rat, SP neurons originating in the post-infundibular arcuate nucleus synapse with GnRH somata in the septal-preoptic area (Tsuruo et al., 1991). SP stimulates GnRH release from the rat medial basal hypothalamus in vivo in a dose-dependent manner (Ohtsuka et al., 1987). Moreover, intraventricular injection of SP into castrated rats increases LH release (Vijayan and McCann, 1979; Arisawa et al., 1990) and injection of SP antisera or antagonists suppresses LH release (Dees et al., 1985; Arisawa et al., 1990). LH is released in a pulsatile fashion, which corresponds to pulses of GnRH released into the portal system (Carmel et al., 1976; Clarke and Cummins, 1982). Pulsatile GnRH secretion, in turn, appears to be driven by synchronous multiunit activity in the medial basal hypothalamus (Wilsonet al., 1984). Interestingly, the hypertrophied neurons are located within the region of the “GnRH pulse generator” (Silverman et al., 1986). Therefore, it is conceivable that the hypertrophied tachykinin neurons could influence gonadotropin release by modifying the pattern of pulse activity. Another fascinating possibility is that the tachykinin neurons are the source of the synchronous multiunit activity in the medial basal hypothalamus. Alternatively, SP may modulate gonadotropin release at the level of the anterior pituitary gland. In this case, GnRH secretion need not be elevated to explain the increased plasma gonadotropins in postmenopausal women. SP could be secreted into the portal circulation via terminals ending on the primary capillary plexus (Hokfelt et al., 1978; Tsuruo et al., 1983; Makara et al., 1986; Palkovits et al., 1989) and bind to specific SP receptors in the anterior pituitary gland (Wormald et al., 1989). Comparable information is not available for NKB. We also hypothesize that the increased activity of the tachykinin system in post-menopausal women may be related to the onset of menopausal flushes (Rance and Young, 1991). Menopausal flushes are induced by estrogen withdrawal (Gambone et al., 1984; Casper and Yen, 1985). Each flush is closely

associated with an LH pulse, indicating a link between the control of GnRH release and the onset of flushes (Casper et al., 1979; Tataryn et al., 1979). How could the altered tachykinin system produce menopausal flushes? Tachykinin neurons projecting to the medial preoptic area (Tsuruo et al., 1991) may directly influence thermoregulation in addition to their proposed effect on GnRH neurons. An alternate hypothesis is that tachykinins may be periodically released into the portal system in sufficient amounts to have systemic effects. Tachykinins are potent systemic vasodilators (Pernow, 1983). Intravenous infusion of SP into the brachial artery dramatically increases forearm blood flow (Eklund et al., 1977). Bright red flushing ensues, particularly in the head and neck, and the subjects report a feeling of warmth (Eklund et al., 1977). In addition, systemic infusion of substance P in normal men causes flushing in a dose-dependent manner (Schaffalitzkey de Muckadell et al., 1986). With small doses, flushing of the face and neck is observed (Schaffalitzkey de Muckadell et al., 1986). As the dose is increased, flushing is observed in the face, neck and upper extremities in a similar distribution as menopausal flushes (Casper and Yen, 1985). In support of this theory is the documentation that carcinoid flushes are associated with increased circulating tachykinins in peripheral blood (Norheim et al., 1986; Conlon et al., 1987; Balks et al., 1989; Oberg et al., 1989). A critical piece of information missing is the blood level of plasma tachykinins at the time of a menopausal flush. Summary and conclusions Neuronal hypertrophy occurs in a subpopulation of neurons in the infundibular nucleus of post-menopausal women. The hypertrophied neurons contain NKB, SP and estrogen receptor gene transcripts. Although associated with reproductive aging, postmenopausal neuronal hypertrophy is not a sign of central nervous system degeneration. Quite the opposite, because the hypertrophy is accompanied by marked increases in tachykinin gene expression, reflecting increased neuronal activity. We have pro-

23 1

posed that infundibular neurons containing NKB, SP and estrogen receptor mRNAs participate in the hypothalamic circuitry regulating estrogen negative feedback on gonadotropin release in the human. In addition, there is evidence to suggest that the hypertrophied tachykinin neurons may be involved in the initiation of menopausal flushes. Because menopause affects a well characterized system, and has consistent and substantial changes in hormone levels, we have had the rare opportunity to correlate changes in hormone secretion with structural and neurochemical changes in the human hypothalamus. We suspect that future studies of the hypothalami of post-menopausal women will continue to be a fruitful avenue for investigating neuroendocrine regulation in the human. Acknowledgements The author gratefully acknowledges the collaboration of Drs. N.T. McMullen, W.S. Young, 111, D.L. Price, J.E. Smialek, and B.O. Parks. Supported by NIH Grant AG-09214 and the Arizona Disease Research Control Commission Grant 0-029. References Adler, S., Waterman, M.L., HeXi and Rosenfeld, M.G. (1988) Steroid receptor-mediated inhibition of rat prolactin gene expression does not require the receptor DNA-binding domain. Cell, 52: 685. Akesson, T.R. and Micevych, P.E. (1988) Estrogen concentration by substance P-immunoreactive neurons in the medial basal hypothalamus of the female rat. J. Neurosci. Res., 19: 412 - 419. Amoss, M., Burgus, R., Blackwell, R., Vale, W., Fellows, R. and Guillemin, R. (1971) Purification, amino acid composition and N-terminus of the hypothalamic luteinizing hormone releasing factor (LRF) of ovine origin. Biochem. Biophys. Res. Commun., 44: 205 - 210. Antonowicz, U . , Jakubowska-Naziemblo, B., Cannon, D. and Powell, D. (1982) Immunoreactive substance P content in median eminence and pituitary gland during oestrus, dioestrus and after anterior hypothalamic deafferentation. Endokrinologie, 79: 25 - 34. Arimura, A., Kastin, A.J., Gonzalez-Barcena, J.S., Siller, J., Weaver, R.E. and Schally, A.V. (1974) Disappearance of LHreleasing hormone in man as determined by radioimmunoassay. Clin. Endocrinol., 3: 421 - 425.

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Discussion N. Kopp: Besides GFAP, there are other specific markers of glia. There is a great need for mapping of glia in man. Was there any problem of distortions while freezing? Was there any lefthight asymmetry? N.E. Rance: There are definitely freezing artifacts which is why we have tried to do complementary studies using formalin-fixed material whenever possible. However, we prefer to use frozen tissue for in situ hybridization because our labeling is so good using this method. Explosion of the cells by the formation of ice crystals can be avoided by rapid freezing of small tissue blocks. When we freeze our sagittal sections with the third ventricle side adjacent to a slide, we introduce a mild amount of distortion because the third ventricle is flattened. However, this method does have the advantage of allowing us to cut a full block face without wasting much tissue, and it standardizes our plane of section. We have not examined whether there is any asymmetry to post-menopausal neuronal hypertrophy. E. Braak: Are there any Golgi impregnations of the enlarged neurons in the infundibular nucleus of post-menopausal women to examine dendrites or axons in respect to plasticity. N.E. Rance: No. I am not awareof studies of Golgi-impregnated neurons of the hypothalami of post-menopausal women. I believe these studies would be extremely interesting because I would expect the marked hypertrophy to be accompanied by changes in the dendritic tree. This is supported by recent studies showing that changes in estrogen levels affect the number of dendritic spines in the rat hippocampus (Gould et al., 1990). D.F. Swaab: Our hypothesis is that activation of neurons would help them to survive during aging. This would mean for the activated infundibular nucleus, that there was no neuronal loss in aging (Swaab, 1991). I know from the literature (ule et al., 1983) that the activated neurons were still present in the infundibular nucleus of women of over 100 years of age. However, do you know of total neuronal counts of the infundibular nucleus in aging? Your data are based upon cell density and not on total cell numbers. N.E. Rance: We have not counted total cells or measured the volume of Nissl-stained neurons in the human infundibular nucleus because it is not clear where the exact boundaries of this nucleus are in Nissl sections. In addition, measuring the total cell

numbers may not give us an accurate representation of what is happening to our specific subpopulation. The total number of neurons labeled with substance P (SP) and neurokinin B (NKB) probes is markedly increased in the post-menopausal infundibular nucleus. However, we do not believe that measuring the volume occupied by the NKB and SP neurons, or the number of Nissl-stained neurons within the area occupied by tachykinin neurons is an appropriate reflection of the entire infundibular nucleus. The infundibular nucleus is extremely complex, with many neuropeptides and neurotransmitters. Therefore, one cell type is not representative of the whole. H. Braak: A frequent finding in the infundibular nucleus of aged women is the presence of protrusions from the cytoplasm into the nuclei. Did you observe such a phenomenon? N.E. Rance: This finding was described by Ule et al. (1983). We have not observed protrusions of the cytoplasm into the nuclei, but to be honest we haven’t specifically looked for it. D.R. Repaske: Women who have never seen estrogen, in Turner syndrome for instance, do not have hot flushes, yet they have elevated LH and presumably, LHRH. Why do they not exhibit hot flushes? N.E. Rance: Yes, it is true that it is not the lack of estrogen per se which produces the hot flushes but withdrawal of estrogen. In fact, the most severe flushes are experienced by young ovariectomized women who have the most rapid changes in estrogen levels. The reasons for this phenomenon are unclear. J.J. Legros: Comment: I fully agree that some old men with low testosterone suffer from “hot flushes”. The term “andropause” should be restricted to that kindof man! Further, I agree that hot flushes may be observed in androgen-deficient males with low LH due to hypophyseal insufficiency. This confirms the absence of a role of LH itself in the pathogenesis of hot flushes in man. J.B. Martin: Have you had the opportunity to examine the anterior pituitary? Substance P is colocalized with gonadotropins in the anterior pituitary. N.E. Rance: No. As of this time, we have not examined the anterior pituitary. C.B. Saper: Have you had the opportunity to look at the hypothalamus from men who had been castrated, for example in the successful treatment of prostatic carcinoma. N.E. Rance: No, we have not had the opportunity to do so, but clearly this would be an interesting project. However, interpretation of such a study might be more complicated because this group would have the confounding variable of chronic illness.

References Gould, E. Woolley, C.S., Frankfurt, M. and McEwen, B.S. (1990) Gonadal steroids regulate dendritic spine density in hippocampal pyramidal cells in adulthood. J. Neurosci., lO(4): 1286-1291. Swaab, D.F. (1991) Brain aging and Alzheimer’s disease, “wear

236 and tear” versus “use it or lose it”. Neurobiol. Aging, 12: 317-324. Ule, G . , Schwechheimer,K. and Tschahargane, C. (1983) Morphological feedback effect on neurons of the nucleus arcuatus

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D.F. Swaab, M.A. Hofman, M. Mirmiran, R. Ravid and F.W. van Leeuwen (Eds.) Progress in Brain Research, Vol. 93

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0 1992 Elsevier Science Publishers B.V. All rights reserved.

CHAPTER 16

The human hypothalamo-neurohypophysealsystem in relation to development, aging and Alzheimer’s disease Elmer Goudsmit, Angela Neijmeijer-Leloux and Dick F. Swaab Netherlands Institute for Brain Research, 1105 AZ Amsterdam, The Netherlands

Introduction The hypothalamo-neurohypophyseal system (HNS) is one of the best studied neuroendocrine systems both in experimental animals and in humans. The system consists of hypothalamic magnocellular neurosecretory neurons located in the supraoptic nucleus (SON) and in the paraventricular nucleus (PVN) (Fig. 1). These neurons project to the posterior lobe of the pituitary. In addition to the SON and PVN, clusters of magnocellular neurons belonging to the HNS are found in the hypothalamic gray in between these two nuclei. These clusters are referred to as accessory neurosecretory nuclei (Dierickx and Vandesande, 1977). Immunocytochemical studies have shown.that the peptides vasopressin and oxytocin are present in mutually exclusive sets of neurons (Vandesande and Dierickx, 1975). Transport of neurosecretory material from magnocellular neurons to the neural lobe of the pituitary and the release of this material into the blood stream had already been demonstrated by Bargmann in 1949 (Bargmann, 1949). The magnocellularvasopressin and oxytocin neurons in the SON are considered to project exclusively to the neural lobe of the pituitary (Sofroniew, 1985), although central projections originating in this nucleus were also reported in the rat (Alonso et al., 1986). In contrast to the SON, the PVN not only contains magnocellular vasopressin and oxytocin cells, but also parvocellular neurons which are im-

munoreactive for these peptides. In the rat these parvocellular neurons are situated in distinct parvocellular subdivisions of the nucleus which have been shown to project to central brain regions where the peptides probably act as neurotransmitters or neuromodulators (Buijs, 1983; Sofroniew, 1985). An additional group of parvocellular vasopressin neurons was shown to project to the median eminence in the rat (see Sofroniew, 1985). In these cells vasopressin is colocalized with corticotropinreleasing hormone (CRH) (Whitnall et al., 1985). The human PVN cannot readily be divided into parvocellular and magnocellular subdivisions as in the rat. The nucleus rather consists of a mixture of cells of different sizes (Fig. 2). The vasopressin and oxytocin cell populations in this nucleus are more or less overlapping (Dierickx and Vandesande, 1977). No data are available on the relative distribution of centrally and peripherally projecting vasopressin and oxytocin cells within this nucleus in the human brain. Colocalization of vasopressin with CRH was recently observed in small neurons in the human PVN (Raadsheer et al., 1991) and might be used as a marker for neurons which project to the median eminence. In the peripheral circulation vasopressin and oxytocin act as hormones on several target organs. Vasopressin plays an important role in the regulation of blood pressure and plasma osmolality; it induces water retention and natriuresis in the kidneys and is a potent vasoconstrictor. In addition, effects

The neuronal circuitry involved in the regulation of vasopressin and oxytocin release has not yet been completely disclosed. Projections from a number of brain-stem nuclei, notably the nucleus of the solitary tract, appear to play an important role (Cunningham and Sawchenko, 1988). In addition, evidence has been presented for hormone-, volumeand osmo-receptive elements in periventricular organs like the subfornical organ which project either directly or indirectly to the SON and PVN (Swanson, 1987). Magnocellular neurons themselves also appear to be sensitive to changes in osmolality (Mason, 1980; Oliet and Bourque, 1991). Changes in HNS function during aging: early studies In the older literature, a functional impairment of the rodent HNS was proposed to occur in senescence. This view was based on evidence that vasopressin levels in plasma and urine were reduced in aged animals in combination with a diminished antidiuretic response following osmotic stimulation (Friedman et al., 1956; Turkington and Everitt, 1976).

Fig. 1. Human hypothalamus, frontal section stained immunocytochemicallyfor vasopressin. Vasopressin cells are stained in the supraoptic, paraventricular and suprachiasmatic nuclei. Abbreviations: OC, optic chiasm; PVN, paraventricular nucleus; SCN, suprachiasmatic nucleus; SON, supraoptic nucleus; 111, third ventricle. Bar, 1 mm.

on carbohydrate metabolism in the liver, on platelet aggregation and on blood coagulation have been described (for a recent review, see Cunningham and Sawchenko, 1991). Oxytocin is well known for its role in labor and lactation (Swaab and Boer, 1979). In males oxytocin peaks during ejaculation, acting on the smooth musculature of the vas deferens. Recent work inexperimental animals has shown the involvement of oxytocin in fat and carbohydrate metabolism (for a review, see Cunningham and Sawchenko, 1991).

In the sixties, an impairment in retention in a passive avoidance paradigm was observed in rats following hypophysectomy (De Wied, 1965). This impairment could be restored by both peripheral and central administration of vasopressin and vasopressin analogues which lacked antidiuretic and vasopressor activity (De Wied, 1983). These results were interpreted as an enhancement of memory processes by vasopressin which was proposed to act as a hormone on the brain (for a review, see De Wied, 1983). This hypothesis was of course of great interest in view of the decline in posterior pituitary function which had been observed in aged rats. A similar decrease in HNS function appeared to occur in human aging. Legros (1975) reported a decrease in plasma neurophysin levels in men after the age of 50. These and other studies gave rise to the hypothesis that an age-related decrease in vasopressin secretion might, at least in part, account for cognitive impairments in the elderly (for a review, see De

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Fig. 2. Frontal section through the human paraventricular nucleus stained immunocytochemicallyfor vasopressin. Cells of various sizes are scattered throughout the nucleus. P V N , Paraventricular nucleus; 111, third ventricle. Bar, 250 pm.

Wied and Van Ree, 1982). Subsequently, intranasally administered vasopressin was reported to enhance memory in middle-aged men (Legros et al., 1978) and numerous trials with vasopressin administration in elderly and demented subjects were started with the aim of improving cognitive impairments in these conditions.

Evidence of increased HNS activity during aging

The putative relationship between age-related memory decline and impaired HNS function, which became topical in the early 1980s, also gave rise to the question whether degenerative alterations might take place in the magnocellular neurons in the SON

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and PVN during aging. This question prompted several groups to study the aging HNS from a morphological point of view. In the rat, no changes in cell numbers were observed in the SON and PVN during aging (Hsu and Peng, 1978; Peng and Hsu, 1982; Flood and Coleman, 1983). Absence of cell loss during aging was also observed in the human SON and PVN (Hofman et al., 1988, 1990; Goudsmit et al., 1990a). When the morphology of the vasopressin and oxytocin cells in human postmortem tissue was studied in more detail using immunocytochemical and morphometric techniques, some unexpected results were obtained. Fliers et al. (1985) observed an increase in the size of the vasopressin cells in the human SON and PVN in subjects older than 80 years. In contrast, the oxytocin cells in these nuclei showed no such change. In order to find out whether this increased vasopressin cell size was due to hypertrophy or to degenerative changes (e.g., accumulation of age pigments), Hoogendijk et al. (1985) determined the size of the nucleoli in the vasopressin and oxytocin cells in the same material. An increase in nucleolar size was observed in the vasopressin cells in the SON and PVN in senescent subjects, but not in the oxytocin cells, suggesting an increase in peptide synthesis in vasopressin cells, but not in oxytocin cells in senescence. Studies in Wistar rats also showed morphological signs of increased neurosecretory activity in the SON and PVN with aging (Fliers and Swaab, 1983). The results of these morphological studies clearly disagreed with the earlier data on decreased plasma levels of vasopressin and neurophysin in aged humans and rodents (see above). However, when Legros extended his studies on plasma neurophysin levels in aging humans to men aged 60 - 100 years, he observed a secondary rise in plasma neurophysin levels after 70 years of age (Legros et al., 1980). Subsequently, other investigators also presented evidence for a gradual increase in plasma vasopressin levels in aging humans and rodents (Frolkis et al., 1982; Kirkland et al., 1984; 0 s et al., 1985). In addition, an increase in vasopressin secretion upon osmotic stimulation was observed in elderly subjects

as compared to young controls (Helderman et al., 1978; Robertson and Rowe, 1980; Philips et al., 1984). In the early studies, which showed declining vasopressin levels in aging rodents, the peptide had been extracted with phenol and measured in a bioassay in which the antidiuretic effect of the extracts was determined in young male animals. This might explain part of the discrepancies with more recent studies which make use of radioimmunoassays. However, the literature on age-related changes in rodent HNS function has remained controversial. The reported data change from marked decreases in vasopressin synthesis, secretion and excretion in senescence to marked increases. Strain differences between the animals used in the various studies might offer an explanation for these discrepancies. In addition, the data are confounded by the use of animals which fail to meet the 50% survival criterion for senescence (cf., Zurcher et al., 1982). When studies with animals which were not old enough according to this criterion are disregarded, decreases in HNS activity and responsiveness were only found in 30 months-old Fisher 344 and Sprague-Dawley rats (Sladek et al., 1981; Zbuzek and Wu, 1982; Zbuzek et al., 1983,1987). Increases in HNS activity were reported in 33 - 34-months-old Brown-Norway rats (Goudsmit et al., 1988), in 32-months-old Wistar rats (Fliers and Swaab, 1983) and in 20 - 23months-old Long-Evans rats (Miller, 1985, 1987). These data suggest the presence of strain differences in HNS aging between Fisher 344 and SpragueDawley rats on the one hand and Brown-Norway, Wistar and Long-Evans rats on the other. All five strains were found to develop an increase in urine production and water intake during aging (Bengele et al., 1981; Beck and Yu, 1982; Miller, 1985; Corman and Michel, 1987; Goudsmit et al., 1988; Phelps et al., 1989). In Fisher 344 and SpragueDawley rats this decline in renal concentrating ability goes together with pronounced histopathological changes in the kidneys (Coleman et al., 1977; Gray et al., 1982). In contrast, histopathological changes in the kidneys of senescent Brown-Norway and Wistar rats are only moderate (Burek, 1978; Gray et

24 1

al., 1982; Ravid et al., 1987). Yet, these two strains show a marked age-related loss in the number and affinity of renal binding sites for vasopressin (Swaab et al., 1986; Ravid et al., 1987; Herzberg et al., 1989). In view of the absence of major renal histopathology these changes might be considered to be due to normal aging rather than to pathology. A loss in renal vasopressin receptors might be expected to result in a decreased renal sensitivity to vasopressin. This appears indeed to be the case since cyclic-AMP production in response to vasopressin was found to be diminished in renal medullary cells of senescent mice as compared to tissue of younger animals (Goddard et al., 1984). Hence, the decreased urinary concentrating ability of aged rodents might be due to a decrease in renal sensitivity to vasopressin. The observation that aged rats drink more than young animals and produce more vasopressin suggests a feedback action mediated by osmo- and/or volume-receptors in order to compensate for the polyuria which occurs in these animals (Fig. 3). Impaired urinary concentrating ability and decreased renal responsiveness to vasopressin also occur in human aging (Miller and Shock, 1953; Rowe et al., 1976). Therefore, an increase in vasopressin production in the human SON and PVN in senes24h Urine volume

Urine osmolality

5

5

1000

$

24h WateP intake

800

3

4

600

0"

400

z *

200

1

4

n

g -- 3

E

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8.' E

2 1

young old

Fig. 3. Urine osmolality, urine volume and water intake in young (4 months) and old (34 months) Brown-Norway rats. Urine osmolality was significantly reduced in aged animals, while urine volume and water intake were significantly increased. Since vasopressin excretion was also increased in the aged animals, these results indicate the development of a moderate renal diabetes insipidus with aging and a subsequent stimulation of drinking behavior and vasopressin release. Bars represent means k S . E . M . ; * , P < O.O5;**,P < 0.01.(FromGoudsmitetal., 1988, with permission from the publisher.)

"- 30 40

I Ctr c60

Ctr >60

Alz >60

Fig. 4. Vasopressin (light bars) and oxytocin (dark bars) cell numbers in the human PVN in young (< 60 years) and old (> 60 years) controls (Ctr) and in Alzheimer patients (Alz). Vasopressin cell numbers increase with age in controls. Alzheimer patients fail to show a similar increase. Oxytocin cell numbers remain unaltered during aging and in Alzheimer's disease. *, Different from young controls and from Alzheimer patients ( P < 0.05). Redrawn using data from Wierda et al. (1991) and Van der Woude et al. (1992).

cence might also be due to renal changes. Recently, we observed a gradual increase in the number of neurons which were immunoreactive for vasopressin in the human PVN with aging (Fig. 4)(Van der Woude et al., 1992). This finding is in line with the morphological and physiological evidence of an increase in vasopressin synthesis in this nucleus during aging (see above). The number of oxytocin-immunoreactive neurons in the PVN was found to remain constant during aging (Fig. 4) (Wierda et al., 1991) which is in line with the absence of morphological signs of activation in these cells in senescence (Fliers et al., 1985; Hoogendijk et al., 1985). The evidence for an activation of the vasopressin cells in the human HNS in senescence might be corroborated by the application of mRNA in situ hybridization to human hypothalamic tissue obtained post-mortem (see Mengod et al., this volume). Increased levels of vasopressin mRNA were demonstrated in patients with clinical evidence of antemortem dehydration (Rivkees et al., 1989). In this small study, the highest levels of vasopressin mRNA were found in the oldest subjects, suggesting that vasopressin synthesis is indeed increased in senescence.

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Alzheimer’s disease

Alzheimer’s disease is the most common form of dementia in the elderly. The pre-senile form of the disease is much less common but tends to be more severe (Hansen et al., 1988). The disease is characterized by a progressive impairment of cognitive function and the presence of characteristic neuropathological changes observed at autopsy (McKhann et al., 1984). Neurochemical research into the nature of this disease has revealed disturbances in numerous transmitter systems, including cholinergic, monoaminergic, peptidergic and amino acid transmitter systems which exceed the changes seen during normal aging (for a review, see Goudsmit et al., 1990b). Decreased levels of vasopressin have been reported in plasma, cerebrospinal fluid and brain tissue in Alzheimer’s disease, suggesting an involvement of both centrally and peripherally projecting vasopressin cells in this disease (Sarensen et al., 1983, 1985; Sundquist et al., 1983; Mazurek et al., 1986a,b; Raskindet al., 1986). Ofcourse, thesefindings were of interest in view of the presumed role of declining vasopressin levels in age-related memory decline (see above). In a recent study (Van der Woude et al., 1992), we found that the number of vasopressin-immunoreactive cells in the PVN of Alzheimer patients was 37% lower than in agematched controls, although values did not drop below values of young controls (Fig. 4). This finding suggests a failure of the vasopressin cells in Alzheimer’s disease to respond to the increased demand for vasopressin in senescence (see above) and appears to support the data on reduced vasopressin levels in Alzheimer patients. In the same study, the nuclei of the vasopressin cells in the PVN of Alzheimer patients were found to be enlarged, suggesting hypertrophy of the unaffected cells, or, alternatively, a selective loss of staining in small vasopressin neurons. Whether a disturbance of vasopressin synthesis in Alzheimer’s disease has any clinical relevance seems doubtful. The results of trials with vasopressin administration to elderly and demented subjects have been rather disappointing (e.g., Wolters et al., 1990)

and at present there remains little evidence that vasopressin administration is an effective treatment for age-related memory disorders, although the peptide apparently enhances certain aspects of cognitive function in healthy volunteers (for a review, see Jolles, 1986). Suprachiasmatic nucleus

In close vicinity to the SON and PVN lies another hypothalamic nucleus which contains vasopressinimmunoreactive cells, viz. the suprachiasmatic nucleus (SCN) (Dierickx and Vandesande, 1977) (Fig. 1). Neurons in the rat SCN have been shown to project exclusively to central brain regions (Watts and Swanson, 1987; Watts et al., 1987). Animal experiments have shown that the SCN is the principal circadian pacemaker of the mammalian brain (Rusak and Zucker, 1979). The rodent SCN receives information about the environmental light-dark cycle from the retina via the retinohypothalamic tract (Moore, 1973) and indirectly via projections of neuropeptide Y-containing neurons in the intergeniculate leaflet (Card and Moore, 1989). Data on the organization and connections of the primate SCN give roughly the same picture, although the presence of a geniculate-hypothalamic projection in the human brain is uncertain (for a review see Moore, this volume). During aging, the amplitude of circadian rhythmicity declines in both rodents and humans (for reviews, see Van Goo1 and Mirmiran, 1986, and Mirmiran et al., this volume). This impairment is even more pronounced in Alzheimer patients (Witting et al., 1990). Cell counts in the SCN of both rats and humans revealed a decrease in the number of vasopressin-immunoreactive neurons in this nucleus during aging (Fig. 5 ) (Swaab et al., 1985, 1987; Roozendaal et al., 1987). In Alzheimer patients this cell loss was even more pronounced, amounting to a 75% reduction in the number of vasopressin cells in this nucleus (Swaab et al., 1985, 1987). Since the integrity of the SCN has been shown to be directly reIated to the expression of its pacemaker qualities (Pickard and Turek, 1983), these degenerative

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input via osmo- and/or volume-receptors (see above). In view of the different aging patterns of the SCN on the one hand and the SON and PVN on the other, the stability of these latter nuclei might be related to the fact that they contain neurons which remain highly active throughout the aging process (Swaab, 1991). Evidence that neuronal activation might also prevent or postpone cell death in other neuronal systems (paraphrased as ”use it or lose it”) was presented in a recent review (Swaab, 1991).

SUPRACHIASMATIC NUCLEUS

Fetal development 20

60 Age (years )

40

00

100

Fig. 5 . Linear regression between vasopressin cell numbers in the human SCN and age. A statistically significant decrease was observed in controls after 60 years of age (P < 0.05). Triangles represent males and circles represent females. Values of Alzheimer patients (closed symbols) are delineated by a minimum convex polygon and were reduced as compared to agematched controls (P< 0.01). Redrawn using data from Swaab et al. (1985, 1987).

changes might form the anatomical basis for disturbances in circadian rhythmicity in Alzheimer’s disease. Degenerative changes have also been reported in the retina and optic tract in Alzheimer’s disease (Hinton et al., 1986; Katz et al., 1989). In addition, Alzheimer patients are generaly exposed to lower light intensities than controls (Campbell et al., 1988). Since the input to the SCN via the retinohypothalamic tract is essential for the entrainment of circadian rhythmicity (Johnson et al., 1988), these changes might cause a decrease in the input which is required for proper functioning of the SCN in Alzheimer’s disease. Hence, the degenerative changes which occur in the SCN in this disease might be secondary to changes in regulatory input to this nucleus. The changes in the SCN during aging and in Alzheimer’s disease are quite distinct from the aging pattern seen in the SON and PVN. The SON and PVN show a striking absence of cell loss during normal aging. The vasopressin cells in these nuclei are probably activated due to an increased stimulatory

The stability of the SON and PVN is not limited to the aging process, but appears to encompass the entire life span, including the fetal period. Cell counts in premature and mature fetuses revealed that adult numbers of vasopressin and oxytocin cells are already present from 26 weeks of gestation onwards (Fig. 6) (Neijmeijer-Leloux et al., in preparation). However, the maturation of the nuclei appeared to continue well beyond this timepoint, since the volume of the vasopressin and oxytocin cell populations was found to increase rapidly from 26 weeks gestation towards term. At term, mean volumes were still only about half of the values observed in the adult (e.g., 1.79 f 0.14 mm3 for the volume of the oxytocin cell population in the PVN in mature infants as compared to 4.04 f 0.21 mm3 in adults),

40

I

5 t 7

30

1

Premature

Mature

Adults

Fig. 6 . Vasopressin (light bars) and oxytocin (dark bars) cell numbers in the PVN of premature (26 - 37 weeks) and mature (37-42 weeks) fetuses and in adults. Adult numbers were already present around 26 weeks gestation (preliminary results from Neijmeijer-Leloux et al., in preparation; for details see text).

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suggesting maturation of this cell group does not stop at birth, but continues post-natally. Data on vasopressin and oxytocin concentration and secretion during the fetal period are scant, but tend to support a gradual maturation of the HNS. Vasopressin and oxytocin can be detected in the human hypothalamus from 11 weeks gestation onwards and concentrations were shown to increase gradually between 11 and 28 weeks gestational age (Burford and Robinson, 1982). Vasopressin levels in the umbilical blood were shown to correlate positively with gestation length (Oosterbaan and Swaab, 1989). From these data it might be inferred that vasopressin and oxytocin neurons are already present and functioning during early fetal life, but continuetomaturethroughoutthefetalperiodandprobably also post-natally. Determination of the nuclear diameter of the vasopressin and oxytocin cells in the SON and PVN during fetal development revealed a rapid increase in nuclear diameter of oxytocin cells in the PVN between 26 and 42 weeks gestation (Neijmeijer-Leloux et al., in preparation), suggesting an increase in neurosecretory activity during this period (cf., Palkovits and Fischer, 1968). The oxytocin cells in the SON showed a similar trend. The vasopressin cells in these nuclei failed to show consistent changes in nuclear diameter during this period. An activation of oxytocin synthesis and release towards term would be in line with a role of oxytocin produced by the fetus in the onset and progression of labor (Swaab and Boer, 1979). Summary and conclusions

The research reviewed in the present paper indicates that vasopressin and oxytocin cells in the human HNS constitute an extremely stable population of neurons throughout the human life span. Increases in the activity of these cells, which are probably related to maturation of the system were observed during fetal development and probably extend well beyond term. During senescence an increase in the activity of the vasopressin cells in the human HNS was observed which is probably a compensation for age-related changes in kidney function. These data

do not support a role of declining vasopressin secretion in age-related memory decline. Although there is some evidence for an impairment of vasopressin synthesis and release in Alzheimer patients, vasopressin cell numbers in Alzheimer’s disease do not fall below values observed in young controls. Furthermore, peripheral administration of vasopressin or vasopressin analogues to AD patients have not yielded consistent results.

Acknowledgements

The secretarial help of Ms. T. Eikelboom in preparing the manuscript is gratefully acknowledged. Expert technical assistance was provided by Mr. B. Fisser. We thank Mr. H. Stoffels and Mr. G. Van der Meulen for preparing the illustrations. Human brain tissue was obtained from the Netherlands Brainbank (Coordinater: Dr. R. Ravid). This work was supported by the Sandoz Foundation for Gerontological Research, The Innovation Fund of the Royal Netherlands Academy of Sciences and the fund ‘‘Beoefening Wetenschap” from the Institute for Obstetrics and Gynecology, St. Radboud ziekenhuis, Nijmegen. References Alonso, G . , Szafarczyk, A. and Assenmacher, I. (1986) Radioautographic evidence that axons from the area of supraoptic nuclei in the rat project to extrahypothalamic brain regions. Neurosci. Lett., 66: 251 - 256. Bargmann, W. (1949) Ueber die neurosekretorische Verknupfung von Hypothalamus und Neurohypophyse. Z . Zellforsch. Mikrosk. Anat., 34: 610-634. Beck, N. and Yu, B.P. (1982) Effect of aging on urinary concentrating mechanism and vasopressin-dependent CAMPin rats. Am. 1.Physiol., 243: F121 -F125. Bengele, H.H., Mathias, R.S., Perkins, J.H. and Alexander, E.A. (1981) Urinary concentrating defect in the aged rat. Am. J. Physiol., 240: F147-Fl50. Buijs, R.M. (1983) Vasopressin and oxytocin - their role in neurotransmission. Pharmacol. Ther., 22: 127 - 141. Burek, J.D. (1978) Pathology of Aging Rats, CRC Press, Boca Raton, FL. Burford, G.D. and Robinson, I.C.A.F. (1982) Oxytocin, vasopressin and neurophysins in the hypothalamo-neurohypophyseal system of the human fetus. J. Endocrinol., 95: 403 - 408.

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247 Serrensen, P.S., Gjerris, A. and Hammer, M. (1985) Cerebrospinal fluid vasopressin in neurological and psychiatric disorders. J. Neurol. Neurosurg. Psychiatry, 48: 50-57. Sundquist, J., Forsling, M.L., Olsson, J.E. and Akerlund, M. (1983) Cerebrospinal fluid arginine vasopressin in degenerative disorders and other neurological diseases. J. Neruol. Neurosurg. Psychiatry, 46: 14- 17. Swaab, D.F. (1991) Brain aging and Alzheimer’s disease “wear and tear” versus “use it or lose it”. Neurobiol. Aging, 12: 317-324. Swaab, D.F. and Boer, K. (1979) Function of pituitary hormones in human parturition - a comparison with data in the rat. In: M.J.N.C. Keirse, A.B.M. Anderson and J. Bennebroek Gravenhorst (Eds.), Human Parturition, Martinus Nijhoff, The Hague, Boston, London, pp. 49 - 71. Swaab, D.F., Fliers, E. and Partiman, T.S. (1985) The suprachiasmatic nucleus of the human brain in relation to sex, age and senile dementia. Brain Res., 342: 37 - 44. Swaab, D.F., Fliers, E. and Ravid, R. (1986) The vasopressin neuron in the aging human and rat brain. In: G.B. Stefano (Ed.), CRC Handbook of Comparative Opioid and Related Neuropeptide Mechanisms, Vol. 2., CRC Press, Boca Raton, FL, pp. 221 -230. Swaab, D.F., Roozendaal, B., Ravid, R., Velis, D.N., Gooren, L. and Williams, R.S. (1987) Suprachiasmatic nucleus in aging, Alzheimer’s disease, transsexuality and Prader-Willi syndrome. In: E.R. De Kloet, V.M. Wiegant and D. De Wied (Eds.), Progress in Brain Research, Vol. 72, Elsevier, Amsterdam, pp. 301 -310. Swanson, L.W. (1987) The hypothalamus. In: A. Bjorklund, T. Hokfelt and L.W. Swanson (Eds.), Handbook of Chemical Neuroanatomy, Vol. 5. Integrated Systems of the CNS, Part I, Elsevier, Amsterdam, pp. 1 - 124. Turkington, M.R. and Everitt, A.V. (1976) The neurohypophysis and aging with special reference to the antidiuretic hormone. In: A.V. Everitt and J.A. Burgess (Eds.), Hypothalamus, Pituitary and Aging, Charles C. Thomas, Springfield, IL, pp. 123 - 136. Van der Woude, P.F., Goudsmit, E., Wierda, M., Purba, J.S., Hofman, M.A., Bogte, H. andSwaab, D.F. (1992) Increasein the number of vasopressin immunoreactive cells in the human paraventricular nucleus with normal aging, but not in Alzheimer’s disease. (In preparation.) Vandesande, F. and Dierickx, K. (1975) Identification of the vasopressin producing and of the oxytocin producing neurons in the hypothalamic magnocellular neurosecretory system of the rat. Cell Tissue Res., 164: 153 - 162. Van Cool, W.A. and Mirmiran, M. (1986) Aging and circadian rhythms. In: D.F. Swaab, E. Fliers, M. Mirmiran, W.A. Van Gool and F. Van Haaren (Eds.), Aging of the Brain and Alzheimer’s disease - Progress in Brain Research, Vol. 70, Elsevier Amsterdam, pp. 255 -277. Watts, A.G. and Swanson, L.W. (1987) Efferent projections of

the suprachiasmatic nucleus: 11. Studies using retrograde transport of fluorescent dyes and simultaneous peptide immunohistochemistry in the rat. J. Comp. Neurol., 258: 230 - 252. Watts, A.G., Swanson, L.W. and Sanchez-Watts, G . (1987) Efferent projections of the suprachiasmatic nucleus: I. Studies using anterograde transport of Phaseolus vulgaris leucoagglutinin in the rat. J. Comp. Neurol., 258: 204-229. Whitnall, M.H., Mezey, E. andGainer, H. (1985)Colocalization of corticotropin-releasing factor and vasopressin in median eminence neurosecretory vesicles. Nature, 317: 248 - 250. Wierda, M., Goudsmit, E., Van der Woude, P.F., Purba, J.S., Hofman, M.A., Bogte, H. and Swaab, D.F. (1991) Oxytocin cell number in the human paraventricular nucleus remains constant with aging and in Alzheimer’s disease. Neurobiol. Aging, 12: 511-516. Witting, W., Kwa, J.H., Eikelenboom, P., Mirmiran, M. and Swaab, D.F. (1990) Alterations in the circadian rest-activity rhythm in aging and Alzheimer’s disease. Biol. Psychiatry,27: 563 - 572. Wolters, E.Ch., Riekkinen, P.. Lowenthal, A,, Van der Plaats, J.J., Zwart, J.M.T. and Sennef, C. (1990) DGAVP (Org 567) in early Alzheimer’s disease patients: an international doubleblind, placebo-controlled, multicenter trial. Neurology, 40: 1099-1101. Zbuzek, V., Fuchs, A., Zbuzek, V.K. and Wu, W.-H. (1988) Neurohypophyseal aging: differential changes in oxytocin and vasopressin release, studied in Fischer 344 and SpragueDawley rats. Neuroendocrinology, 48: 619- 626. Zbuzek, V.K. and Wu, W.-H. (1982) Age-related vasopressin changes in rat plasma and the hypothalamo-hypophyseal system. Exp. Gerontol., 17: 133 - 138. Zbuzek, V.K., Zbuzek, V. and Wu, W.-H. (1983) The effect of aging on vasopressin system in Fischer 344 rats. Exp. Gerontol., 18: 305-311. Zbuzek, V.K., Zbuzek, V. and Wu, W.-H. (1987) Age-related differences in the incorporation of ’H-arginine into vasopressin in Fischer 344 rats. Exp. Gerontol., 22: 113 - 125. Zurcher, C . ,VanZwieten, M.J., Solleveld, H.A. andHollander, C.F. (1982) Aging research. In: H.L. Foster, J.D. Small and J.G. Fox (Eds.), The Mouse in Biomedical Research, Vol. 4, Academic Press, New York, pp. 11 - 35.

Discussion M. Mirmiran: You have made an important point by saying that the differences in stability between certain hypothalamic nuclei (such as the supraoptic and paraventricular vs. the suprachiasmatic nucleus) are based on differential effects of the input (no change for the SON/PVN and a decrease for the SCN). Do you think that the changes found in the development of the SON/PVN in premature infants can be the result of dehydration or changes in the osmolality in these infants? E. Goudsmit: Our data show a gradual increase in the volumes

248 of the vasopressin and oxytocin cell populations in the SON and PVN during fetal development. At term the volumes are still only half of the volumes observed in adults. Therefore, it is likely that these volume changes are related to a gradual process of maturation which extends well into post-natal life. As far as I know, there is no evidence for sustained hyperosmolahty during fetal and early post-natal life. The differences in nuclear diameters between premature and mature infants are probably not due to changes in plasma osmolality since these changes occurred only in oxytocin cells and not in vasopressin cells. W.A. Scherbaum: Osmotic stimulation of vasopressin secretion is regulated minute by minute. Most patients during the last hours before death will have a certain degree of dehydration so that I would expect that vasopressin and its mRNA are increased in all your hypothalami. Therefore, I would suggest that you collect data on plasma and urine osmolality in these individuals

before death in order to correlate your mRNA values with the hydration state. E. Goudsmit: I wonder whether dehydration really is a general phenomenon indying subjects. Fluid and electrolyte equilibrium are usually carefully controlled in critically ill patients. Moreover, vasopressin mRNA levels appear to rise more in terms of days than in terms of hours following dehydration (Zingg el al., 1986). Therefore, I expect that elevated levels will only be observed in cases of chronic osmotic stimulation.

Reference Zingg, H.H., Lefebvre, D. and Almazan, G. (1986) Regulation of vasopressin gene expression in rat hypothalamic neurons. J. Biol. Chem., 261(28): 12956- 12959.

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0 1992 Elsevier Science Publishers B.V. All rights reserved.

CHAPTER 17

The hypothalamic lateral tuberal nucleus: normal anatomy and changes in neurological diseases H.P.H. Kremer Department of Neurology, Academic Hospital, 2333 A A Leiden, The Netherlands

Introduction

Although the hypothalamic lateral tuberal nucleus (nucleus tuberis lateralis; NTL) is well known since theendofthenineteenth (1896, Koelliker; referenced in Diepen, 1962) an early twentieth century (Malone, 1910), it escaped the attention of the modern neuroscientific community. This cannot be for lack of size: the complex occupies a prominent place in the lateral hypothalamus. Yet, investigators have focused on the medial nuclei that participate in brain-endocrineinteractions. As the NTL can be unambiguously delineated in man and higher primates only, it was lost to those working with rats. The dogma became established that the lateral hypothalamic area is practically coextensive with the medial forebrain bundle; its interspersed neurons constitute the bed nucleus of the medial forebrain bundle (Nieuwenhuys et al., 1982). But even those studying the human hypothalamus can be misled if closely spaced serial sections are not used. A traditional presentation in which a coronal section through the mid-tuberal area is followed by a coronal section through the middle part of the mammillary body leads to an underestimation of the true extent of the NTL (Nauta and Haymaker, 1969). As the most complete descriptions can be found in the German anatomical literature (Feremutsch, 1955; Diepen, 1962; Strenge, 1975), information may have been difficult to obtain. Still, some excellent English reviews are available (e.g., Le Gros Clark, 1936,

1938; Wahren, 1959; Christ, 1969; Daniel and Prichard, 1975; Saper, 1990). At present, the NTL is an enigmatic structure, whose neurotransmitter(s), connections, and function are unknown, and whose interspecies homologies are disputed. Yet, the nucleus is involved in a variety of human neurodegenerative diseases, making renewed attention to this structure worthwhile. The purpose of this review is to summarize the fragmentary knowledge of the NTL. Details can be found elsewhere in this volume (Braak and Braak, this volume).

Normal anatomy

Location The features that can be used to define the NTL in the human are its position in the lateral tuber cinereum and the morphological appearance of its constituent neurons. The nucleus is found in two or more discrete subdivisions in the basal lateral tuberal hypothalamic region. The bulk lies caudally and is contained within a space which is bordered laterally by the optic tract, dorsolaterally by the internal capsule and ansa lenticularis, dorsomedially by the descending fornix, mediocaudally by the mammillary body, and ventrally by the pia mater of the tuber cinereum (Fig. 1). Almost always a smaller complex can be found situated medioventrally and slightly rostrally from the major lateral part. This division may ex-

250

tend medially to the arcuate (infundibular) nucleus. Small aggregates can be found just caudally from the optic chiasm (Braak and Braak, 1987). In general, the nucleus only appears in coronal sections when the supraoptic nucleus is no longer visible. Macroscopically the presence of the NTL is revealed by the so-called lateral eminence on the ventral surface of the tuber cinereum (Le Gros Clark, 1936, 1938; Diepen, 1962; Daniel and Prichard, 1975). Many authors consider the subdivision of the NTL in two, three, or even more discrete bodies to be characteristic of the nucleus. As Feremutsch (1948) already pointed out, these subdivisions are extremely variable in different individuals. Even if the alterations in outlines of the complex in different sections are taken into account, the variations in form may be such, that in one individual the left NTL is differently shaped from the right. However,

For legend, seep. 251.

NTL cells lying laterodorsally from the fornix in the mid-tuberal plane, as mentioned by Strenge (1975) have never been observed in our own material.

Cytoarchitecture NTL neurons can be easily recognized and are readily distinguished from the neurons of the surrounding nuclear formations. They are triangular, polygonal, or rounded, with a diameter of about 25 pm (Kremer et al., 1990). One or two apical dendrites may be present. The nucleus is situated eccentrically, close to one of the apices, and often shows an indented nuclear membrane. It accounts for onethird to one-half of the total neuronal surface on section. Its nucleolus measures 1 - 2 pm in diameter. The perikaryon is smoothly limited and completely filled with a fine-grained pigment which is stained by lipophilic (lux01 fast blue; Sudan black B) and PAS

25 1

Fig. 1. Normal anatomy of the lateral tuberal nucleus (NTL). A . Overview: the borders of the NTL are indicated. Note position relative to fornix (upper left) and optic tract (to the right). Kluver-Barrera, x 20. B. NTL neurons, in the lower two-thirds of the photo, and tuberomammillary neurons, upper part. The sharp demarcation between the two areas is clearly visible. Kluver-Barrera ( x 100). C. Individual NTL neurons. H and E, x 400.

dyes. The Nissl substance is evenly dispersed over the cytoplasm. Lipofuscin accumulates with increasing age (Wahren, 1964; Daniel and Prichard, 1975; Strenge, 1975). Beautiful staining can be achieved in thick sections (100 pm or thicker) by pigment stains like aldehydefuchsin plus darrow red or gallocyanin chrome alumn (Braak and Braak, 1987, this volume). The neurons are not identical in every part of the nucleus, however. A layer adjacent to the pial surface of the tuberal floor contains' closely packed smaller, darker, more compact and triangular cells. These same characteristics can be found in the neurons making up the medioventral subdivision mentioned above. Whether this represents true cytoarchitectonic (and functional?) heterogeneity, or just artefactual changes due to agony or subsequent handling of the brain remains to be determined.

The NTL is partially surrounded by the large neurons of the tuberomammillary nucleus (TM). The intimate relationship with the tuberomammillary nucleus is stressed by all authors (Ingram, 1940; Christ, 1951; Feremutsch, 1955; Wahren, 1959; Diepen, 1962)and can be used for closer delineation of the NTL. TM neurons can be easily distinguished from NTL neurons: they are larger (up to 40 pm), have an irregularly limited perikaryon, coarse patches of Nissl substance, and a centrally placed nucleus with a large nucleolus. Particularly in the posterior tuberal region TM neurons lie closely apposed to the main mass of the NTL, demarcating it from the rest of the lateral hypothalamic area. The smaller rostra1 NTL extensions are usually not bordered by these large characteristic ganglion cells. Occasional TM neurons of typical appearance may be found lying isolated in the NTL. Furthermore, its

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several subdivisions, if present, are separated by tissue bands containing TM neurons. The NTL’s neuropil shows a homogeneous structure with hardly any myelinated fibers, some vessels, and relatively few glial cells. The fibrous capsule mentioned by several authors (Nauta and Haymaker, 1969; Fujii, 1982) is, according to our observations, not a distinct structure, but formed by extensions of adjoining myelinated structures like the medial forebrain bundle, the internal capsule, and the pes pedunculi. Yet, these myeloarchitectural features facilitate the identification of the NTL as a myelin-poor structure in the myelin-rich environment of the lateral hypothalamus. They explain the excellent results of the Kliiver-Barrera stain (luxolfast blue and cresyl-violet)in delineating the NTL in serial sections.

Neurotransmitters, connections and function As yet, nothing is known about whichneurotransmitters are contained by NTL neurons. Some clues may be scraped from the literature. Najimi et al. (1989), investigating the infant hypothalamus, found a dense to moderately dense group of smallsized somatostatin-immunoreactive cell bodies in what they called the lateral and medial tuberal nuclei, particularly in the ventral and medial parts. We were unable to find somatostatin immunoreactivity in the adult NTL (unpublished data). Somatostatin expression may be a transient phase in the development of the NTL. Galanin-immunoreactive fine caliber fibers were sparsely found in the NTL (Gai et al., 1990). In marmoset monkey brains we observed neuropeptide Y-immunoreactive fibers, although in a lower density than in the surrounding structures (unpublished results). Some LHRH fibers may occur in infancy (Najimi et al., 1990). Some histamine-immunoreactive perikarya apparently occurred within the confines of the nucleus (Panula et al., 1990; Airaksinen et al., 1991), but these may rather represent the isolated TM neurons mentioned in the preceding paragraph, as the TM neurons are generally considered to constitute the major, if not the sole histamine-containing neuronal population in rodents (Steinbusch and Mulder,

1984; Watanabe et al., 1984) as well as in man (Panula et al., 1990; Airaksinen et al., 1991). More is known about receptor distribution in the NTL. These data were all obtained by autoradidgraphy, using various radioligands. Millan et al. (1986) found a very high concentration of corticotropin-releasing factor in cynomolgus monkeys. In adult human post-mortem material the NTL was found to contain high densities of somatostatin binding sites (Reubi et al., 1986; Najimi et al., 1991) and very high densities of muscarinic cholinergic receptors (CortCs et al., 1987). In this, the NTL stood clearly out from the other hypothalamic nuclei. Somatostatin binding in the NTL of infant brains was less conspicuous (Najimi et al., 1991). Moderate densities of benzodiazepine receptors were found in the NTL, in the posterior nucleus, and in the preoptic area of adults, contrasting with high or very high densities in the ventral and medial mammillary nuclei, respectively (Zezula et al., 1988). [3H]-Flunitrazepam binding sites of the NTL were considered to represent almost exclusively the benzodiazepine type I1 receptors (Zezula et al., 1988). Recently, R. Albin demonstrated moderate densities of N-methyl-D-aspartate (NMDA) receptors in the NTL of adults (unpublished observation), while surrounding hypothalamic structures almost completely lacked [3H]-glutamate binding. Although these data on neurotransmitter and receptor contents might be related to the afferent and efferent connections of the NTL, up to now surprisingly little is known about the connections of the NTL with other parts of the brain. In 1925, using myelin stained serial sections, Greving noted a bundle that joined the ansa peduncularis and probably reaches the basal nucleus of Meynert (NBM) and the striatum. Also, thalamic and lower brain-stem connections were described. These findings have not been confirmed by the use of modern anatomical tracing techniques. Considering these basal lacunae in our knowledge of the NTL, it comes as no surprise that we know nothing about its function, or the clinical signs on dysfunction. In the discussion of this review a hypothesis will be proposed.

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Interspecies homologies The lateral tuberal nucleus can be well recognized in man and in simian primates such as Callithricidae, as well as in some prosimiae such as Lemuriformes, but not in Lorisiformes, Tarsiformes, or Tupaiiformes (Fuji, 1982). There used to be considerable controversy as to its presence in other mammals (Diepen, 1962; Strenge, 1975). The problem apparently is related to how we identify the NTL. In Callithrixjacchus, for example, the NTL neurons only remotely resemble those in man, but they are surrounded bymuchlarger (tuberomammillary) neurons, like in man. The cell mass is well demarcated by fibers and situated correctly in the lateral part of the tuberal and pre-mammillary hypothalamus (Stephan et al., 1980). Thus the existence of the NTL should be supported in this species. In a well known hypothalamic atlas of the rat the NTL is mentioned (Bleier et al., 1979). The criteria used, however, are obscure. We tried but failed to identify the NTL in these animals. The issue should be resolved if the appropriate neurotransmitter is found, or if NMDA receptor dense neurons in an animal’s lateral hypothalamus are identified. NTL changes in normal aging and neurological diseases

Normal development and aging No systematic knowledge is available on the normal development of this nucleus. Examining an at term neonatal brain, we found poorly differentiated neurons that were evidently different from those in adult brains. Sometimes it was difficult to discern NTL neurons from tuberomammillary neurons. In a 2.5-year-old infant the characteristic neurons were already recognizable. To study the fate of the NTL in normal aging and in various diseases, we started to count its neurons. The methods employed have been described previously (Kremer et al., 1990, 1991a,b). In our series of 15 adult controls the normal neuronal number was about 60000 k 10000 (mean 2 S.D.). At what age the adult complement of cells is reached is unknown; the number may

*

gradually decline with age, especially in the eighth or ninth decade (Fig. 2). As noted before (Wahren, 1964; Daniel and Prichard, 1975; Strenge, 1975), NTL neurons have their lipofuscin content increased with increasing age.

Alzheimer ’ disease In Alzheimer’s disease (AD) patients the numbers of neurons in the NTL were not different from controls (Fig. 2). In silver and thioflavin-S stains neurofibrillary tangles or abnormal (dystrophic) neurites were rarely visible, and may in fact have been localized in interspersed TM neurons, instead of NTL neurons. A few anti-A4//3-protein immunostaining plaques were present in the AD patients, but they were almost exclusively of the amorphous non-neuritic variety. These amyloid depositions were not different from what was found in the rest of the brain. Thus, it would appear that the NTL shows no special vulnerability in AD. Yet when we used Alz-50 for immunostaining, a completely different picture emerged (Fig. 3). Alz-50 is a monoclonal antibody which recognizes an epitope on an Alzheimer’s disease-associated intraneuronal 68 kDa microtubule MAP T (Wolozin et al., 1986; Ksiezak-Reding et al., 1988; Nukina et al., 1988). Abnormally phosphorylated (Grundke-Iqbal et al., 1986), primitive (Kosik et al., 1989) or otherwise altered 7 proteins are part of the paired helical filaments of the neurofibrillary tangles in AD. They may occur in very early stages of a development that ultimately leads to the formation of neurofibrillary tangles. The cell bodies and the neuropil of the NTL showed such outspoken Alz-50 reactivity that in most cases it clearly stood out from the rest of the hypothalamus and could be recognized by the naked eye (Kremer et al., 1991a). Only the adjoining tuberomammillary nucleus was affected in an equally strong manner. The oldest controls (over age 60) sometimes showed slight immunoreactivity to Alz50. Only a few positive neurons and neurites were recognized. Apparently, the NTL represents a brain area in which AD affects the neurons in a distinct way. The neuronal cytoskeleton is diseased, but the

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Fig. 2. Neuron number counts of the NTL in normal controls and neurological diseases. AD, Alzheimer's disease; PD, Parkinson's disease; HD, Huntington's disease; AIDS: acquired immunodeficiency syndrome. Linear regression analysis of neuronal numbers vs. age: for controls only (n = 15): neuron number = 71955 - (age .237.92), r = - 0.38, P = 0.15; for the total group of contiols, AD, PD and AIDS (n = 32): neuron number = 79126 - (age . 315.19), r = -0.48, P = 0.0053.

Fig. 3. MTL in Alzheimer'sdisease. A . Overview of the NTL.F,Fornix; 0,optic tract. Alz-50, x 20. B. Detail ofA. Prominent staining of neurons as well as neuropil. Alz-50, x 100.

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changes do not progress to the ultimate stage of silver staining tangles and neuronal loss. Subsequent staining of AD hypothalami with anti-r monoclonal antibody, polyclonal antibody against paired helical filaments, and anti-ubiquitin monoclonal, showed about the same density of immunostaining NTL neurons, but far less neuritic staining, compared to Alz-50 (Swaab et al., 1992). The AD pattern of Alz-50 staining in the NTL was also encountered in patients with Down’s syndrome.

Parkinson 3 disease Again, in the NTL of Parkinson’s disease patients we found no neuronal loss (Fig. 2). In accordance with earlier work (Langston and Forno, 1983)’a few Lewy bodies were found in this structure (Kremer et al., submitted). In fact, more of these inclusions could be found than Langston and Forno suggested.

Intracytoplasmatic as well as neuritic forms were recognizable, and sometimes they appeared to lie freely in the neuropil (Fig. 4). Most of them were found in parts of the NTL that are adjacent to the TM. As TM neurons are known to display many Lewy bodies (Von Buttlar-Brentano, 1954; Den Hartog Jager and Bethlem, 1960; Ohama and Ikuta, 1976; Langston and Forno, 1983), it could be argued that at least some Lewy bodies in the NTL in fact represent intraneuritic forms of TM neurons. Yet some NTL neurons do contain these characteristic inclusions in their perikaryon, thus refuting the opinion that Lewy bodies are a sign of neuronal depletion in Parkinson’s disease (Forno, 1982; Gibb and Lees, 1988).

Huntington ’s disease Recently our attention was drawn to atrophy of the NTL in Huntington’s disease (Kremer et al..

Fig. 4. NTL in Parkinson’s disease. Lewy body, apparently free lying in the neuropil. H and E, x 400.

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For legend, see p. 257.

1990). In fact, this was already described by Vogt and Vogt in 1951 and Wahren in 1952. Unfortunately, subsequent authors have neglected their interesting findings. In 18 Huntington’s disease (HD) patients we examined, the NTL showed striking neuronal loss and gliosis (Fig. S), especially in young patients. In elder patients the neuronal depletion was much less clear. Often, a slight increase in apparent glial density was the only indication of pathology. Yet, in all HD cases the remaining number of NTL neurons at death was well below the normal control values and ranged from 28000 to 40600. This wide variation was inversely related to the age-of-death of the patients, but even stronger to the age-of-onset of the

motor symptoms. A late age-at-onset and a late ageat-death were significantly associated with a relative preservation of NTL neurons (Kremer et al., 1991b). Thus, neuronal loss in the NTL may be a good estimator of disease severity, and the NTL may be one of the brain structures that is primarily affected by the deleterious action of the HD gene. According to the hypothesis on excitotoxin-mediated neuronal damage (Meldrum and Garthwaite, 1990) the apparent vulnerability of NTL neurons may be explained by their NMDA receptor content (R. Albin; unpublished data). Interestingly, the surrounding large neurons of the tuberomammillary nucleus are not affected in young nor in old HD cases and they do not contain NMDA receptors.

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(Horn et al., 1988). In a patient with Kallmann’s syndrome (hypogonadotrophic hypogonadism with anosmia) the NTL was “underdeveloped” (Kovacs and Sheehan, 1982). In one case of Pick’s disease we found a normal complement of neurons. Many of these contained Alz-50 staining globose tangles, characteristic of the disease. In patients dying from AIDS and related dementia, in one patient with Prader-Willi syndrome, as well as in a patient with multiple sclerosis, we found no changes. Discussion

Fig. 5. NTL in Huntington’s disease. A . Composition depicting the entire NTL. Note neuronal loss and gliosis of the NTL, with preservation of bordering tuberomammillary neurons. Asterisk indicates detail highlighted in B. Kliiver-Barrera, x 40. B. Detail taken from A. Noted the paucity of NTL neurons, as well as the increase in glial cell density, compared to Fig. 1B. KliiverBarrera, x 100.

Others Pathological changes in the NTL have been described in other diseases. Braak and Braak (1989) noted preferential changes in the NTL in a disease characterized by adult-onset dementia, intraneuronal argyrophilic grains and silver staining coiled bodies containing straight filaments. Selective pyknosis and disintegration of neurons was found in the NTL of a patient with severe depressive illness who died of a malignant neuroleptic syndrome

Apparently, the NTL is a phylogenetically young structure that is affected in a variety of neurodegenerative disorders. The first question to be addressed would be: what makes the NTL so vulnerable to neurodegeneration? Its NMDA receptor content may, through the action of excitotoxic neurotransmitters (Meldrum and Garthwaite, 1990), contribute to its special vulnerability in Huntington’s disease. Yet, the hippocampus and amygdala contain high densities of NMDAreceptors as well (Cortman et al., 1987); these structures seem not to be severely damaged in HD (Bruyn, 1968). In AD the NMDA receptor cannot be related to the occurrence of the cytoskeletal pathology. TM neurons do show Alz-50 reactivity, as well as silver staining neurofibrillary tangles (Ishii, 1966; Simpson et al., 1988; Ulfig and Braak, 1989), but their NMDA receptor density is much lower. The nature of the connections may be a second determinant. If the NTL is indeed connected with the NBM (Greving, 1925) and receives trophic stimulation from this complex, this may contribute to the alterations observed in AD. Obviously, anatomical studies of the NTL connections are of major relevance. What is the clinical significance of the neuropathological alterations? In animals the lateral hypothalamic area is involved in a large variety of behavioral and metabolic functions, e.g., the regulation of feeding and energy metabolism (for review, consult Robbins (1986) and Shimazu (1986)). In rodents ibotenic acid or NMDA lesions of lateral

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hypothalamic interstitial neurons produce longlasting weight loss and feeding and drinking impairment (Winn et al., 1984,1990). The anatomy of the lateral hypothalamus of non-primates is different from that of higher primates. In rodents it is entirely occupied by the medial forebrain bundle (Nieuwenhuys et al., 1982), with interspersed neurons that can only be differentiated by their immunohistochemical characteristics. In primates a well-delineated NTL, a cytologically distinct tuberomammillary nucleus, and a dorsally placed medial forebrain bundle can be discriminated. As noted above, homologies between the NTL of non-primates and primates are uncertain, because immunocytochemical markers for NTL neurons are not yet available. Yet several clinical case reports (White and Hain, 1959; Lewin et al., 1972; Kamalian et al., 1975) describe anorexia-like states with severe weight loss in patients with lesions involving the lower part of the lateral hypothalamus, possibly the NTL. Huntington’s disease patients suffer weight loss during the later stages of their disease, in spite of normal or increased foodintake (Bruyn, 1968; Sanberg et al., 1981; Farrer and Yu, 1985). Weight-loss withnormal food intake in AD patients has recently been documented in several clinical studies (Sandman et al., 1987; Singh et al., 1988; Burns et al., 1989; Franklin and Karkeck, 1989). Moreover, in the disease described by Braak and Braak (1989) the NTL is one of the most affected structures. Of the 28 patients described, 18 were explicitly noted to be cachectic, three were not, and the metabolic status of seven patients was unknown (consult table 2 in their paper). We hypothesize that the NTL may play arole in feeding behavior and metabolism, and that damage to this structure, in Huntington’s and Alzheimer’s diseases, and perhaps in Parkinson’s disease, may impair these functions. Summary and conclusions The lateral tuberal nucleus is a circumscribed cell mass in the lateral posterior part of the hypothalamus, containing about 60000 neurons. It can be recognized in man and higher primates, prob-

ably not in other mammals. Its neurotransmitter content and connections with other parts of the brain are as yet unknown. But receptors for corticotropin-releasing factor and somatostatin, as well as muscarinic cholinergic receptors, benzodiazepine receptors and N-methyl-D-aspartate receptors have been localized within the confines of the nucleus. The lateral tuberal nucleus is affected in a number of human neurodegenerative diseases. Changes in Parkinson’s disease are the least obvious: Lewy bodies appear in small amounts, the majority of them apparently lying outside a neuronal perikaryon. Neuronal loss does not occur. In Alzheimer’s disease the number of neurons seems to be normal as well. Rarely silver staining tangles occur, and the deposition of A4/@-proteinin amorphous plaques is moderate. Yet, NTL neurons stain heavily in Alz-50 immunocytochemistry, while Alz50 staining in NTL neurites is very dense. These changes are interpreted as indicating early Alzheimer-related pathology. In Huntington’s disease the NTL loses neurons. This loss is related to the severity of the disease: patients who first display motor disturbances at an early age will lose more neurons than those who start later. The relation between these clinicalcharacteristics and the severityof neuronal loss is such, that it seems likely that NTL neurons possess a special vulnerability for the effect of the Huntington gene. This could be related to their NMDA-receptor content. It is hypothesized that the NTL is involved in a neuronal network that regulates feeding and metabolism. NTL pathology may explain the peculiar catabolic state of many patients with Alzheimer’s or Huntington’s diseases. Acknowledgements Brains were obtained from the Netherlands Brain Bank in the Netherlands Institute for Brain Research, Amsterdam, coordinator Dr. R. Ravid. Throughout this work, Miss G.M. Dingjan, Department of Neuropathology, Leiden State University, and Mr. B. Fisser, Netherlands Institute for Brain Research, Amsterdam, sectioned and stained literally dozens of hypothalami. Mr. G.J. Van de Gies-

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sen, Department of Neurology, Leiden State University, helped with photography.

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Brain Res., in press. Ulfig, N. and Braak, H. (1989) Amyloid deposits and neurofibrillary changes in the hypothalamic tuberomammillary nucleus. J. Neural Transm. (P.-D. Sect.), 1: 143. Vogt, C. and Vogt, 0. (1951) Precipitating and modifying agents in chorea. J. Nerv. Ment. Dis., 116: 601 -607. Von Buttlar-Brentano, K. (1954) Zur Lebensgeschichte des Nucleus basalis, tuberomammilaris, supraopticus, und paraventricularis unter normalen und pathogenen Bedingungen. J. Hirnforsch., 1: 337-419. Wahren, W. (1952) The changes of hypothalamic nuclei in schizophrenia. In: Proceedings of the First International Congress of Neuropathology 1952, Vol. 3, Rosenberg and Sellier, Turin, pp. 660 - 673. Wahren, W. (1959) Anatomy of the hypothalamus. In: G. Schaltenbrand and P. Bailey (Eds.), Introduction to Stereotaxis with an Atlas of the Human Brain, Georg Thieme, Stuttgart, pp. 119- 151. Wahren, W. (1964) Zur Pathoklise des Nucleus tuberis lateralis. In: Progress in Brain Research, Vol. 5, Elsevier, Amsterdam, pp. 161 - 170. Watanabe, T., Taguchi, Y., Inagaki, S., Tanaka, J., Kubota, H., Terano, Y., Tohyama, M. and Wada, H. (1984) Distribution of the histaminergic neuron system in the central nervous system of rats: a fluorescent immunohistochemical analysis with histidine decarboxylase as a marker. Brain Res., 295: 13 -25. White, L.E. and Hain, F. (1959) Anorexia in association with a destructive lesion of the hypothalamus. Arch. Pathol., 68: 275 - 281. Winn, P., Tarbuck, A. andDunnett, S.B. (1984) Ibotenic acidlesions of the lateral hypothalamus: comparison with the electrolytic lesion syndrome. Neuroscience, 12: 225 - 240. Winn, P., Clark, A., Hastings, M., Clark, J., Latimer, M., Rugg, E. and Brownlee, B. (1990) Excitotoxic lesions of the

lateral hypothalamus made by N-methyla-aspartate in the rat: behavioural, histological and biochemical analyses. Exp. Brain Res., 82: 628 - 636. Wolozin, B.L., Pruchnicki, A., Dickson, D.W. and Davies, P. (1986) A neuronal antigen in the brains of Alzheimer patients. Science, 232: 648 - 650. Zezula, J., Cortks, R., Probst,A. andpalacois, J.M. (1988)Benzodiazepine receptor sites in the human brain: autoradiographic mapping. Neuroscience, 25: 771 - 795.

Discussion C.B. Saper:Two questions about the Alz-50 staining. First, I was impressed by the staining in Alzheimer’s disease. In staining of normal brains in our laboratory, the lateral tuberal nucleus has the lowest staining in the hypothalamus. Is the staining in Alzheimer’s disease an early and consistent finding? Second, did you find Alz-50 staining in the lateral tuberal nucleus in Huntington’s disease? H.P.H. Kremer: Alz-5Ostainingof theNTLwas aconsistent finding in the Alzheimer’s disease brains we examined. It should be noted, however, that these constituted a selected group of cases with advanced disease and clear-cut clinical and post-mortem findings. As yet, I am unable to answer the question whether Alz50 staining of the NTL is an early finding. Neither in Huntington’s disease patients, nor in Parkinson’s disease patients, Alz-50 staining could be observed. J.M.B.V. de Jong: Do you intend to study other diseases leading to cachexia? H.P.H. Kremer: Yes, we would like to. Due to the interspecies differences in lateral hypothalamic anatomy, it will be difficult to extrapolate from animal experiments the clinical relevance of these findings. One of the strategies may be to compare the condition of the NTL in different human diseases. For example, brain from anorexia nervosa patients might offer interesting insights.

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CHAPTER 18

Galanin tuberomammillary neurons in the hypothalamus in Alzheimer’s and Parkinson’s diseases V. L. Chan-Palay and B. Jentsch Neurology Clinic, University Hospital, Zurich 8091, Switzerland

Introduction The hypothalamus is central to the neuroendocrine axis, positioned to integrate the central and peripheral endocrine, autonomic and behavioural responses through brain and pituitary interactions. Within the hypothalamic complex of nerve cell groups, one in particular will be the subject of this review - the tuberomammillary nucleus (TM), particularly the galanin-containing neurons of the TM. The TM is of major interest in the study of brain neurotransmitter systems as it is one of the “papal” systems - so named because a small number of neurons utilising histamine as a major transmitter (Pannula et al., 1990), provide neuronal innervation of large areas of cerebral cortex, hippocampus, hypothalamus and brain-stem. The galanin TM neurons are a part of this cell group and as such retain considerable interest. The other “papal” systems of the brain are the familiar cholinergic basal forebrain neurons, the noradrenergic locus coeruleus neurons, and the serotonergic raphe neurons which provide their respective innervations of large areas of cerebral cortex, hippocampus, hypothalamus and remaining brain regions. Certain degenerative diseases of the central nervous system, such as Alzheimer’s and Parkinson’s diseases, are characterised by destruction and pathology in one or more of these cell groups, such as Alzheimer’s and Parkinson’s diseases. Reports exist in the literature (Saper and German, 1987) that the TM may be in-

volved in the neurofibrillary degeneration of Alzheimer’s disease, and that Lewy body formation in TM hypothalamic neurons in Parkinson’s disease may in part underlie hypothalamic dysfunction (Sandyk et al., 1987). Others (Wallin and Gottfries, 1990) have reported an increase in galanin in the hypothalamus of Alzheimer patients; whereas, a study of the supraoptic and paraventricular nuclei showed a stable unchanged cell population both in aging and Alzheimer’s disease (Swaab et al., 1986). A recent study (Chan-Palay and Jentsch, 1991) has demonstrated that alterations in TM galanin neurons occur but in a highly variable manner from one Alzheimer patient to another. This is in line with the fact that reports exist in the literature describing both increases and decreases in brain histamine in Alzheimer patients (Cacabelos et al., 1989, vs. Mazurkiewicz-Kwilecki and Nsonwah, 1989). The TM has been extensively investigated in rats. The interest in the nucleus is heightened by the fact that TM neurons have been demonstrated to contain multiple neurotransmitters and neuropeptides in coexistence within individual neurons. Thus, although the major neurotransmitter appears to be histamine, other neuroactive substances such as galanin and gamma-aminobutyric acid (GABA) are also present differentially within the cell groups (Kohler et al., 1986; Ericson et al., 1987, 1989). The TM in rats consists of small, distinct clusters of neurons situated in the tuberal region of the hypothalamus embedded between the mammillary

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nuclei and the third ventricular surface (Bleier et al., 1979; Ericson et al., 1987). In primates and man, there is one main cluster of neurons lying against the tuberal third ventricle wall, close to the mammillary bodies (Diepen, 1962). In rats, the efferent projections of these TM neurons include olfactory regions, hippocampus, caudate, hypothalamic nuclei, cerebellum, medulla, spinal cord and extensive areas of cortex (see Staines et al., 1987a,b). Of the regions of the rat brain that in turn innervate TM neurons, there appear to be subcortical and cortical areas as well as those which arrive from the near vicinity of the hypothalamus itself. The monoaminergic inputs to the TM appear to be from non-locus coeruleal adrenergic and noradrenergic cell groups C1- C3 and A1 - A2, and from the rostra1 and dorsal raphe groups (Ericson et al., 1989), with greater emphasis on the latter inputs. The conclusion that the TM receives mainly innervation from the medullary catecholamine neurons rather than the coeruleal ones supports the fact that the TM may be more concerned with visceral and autonomic functions rather than with external stimuli (Ericson et al., 1991a,b). The 29 amino-acid peptide galanin, named from its N-terminal glycine and C-terminal amidated alanine (Tatemoto et al., 1983), was shown to be present in neurons and axons of the hypothalamic neurons in animals. (Skofitsch and Jacobowitz, 1985; Melander et al., 1986a,b; Palkovits et al., 1987) and in man (Gentleman et al., 1989). In situ hybridization studies have been performed with an oligonucleotide complementary to rat galanin demonstrating hypothalamic galanin neurons (not including the TM) in rat and human post-mortem brain (Bonnefond et al., 1990). In the human brain, galanin is also contained in cholinergic projection neurons as well as in the non-cholinergic local interneurons (Chan-Palay, 1988a,b). These interneurons have been shown to form extensive terminal networks which innervate the somata and dendrites of cholinergic cells. In brains from Alzheimer patients a hyperinnervation of the cholinergic projection neurons by galanin-immunoreactive (GAL-i) axons has been found (Chan-Palay, 1988a,b). The receptors for galanin have been demonstrated to be

preserved in Alzheimer’s disease in the nucleus basalis of Meynert neuron regions despite serious loss of cholinergic Meynert cells (Kohler and ChanPalay, 1990a,b), and there is an increase of tissue specific expression of the galanin gene in this region (Chan-Palay et al., 1990). The present paper will provide some data and quantitate the extent of involvement of galanin-immunoreactive (GAL-i) neurons in the TM in normal controls, as compared in patients with Alzheimer’s disease and Parkinson’s disease. The Zurich study The Zurich study on dementia is an interdisciplinary program to follow longitudinally a group of demented patients and their age-matched nondemented cohorts permanently residing in chronic care hospitals for the elderly. The patients are selected on the basis of their clinical symptomatology and neurological findings, regardless of exact age and sex. However, the majority of our cases are between the ages of 70 and 100 and the proportion of females is higher than males, reflecting the general demographic trends in the society. The relationship between depression and Alzheimer’s disease with the LC norepinephrine neurons and lowered norepinephrine synthesis has been recently described (Chan-Palay, 1990), as has the relationship between depression and dementia in Parkinson’s disease with the LC norepinephrine neurons (Chan-Palay, 1991). The cases are diagnosed following DSM-IIIR criteria. The patients are clinically folIowed for the total period of hospitalization (from 1.5 to 5 years) to exitus. In this period a psychometric test battery consisting of lateralization index, activities of daily living, socialization scale, mini-mental (Zurich variant) status, Hamilton depression scale, involuntary movements and extrapyramidal movements scale, and memory tests are administered every 6 months in addition to complete neurological examinations. After exitus, all cases go to autopsy. This paper will concern a number of selected patients taken from the larger longitudinal program consisting of several hundred

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TABLE 1 Galanin neurons in the tuberomammillary nucleus Case

1 2 3 4 5 6 7 a

Diagnosis

Control Control Controla Alzheimer Alzheimer Parkinsonb Parkinson‘

Age (Years)

75 82 83 84 91 66 83

With severe arteriosclerosis;

Sex

m m m f f f f

GAL4 cells (left) ( X 103)

GAL4 cells (right) ( X 103)

Galanin cells total ( X I@)

Brain weight (g)

Plaquedtangles CAI hippocampus

13.22 9.39 9.25 11.61 5.96 14.61 5.89

15.05 9.69 9.94 13.64 6.81 17.99 7.06

28.27 19.07 19.19 25.25 12.76 32.6 12.95

1415 1345 1600 965 1100 1202 1150

o/o 010

o/o

***/+ ***/+

f f

+ +

o/o */

+

with concurrent depression; ‘with concurrent dementia.

patients over the last 4 years described briefly above. The vital statistics on each case are summarised in Table I. Each patient was selected for this paper based upon the fact that thecase summarised best the critical features, and was consistent with the results of other similar cases in the longitudinal study and post-mortem neurotransmitter studies. It has been previously demonstrated that the reactions with antisera against galanin allow detailed analyses of the neuronal morphology of galanin neurons in the cholinergic nucleus of Meynert in controls, Alzheimer’s and Parkinson’s disease patients (ChanPalay, 1988a,b). A remote possibility exists that the immunoreactivity observed may be due to the recognition of molecules other than galanin peptide; however, for practical purposes we believe that positive neuronal immunoreactivity indicates the presence of the peptide. Estimates of neuritic plaques and neurofibrillary tangles were made on Bodian silver-stained preparations at a magnification of x 250 on a total of 20 separate visual fields of the CAI hippocampal region as an index of the extent of the Alzheimer pathology (see Chan-Palay, 1990) as compared to normally aged and Parkinson’s disease patients. The methods used for the localization of galanin peptide have been published in detail (Chan-Palay, 1988a,b). A total of 22 cases were studied, and full computerized reconstructions were obtained for seven

cases consisting of 3 controls, 2 Alzheimer’s and 2 Parkinson’s disease patients. The ages ranged from 66 years to 91 years with 3 males and 4 females. At autopsy all brains were perfused through the basilar and carotid arteries with 4% paraformaldehyde, post-fixed overnight, and washed extensively in buffer. 80pm thick serial sections were cut on a cryostat from the region of the mammillary bodies to the pituitary stalk. A complete series of one in five sections were prepared for Nissl staining for cytomorphology, the next series were reacted with galanin antibodies, and a third series for parvalbumin antibodies. The galanin antibodies were used at 1:1000 dilution with a 7-day incubation and the parvalbumin antibodies (rabbit anti-rat muscle Baimbridge R 301; Baimbridge and Miller, 1982) were used at a dilution of 1:5000 with a4-day incubation (see Zetzsche et al., 1990). The reactions were visualized by the immunogold silver staining method (IGSS, Chan-Palay, 1987). All sections were analysed with a computer-assisted quantitative program and reconstructions in the third dimension of the entire tuberomammillary neurons on both sides of the hypothalamus were made. Computer-assisted quantitative morphological analyses The computer system and the recording procedures

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used have been described in detail (Chan-Palay and Asan, 1989a,b). Briefly, the immunocytochemically stained serial brain-stem sections were reassembled in the correct anatomical order. Outlines of individual cell somata and dendritic arbors were recorded for calculations of soma areas and dendritic arbor length (Cellmate, Bioquant, TN). Plots of these recordings served to illustrate alterations in individual neuron morphology. To ensure comparability all quantitative measurements of neuronal parameters were carried out on immunoreactive neurons stained with the IGSS (immunogold silver staining) method.. Cell counts were performed on all reassembled sections of the galanin or parvalbumin-immunoreacted series of sections on both sides by cursor-marking the localization of whole cell bodies in all focus levels throughout the entire extent of the hypothalamus. Neuron numbers on partially damaged sections were approximated either from the numbers counted on immediately adjacent galanin-immunoreacted sections or from those counted in the contralateral hypothalamus of the same section and the ipsilateral hypothalamus of the preceeding and following sections of the same series. Total neuron numbers were calculated for the entire nucleus in all sections. The recordings of the reassembled sections were then aligned to match as closely as possible the situation in the intact brains, and images of the three-dimensional distribution of the neurons were created by the computer. For the surface morphology of the tuberomammillary area the comparisons should be made with a photomicrograph of the surface landmarks of the tuberomammillary region (Duvernoy, 1988). The reader is referred to Chan-Palay and Jentsch (1991) for figures illustrating the changes detected in Alzheimer’s disease. Only comparative figures illustrating Parkinson’s disease will be given here (Figs. 1 - 3).

Galanin neurons The GAL-i are immunoreactive and lie in a clearly defined group within the tuberal por-

Fig. 1. Ventralviewofthetuberal hypothalamicregion fromthree dimensional computer reconstructions of the TM nuclei in one Parkinson’s disease case (case 7, Table I); a represents a solid model of the TM nuclei (TM, shown in yellow) and b represents a wire computer diagram of the TM nuclei (yellow). See text for details. The mammillary bodies (mb) and the third ventricle (111) are indicated.

261

Fig. 2. Non-adjacent serial sections (coronal) in sequence from rostral through caudal through the hypothalamus regions containing the TM nuclei (arrows) to the level of the mammillary bodies (mb). The series is from a Parkinson’s patient (case 7, Table I).

Fig. 3. Photomicrographs of GAL4 neurons in the TM nuclei of a Parkinson’s disease patient (case 7, Table I). (Magnification: x 125.)

tion of the hypothalamus in the brains of control patients. The rostral boundary of the nucleus is the median eminence and the hypophyseal stalk. The caudal boundary is formed by the mammillary nuclei. The TM group abutts into the wall of the third ventricle. The TM is a single well-defined parvocellular cluster of small to medium sized neurons, mostly bipolar with extensivelengths of aspinous dendrites. The larger neurons, encountered less often, have somatic areas of approximately 350 - 470 pm2 and dendritic arbor lengths of 380 - 450 pm. The small neurons have somatic areas of 120- 270 pm2 with dendritic arbor lengths of 200 - 460 pm. There are no magnocellular neurons in the nucleus. Half of these TM neurons are GAL-1. Remaining neurons of similar description in this parvocellular neuropil are not GAL-i. Within the TM neuropil, there are numerous axonal boutons as well as varicose axonal segments visualized. GAL4 neurons abutt directly against the glia limitans which envelop the ventricular surface of the brain. A comparison of cytological differences between GAL4 cells of TM controls with TM of Alzheimer brains indicates that the latter GAL4 neurons, though reasonably numerous in number, are altered. Their somatic areas are larger in the larger bipolar and multipolar neurons (from 350 to 490 pm2) and the dendritic arbors are shorter, with lengths from 200 to 320 pm. GAL-i cells appear plumper with stumpy thick dendrites in the Alzheimer TM (Fig. 3). The smaller group of bipolar and multipolar neurons have a similar disposition with cell bodies from 150 to 180 pm2 in somatic areas and dendritic lengths from 105 to 300 pm. Axonal varicosities in Alzheimer TM are larger and more irregular in shape and size than the fine dustlike varicosities in the control cases. Comparing measurements of representative neurons taken from the Parkinson’s disease patients with those of the control patients and Alzheimer patients described above, there appears to be considerable variation. The neurons are generally larger in somatic size with longer dendritic arbors. For the larger bipolar and multipolar neurons the somatic sizes range from 300 to 545 pm2 and dendritic

268

lengths from 257 to 834 ym. The smaller bipolar and multipolar neurons range from 186 to 259 ym2 in the somatic area and in dendritic lengths from 135 to 507 pm. The immunoreactions have a lighter intensity compared to the controls, and the background neuropil has distinctly fewer immunoreactive axon terminals. Table I summarises the vital information of seven patients from whose brains the entire rostral to caudal extent of the TM galanin neuron group in the hypothalamus were reconstructed. The total number of GAL4 neurons on left, right and both sides are indicated, as are the age, sex and brain weights of each patient, and the relative numbers of neuritic plaques and neurofibrillary tangles as an index of the Alzheimer changes. Within the groups of controls and Alzheimer brains, the numbers of GAL-i TM neurons varied widely. This is apparently not dependent on the technical handling of the tissues. In terms of neuron numbers, there appears to be no clear consistent trend towards loss of neurons in any disease, although the number of GAL4 neurons tends to decrease with increasing age. In the brains of the three controls studied the total number of TM neurons ranged from 19 x lo3 to 28.3 x lo3. The Alzheimer patients had 12.8 and 25.3 x lo3GAL4 neurons, and the Parkinson’s disease patients had 13 and 32.6 x lo3 neurons, respectively. The topographical distribution of these neurons (dots) is displayed in serial sections taken from a Parkinson’s disease case no. 7 (Fig. 2). Further, with the aid of computerised programs these G A L i neuron groups can be further displayed in the third dimension (Fig. la,b), and rotated in three axes. The TM GAL-i neurons reconstructed in yellow, lie rostral to the mammillary bodies (dark blue) and abutting the ventral border of the third ventricle. The reconstruction is displayed in a view from the ventral aspect of the brain and is made from reconstruction of coronal sections through the hypothalamus. Immunocytochemical staining with antibodies against parvalbumin produced positively reacted neurons with full dendritic arborization in several locations of the hypothalamus (posterior hypothalamic nucleus, mammillary bodies per se) but not

within the TM region. Immunocytochemical reactions with antibodies against histamine, histidine decarboxylase gamma aminobutyric acid (GABA) and glutamic acid decarboxylase were unsuccessful. Thus, it was technically not possible to demonstrate coexistence of histamine and GABA with GAL4 in TM neurons. Conclusions and summary

The present study has presented evidence for the presence of GAL4 neurons in the TM nucleus of the hypothalamus which persist in aging as well as in Alzheimer’s and Parkinson’s diseases. There is, however, a variable number of cells present from patient to patient, dependent to some extent upon age. Higher age seems to be accompanied by smaller numbers of TM galanin neurons. In Alzheimer and Parkinson patients, the GAL4 TM neurons undergo changes in cellular morphology: enlargement of cell somata, shortening and thickening of dendrites and the general neuropil of the TM is less densely populated with axonal and terminal G A L i boutons. These cellular changes are typical and have been described in other systems of neurons with peptide and non-peptide transmitters, for example hippocampal and cortical somatostatin and neuropeptide Y neurons (Dawbarn et al., 1985; Chan-Palay et al., 1986; Chan-Palay, 1987), and locus coeruleus norepinephrine neurons (Chan-Palay and Asan, 1989b). The finding in general that the TM undergoes cellular changes in the neuron and axonal populatiqn but not necessarily drastic changes in cell numbers, provokes the speculation that the afferentation to the TM galanin neurons from other brain regions may be more severely affected than the galanin efferent innervation of other brain areas from the TM. In addition, these hypothalamus peptide neurons are involved in the disease but are not likely the primary affected brain regions. References Baimbridge, K.G. and Miller, J.J. (1982) Immunohistochemical localization of calcium-binding protein in the cerebellum, hip-

269 pocampal formation and olfactory bulb of the rat. Brain Res., 245: 223 - 229. Bleier, R., Cohn, P. and Siggelcow, I.R. (1979) A cytoarchitectonic atlas of the hypothalamus and the hypothalamic third ventricle of the rat. In: P.J. Morgane and J. Panksepp (Eds.), Anatomy of the Hypothalamus, Vol. I , Marcel Dekker, New York, pp. 137 - 220. Bonnefond, C., Palacios, J.M., Probst, A. and Mengold, G. (1990) Distribution of galanin mRNA containing cells and galanin receptor binding sites in human and rat hypothalamus. Eur. J. Neurosci., 2: 629 - 637. Cacabelos, A., Yamatodani, H . , Niigawa, S., Hariguchi, S., Tada, K., Nishimura, T., Wada, H., Brandeis, L. and Pearson, J. (1989) Brain histamine in Alzheimer’s disease. Methods Exp. Clin. Pharmacol., 1l(5): 353 - 360. Chan-Palay, V. (1987) Somatostatin immunoreactive neurons in the human hippocampus and cortex shown by immunogold/silver intensification on vibratome sections: coexistence with neuropeptide Y neurons, and effects in Alzheimer-type dementia. J. Comp. Neurol., 260: 201 -223. Chan-Palay, V. (1988a) Galanin hyperinnervates surviving neurons of the human basal nucleus of Meynert in dementias of Alzheimer’s and Parkinson’s disease: a hypothesis for the role of galanin in accentuating cholinergic dysfunction in dementia. J. Comp. Neurol., 273: 543 - 557. Chan-Palay, V. (198813) Neurons with galanin innervate cholinergic cells in the human basal forebrain and galanin and acetylcholine coexist. Brain Res. Bull., 21: 465 -472. Chan-Palay, V. (1990) Depression and senile dementia of the Alzheimer type: catecholamine changes in the locus coeruleus - basis for therapy. Dementia 1: 253 -261. Chan-Palay, V. (1991) Depression and dementia in Parkinson’s disease: catecholamine changes in the locus coeruleus - basis for therapy. Dementia, 2: 7- 17. Chan-Palay, V. and Asan, E. (1989a) Alterations in catecholamine neurons of the locus coeruleus in senile dementia of the Alzheimer type and in Parkinson’s disease with and without dementia and depression. J. Comp. Neurol., 287: 373 - 392. Chan-Palay, V. and Asan, E. (1989b) Quantitation of catecholamine neurons in the locus coeruleus in human brains of normal young and older adults and in depression. J. Comp. Neurol., 287: 357 - 372. Chan-Palay, V. and Jentsch, B. (1991) Galanin tuberomammillary neurons in the hypothalamus in normal patients, in senile dementia of the Alzheimer’s type and in Parkinson’s disease. Dementia, 2: 95 - 101. Chan-Palay, V., Kohler, C., Haesler, U., Lang, W. and Yasargil, G. (1986) Distribution of neurons and axons immunoreactive with antisera against neuropeptide Y in the normal human hippocampus. J. Comp. Neurol., 248: 360 - 375. Chan-Palay, V., Jentsch, B., Lang, W., Hochli, M. and Asan, E. (1990) Distribution of neuropeptide Y, C-terminal flanking peptide of NPY and galanin and coexistence with cate-

cholamine in the locus coeruleus of normal human, Alzheimer’s dementia and Parkinson’s disease brains. Dementia, l(1): 18-31. Dawbarn, D., DeQuidt, M.E. and Emson, P.C. (1985) Survival of basal ganglia neuropeptide Y-somatostatin neurons in Huntington’s disease. Brain Res., 340: 251 -260. Diepen, R. (1962) Der Hypothalamus. In: W.V. Mollendorf (Ed.), Handbuch der Mikroskopischen Anatomie des Menschen, Springer, Berlin. Duvernoy, H.M. (1988) The Human Hippocampus. An Atlasof Applied Anatomy, J.F. Bergmann, Munich. Ericson, H., Watanabe, T. andKohler, C. (1987) Morphological analysis of the tuberomammillary nucleus in the rat brain: delineation of subgroups with antibody against L-histidine decarboxylase as a marker. J. Comp. Neurol., 263: 1-24, Ericson, H., Blomqvist, A. and Kohler, C. (1989) Brain-stem afferents to the region of the tuberomammillary nucleus in the rat brain with special references to monoaminergic innervation. J. Comp. Neurol., 3 11: 45 - 64. Ericson, H., Blomqvist, A. and Kohler, C. (1991a) Origin of neuronal inputs to the region of the tuberomammillary nucleus of the rat brain. J. Comp. Neurol., 3 11: 45 - 64. Ericson, H., Kohler, C. and Blomqvist, A. (1991b) GABA-like immunoreactivity in the tuberomammillary nucleus: an electron microscopic study in the rat. J. Comp. Neurol., 305: 462 - 469. Gentleman, S.M., Falkai, P., Bogerts, B., Herrero, M.T., Polak, J.M. and Roberts, G.W. (1989) Distribution of galanin-like immunoreactivity in the human brain. Brain Res., 505: 311-315. Kohler, C. and Chan-Palay, V (1990a) Galanin receptors in the post-mortem human brain. Regional distribution of 9galanin binding sites using the method of in vitro receptor autoradiography. Neurosci. Lett., 120: 179- 182. Kohler, C. and Chan-Palay, V. (1990b) Preservation of galanin receptors in the human basal nucleus of Meynert in senile dementia of the Alzheimer type. Dementia, 1: 82 - 89. Kohler, C., Ericson, H., Watanabe, T., Polak, J., Palay, S.L., Palay, V. and Chan-Palay, V. (1986) Galanin immunoreactivity in hypothalamic histamine neurons: further evidence for multiple chemical messengers in the tuberomammillary nucleus. J. Comp. Neurol., 250: 58 - 64. Mazurkiewicz-Kwilecki, I.M. and Nsonwah, S. (1989) Changes in the regional brain histamine and histidine levels in postmortem brains of Alzheimer patients. Can. J. Physiol. Pharmacol., 67: 75 -78. Melander, T., Hokfelt, T. and Rokaeus, A. (1986a) Distribution of galanin-like immunoreactivity in the rat central nervous system. J. Comp. Neurol., 248: 475 - 517. Melander, T., Hokfelt, T., Rokaeus, A., Cuello, A.C., Oertel, W.H., Verhofstad, A. and Goldstein, M. (1986b) Coexistence of galanin-like immunoreactivity with catecholamines, 5hydroxytryptamine, GABA, and neuropeptides in the rat CNS. J. Neurosci., 6: 3640-3654.

270 Palkovits, M., Rokaeus, A., Antoni, A. and Kiss, A. (1987) Galanin in the hypothalamo-hypophyseal system. Neuroendocrinology, 46: 417 - 423. Pannula, P., Airaksinen, M.S., Pirvola, U. and Kotilainen, E. (1990) A histamine-containing neuronal system in human brain. Neuroscience, 34: 121 - 132. Sandyk, R., Iacono, R.P. and Bamford, C.R. (1987) The hypothalamus in Parkinson’s disease. Ital. J. Neurol. Sci., 8(3): 227 - 234. Saper, C.B. and German, D.C. (1987) Hypothalamic pathology in Alzheimer’s disease. Neurosci. Lett., 74(3): 364 - 370. Skofitsch, G. and Jacobowitz, D.M. (1985) Immunohistochemical mapping of galanin-like neurons in the rat central nervous system. Peptides, 6: 509 - 546. Staines, W.A., Daddona, P.E. and Nagy, J.I. (1987a) The organization and hypothalamic projections of the tuberomammillary nucleus in the rat: an immunohistochemical study of adenosine deaminase-positive neurons and fibers. Neuroscience, 23: 571 - 596. Staines, W.A., Yamamoto, T., Daddona, P.E. and Nagy, J.I.

(1987b) The hypothalamus receives major projections from the tuberomammillary nucleus in rat. Neurosci. Lett., 76: 251 - 262. Swaab, D.F., Fliers, E. and Goudsmit, E. (1986) Differentialcell loss in (peptide) neurons in the anterior hypothalamus with aging’and Alzheimer’s disease: lack of changes in cell density. Brain Rex, 505: 311-315. Tatemoto, K., Rokaeus, A., Jornvall, H . , McDonald, T.J. and Mutt, V. (1983) Galanin - a novel biologically active peptide from porcine intestine. FEBS Lett., 164: 124- 128. Wallin, A. and Gottfries, C.G. (1990) Biochemical substrates in normal aging and Alzheimer’s disease. Pharmacopsychiatry, (S~ppl.2)23: 37-43. Zetzsche, T., Bainbridge, K.G., Mohler, H. and Chan-Palay, V. (1990) Subsets of GABA neurons in the human cerebellum in normal controls and senile dementia of the Alzheimer type patients detected by glutamate decarboxylase, GABA,/benzo diazepine receptor protein, calbindin D-28k and parvalbumin immunocytochemistry. Dementia, 1 : 231 - 252.

SECTION VI

Osmoregulation

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D.F.Swaab, M.A. Hofman, M. Mirmiran, R. Ravid and F.W. van Leeuwen (Eds.) Progress in Brain Research, Vol. 93

0 1992 Elsevier Science Publishers B.V. All rights reserved.

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CHAPTER 19

Animal models for osmoregulatory disturbances F.W. Van Leeuwen Netherlands Institute for Brain Research, 1105 A Z Amsterdam, The Netheriands

Introduction

For terrestrial organisms it is of the utmost importance to conserve water. The water balance is mainly controlled by the neurons of the hypothalamo-neurohypophyseal system (HNS). The HNS is highly conserved throughout the vertebrate classes, which indicates its crucial importance. Within the HNS vasopressin (VP) is synthesized in magnocellular neurons and axonally transported to the neural lobe, where it is released into the blood stream to exert its functions at various sites in the periphery (e.g., the kidneys; Cunningham and Sawchenko, 1991). In humans, the causes of diabetes insipidus (di) can be either neurogenic or nephrogenic or related toprimarypolydipsia. Of all three types subtypes can be distinguished (Robertson, 1988). A failure to synthesize VP or its precursor leads to di of the hypothalamic type, of which two main forms (viz., acquired and familial) have been described (Scherbaum, this volume). Nephrogenic diabetes is caused by disturbances in the kidney itself and can be either acquired or familial (X-linked recessive). Another cause of di is primary polydipsia, in which excessive drinking chronically inhibits the release of VP into the blood. In this acquired disturbance the kidneys are able to respond to VP. For all three types of di animal models are available. In the clinical diagnosis, these three forms of di are differentiated from one another by a number of sophisticated methods of laboratory analysis (for details see Robertson, 1988). A major component of the diagnosis is the water deprivation test, in which

the plasma is made hyperosmotic by withdrawing drinking fluid. If, in response to this treatment, the urinary osmolarity rises to clearly hyperosmotic levels, it may be concluded that VP was secreted from the neural lobe and that the kidneys were able to respond to the hormone. The diagnosis is therefore primary polydipsic diabetes insipidus primary, since polydipsia is the initiating event. If the urine does not become concentrated during the water deprivation test it can be concluded that either the HNS is unable to produce VP (termed neurogenic di) or that the kidneys do not respond to the hormone (called nephrogenic di). If, in response to giving VP, the urine does become concentrated the diagnosis is neurogenic di, whereas if the urine remains hypo-osmolar, di of the nephrogenic type is the cause of the disorder. Primary polydipsic diabetes insipidus

Silverstein et al. (1961) reported on a strain of excessively drinking mice (STR/N) in whom high fluid turnover is first detected at approximately 3 months of age and fully developed by 8 - 9 months. Their mean water intake is 120% of their body weight (in control mice: 20%). Sometimes individual animals even consume up to 120 ml of fluid, or five times their body weight, within 24 h. The inheritance of the trait was suggested to have a recessive character, its mode and the number of genes involved still remain to be determined. On average these mice excrete approximately 45% of their body weight as. urine compared to 2.4% in wild-type strains. When

274

STR/N mice abundant gomori-positive neurosecretory material and VP immunoreactivity have been reported (Silverstein et al., 1961; Hattori et al., 1991). Remarkably few studies have been performed on the brain of this mouse strain. Recently, however, a few reports appeared on the possible cause of this trait. It is known from literature that the anteroperiventricular area is of major importance to induce excessive drinking (Johnson and Buggy, 1987) and that infusion of angiotensin I1 in this area (e.g., into the subfornical organ) can promote drinking (Johnson and Cunningham, 1987). It has also been shown that there is a direct connection between the subfornical organ, the anterior periventricular area and the supraoptic and paraventricular nuclei (Miselis et al., 1979). One of the reasons of polydipsia might therefore be a disturbed metabolism of the

the diseased mice are eating and drinking at libitum, the osmolarity of their urine is hypo-osmolar (298 mosm/kg H20), while it is 1730 mosm/kg H 2 0 in wild-type strains. The plasma osmolarity and sodium concentrations are not significantly different from those of the wild-type strains. The kidneys of the STR/N strain respond to VP by a rise in their urine osmolarity. The STR/N mice were also studied during 15 months of fluid restriction during which their water intake was below wild-type water intake (5-6 ml/day), namely 3.2 ml/day on average. They concentrated their urine to the same degree as wild-type mice. However, the STR/N mice started drinking excessively again immediately after they were offered water ad libitum. After 3 days they increased their intake to 19 ml/day, and subsequently to 30 ml/day after 66 days, whereas urinary osmolarity decreased concomitantly. In the HNS of TABLE I

General idea about the AOGEN-angiotensin cascade. The processing of AOGEN might differ per species and per organ Angiotensinogen* (AOGEN precursor) 453 amino acids (glycopeptide) 1 renin (+ prorenine) PEP ACE

1

1

renin

3-

Angiotensin I (Ang I) Asp - Arg - Val - Tyr - Ile - His - Pro - Phe - His - Leu 1 angiotensin converting enzyme (ACE), 1prolylendopeptidase (PEP) Asp- Arg - Val - Tyr - Ile - His -Pro ...........Angiotensin 1 - 7 Angiotensin I1 (Ang 11) Asp -Arg - Val -Tyr - Ile - His -Pro - Phe 1glutamyl aminopeptidase** (GA) Arg - Val - Tyr - Ile - His - Pro - Phe Angiotensin 111 (Ang 111)

Antagonists: AT,: DUP 753 (non-peptide) AT,: Parke Davis PD 123177 (non-peptide) Ciba-Geigy P 421 12A (peptide) Perifery: Vasoconstriction (arterioles) Aldosteron secretion (adrenal cortex) Central: Thirst Secretion of vasopressin/CRF

* Okhubo et al. (1983). ** GA also stimulates formation of other metabolites (Ang 3-8, 4-8). AOGEN formation inhibitor: perindopril; ACE inhibitors: captopril, perindopril; GA inhibitors: Glu(Asp)thiol, amatastatin; PEP in hibitor: Z-pro-prolinal, amatastatin. Tyr - Ile - His -essential for Ang I1 activity.

21 5

angiotensin I1 precursor angiotensinogen. Support for this idea was recently obtained by Katafuchi et al. (1991), who reported that an inhibition of the angiotensin I converting enzyme, an enzyme of the angiotensinogen-angiotensinI1 cascade (Table I), by captopril resulted in a strong reduction of the polydipsic behaviour. This suggests that the polydipsia in the STR/N mice may involve, at least in part, the angiotensin I1 system in the brain (Hattori and Koizumi, 1990). However, other disturbances cannot be ruled out since lower sensitivity for opioid peptides has also been reported in the hypothalamus of this strain (Hattori et al., 1991). Whether the human form of polydipsia is com-

parable to that of the mice remains to be determined. Nephrogenic diabetes insipidus

With regard to nephrogenic di (NDI) several mouse strains are available, of which the + / + severe mice may be an analogue of the human trait (for anextensive review, see Valtin, 1992). In the hypothalamus of + / + mice VP immunoreactivity is abundantly present within the classical hypothalamo-neurohypophyseal system (Fig. 1). The disease is characterized by the inability to respond to V P in this strain. This is caused by an enhanced activity of CAMP

Fig. 1. Transversal vibratome section of the hypothalamus of an adult male mouse of the + / + severe stram with an average water intake of 0.6 ml/day per gram body weight stained with an anti-vasopressin antibody. The cells in the paraventricular (PVN), supraoptic (SON) and suprachiasmatic nucleus (SCN) are visible. Cells between the PVN and SON forming hypothalamic islands (hi) can be seen as well. From the PVN many beaded fibres running into the direction of the SON and from there towards the neural lobe can be seen: OC, Optic chiasm.

216

phosphodiesterase resulting in subnormal levels of the second messenger, c-AMP, in the kidney (Takeda et al., 1991). Phosphodiesterase activity can be inhibited by the drugs rolipram and cilostamide (Bichet et al., 1991) which minimize the symptoms of NDI. Rolipram, however, is not effective in human NDI, which indicates that this must have a different cause. It also indicates that the mouse model for NDI is only of relative value when it comes to understanding human NDI. Familial neurogenic diabetes insipidus Familial neurogenic di in human is a very rare disease (1 - 3% of di cases (Blotner, 1958; Green et al., 1967). The urine production varies per patient but volumes of up to 20 1 have been reported (e.g., Bravermanet al., 1965; Bahnsen et al., 1992). In the few cases in which autopsy material of the hypothalamus was studied (aged 37, 49 and 72 years) a massive loss of magnocellular VP neurons was found (e.g., Green et al., 1967; Bergeron et al., 1991). Nevertheless, minute quantities of circulating VP must be present (e.g., Bruguier et al., 1981; Blackett et al., 1983; Nagai et al., 1984) since the patients react upon chlorpropamide treatment. This drug exerts its antidiuretic activity only in combination with minute doses of VP, which in itself would not act as such (Uhlich et al., 1971). These minute amounts can be synthetized at various places, including the brain, where parvocellular and a few magnocellular VP neurons were still detectable in the hypothalamus (Bergeron et al., 1991). In contrast to the studies mentioned earlier, Nagai et al. (1984) reported no loss of V P cells in the supraoptic nucleus of a 44-year-old man, whereas in the paraventricular nucleus only a few cells were detectable. This differential loss of VP neurons might be related to the progressive character of di of the familial type which in most cases starts during early or late childhood. In the 44-year-old patient the process may have been delayed in the neurons of the supraoptic nucleus. The progressive character of di is nicely illustrated in two subsequent reports on the same family. One young female (2.5 years old) appeared

to be phenotypically unaffected. However, on the basis of restriction fragment length polymorphism she was expected to suffer from di (Schmale et al., 1991). This did indeed occur 13 years later so that in a subsequent paper (Bahnsen et al., 1992) she was reported to clearly show the symptoms of di. At present it is not clear why these magnocellular V P neurons are reduced in number. In controls this cell population was shown to be remarkably stable in aging with no cell loss being up to the age of 97 years (Goudsmit et al., 1990). One of the explanations of the loss of neurons in familial neurogenic di is that the mutated VP precursor (see below) influences the degree of cell death in the hypothalamus. This idea may be tested in transgenic animals (e.g., Habener et al., 1989; Young et al., 1990). The homozygous Brattleboro (di/di) rat is used as a model for this trait. This animal exhibits a Mendelian inherited di due to a mutation in the single autosomal recessive gene for the VP precursor (Fig. 2). In the neurophysin-encoding exon B of the VP-neurophysin gene a deletion of a single deoxyguanosine has been shown in di/di rats (Schmale and Richter, 1984). This results in a frame-shift by which a V P precursor with a different C-terminus is synthesized (for review see Ivell et al., 1990) (Fig. 3, Table 11). Schmale et al. (1989) haveelegantly shown that this altered VP precursor is arrested within thc membranes of the endoplasmic reticulum. This ir most probably due to the aberrant configuration ol the VP precursor. Consequently the mutant VF precursor cannot be transported towards the Golg: apparatus and packaged in neurosecretory granules. The symptoms of di/di rats are well comparablc with the human neurogenic di, which, in contrast, ic inherited as an autosomal dominant trait (e.g., Lev. inger and Escamilla, 1955; Schmale et al., 1991 Bahnsen et al., 1992). The di/di rat has become i useful model for many disciplines to study this trail (Sokol and Valtin, 1982). As in human hypothala. mic di cases, the di/di rat, which produces largc volumes of urine (approximately 70% of the bodj weight/day), can be treated effectively with VP or 1. deamino[d-Arg*]VP (dDAVP). So far, no loss ir cell number in di/di rats have been reported (Rhoder

277

LYSOSOME-LIKE BODIES SECRETORY GRANULES

0

0

0 [

BRATTLEBORO (di/di)

Fig. 2. Structural organization of the vasopressin gene from wild-type (left panel) and homozygous Brattleboro di/di (right panel) rats: their transcription, translation into a precursor protein that is post-translationally processed (e.g., amidation) in magnocellular neurons. ER, Endoplasmic reticulum; SP, signal peptide; V, vasopressin; NP, neurophysin; CPP, C-terminal glycopeptide of propressophysin; CP 14, stretch of 14 amino acids predicted from the frame-shifted DNA sequence and used to generate mutant-specific antibodies. In the homozygous Brattleboro rat (di/di) the mutant precursor transport from the ER to the Golgi apparatus is disturbed, most probably by incorrect folding of the prohormone (see Schmale et al., 1989). However, a co-expressed peptide such as dynorphin is packaged in secretory granules that are much smaller (100 nm) than usually (Whitnall et al., 1985). (Picture taken with permission from hell et al., 1990.)

TABLE I1 Consequence of G deletion: frame shift mutation Absence of stop codon in mRNA: translation of poly(A) tail polylysine Loss of spacer arginine between neurophysin and glycopeptide (cleavage site) Loss of five out of 14 cysteines in the precursor (abnormal folding) Absence of glycosylation site ( - A m - Ala - Thr - ) +

Impaired translocation +impaired passage of precursor: endoplasmic reticulum +bGolgi apparatus tkneurosecretory granules

278

Preliminary data indicate that through a single base insertion at several sites in the area of the G deletion the correct reading frame returned (Evans et al., 1991). Within this area of the exon B VP gene more alterations have been reported. In the VP-neurophysin moiety of the ostrich at the very place of the G deletion even an amino acid insertion was reported, which indicates that this area could represent a potential “hot-spot recombinational” area (Lazure

Fig. 3. Transversal section of the hypothalamus of a 83-week-old homozygous Brattleboro rat showing intense mutant immunoreactivity (no. CP 14, see Fig. 2). This type of immunoreactivity appeared to be age-dependent (Van Leeuwen van Van der Beek, 1991). In young heterozygous Brattleboro rats this type of immunoreactivity is absent, also after osmotic challenge, and in older heterozygous rats.

et al., 1982) and the cells are hyperactive (e.g., Swaab et al., 1973). An intriguing finding was that during post-natal life a small but linearly increasing number of hypothalamic post-mitotic VP neurons (from 0.1070 up to 3%) of the di/di rat undergo a switch to a genuinely heterozygous phenotype (Van Leeuwen et al., 1989) (Fig. 4a,b), thereby impacting the expression of a number of coexisting peptides and re-initiating axonal transport of the V P precursor (Fig. 5a,b; Ivell et al., 1990; Van Leeuwen et al., 1991; Gabreels et al., 1992). Although these cells show signs of hypertrophy (e.g., cell size and abundant glycoprotein immunoreactivity; Figs. 4a,b and 6), their number is too small to see indications of recovery from di. Interestingly, in one of the first papers on di/di rats VP bioactivity was already detected (Valtin et al., 1965). In the hypothalamus and pituitary gland of some rats unusually high levels of bio-assayable VP were reported. The age of only one di/di rat was reported and appeared to be 21 months, which correlates nicely with our data of an increasing number of VPproducing cells with age. Unfortunately, the other age data of the di/di rats are not available anymore. (H. Valtin, personal communication). The molecular event generating the heterozygous phenotype in di/di rats remains to be established.

Fig. 4. Transversal vibratome sections of the hypothalamus of a homozygous Brattleboro rat showing in (a) the supraoptic nucleus (SON) and in (b)the paraventricular nucleus (PVN). The sections were incubated with anti-glycopeptide ( # C3 final, see Van Leeuwen et al., 1989, for details) and revealed two types of immunoreactivity. (1) Over almost all elements of magnocellular size a low but distinct immunoreactivity was visible. This staining was also obtained with other antibodies raised against glycopeptide preparation from different species (cf. Van Leeuwen et al., 1986). (2) A very intense reactivity that was present not only in the cell body but also in the neurites running towards the neural lobe (cf. Van Leeuwen et al., 1986). These cells occur at random throughout the SON, PVN and hypothalamic islands and their number increases with age (cf., Van Leeuwen et al., 1989).

279

Fig. 5 . Transversal consecutive cryostat sections of the hypothalamus of a homozygous Brattleboro rat incubated with glycopeptide (a) and angiotensin I1 (b)antibody. Note that the same cell is intensely stained for glycopeptide (a) and angiotensin 11. The glycopeptide immunoreactivity that is present all over the cells, as discussedin Fig. 4, was accentuated by the intensification of the diaminobenzidine reaction using nickel ions (cf., Van Leeuwen et al., 1991). Arrowheads point to erythrocytes displaying pseudoperoxidase staining.

Fig. 6 . Solitary cell within the supraoptic nucleus displaying ver! intense immunoreactivity both in the cell body (except for thc nucleus) and the neurites. Note that also the size of the cell body is different from those in the neighbourhood.

et al., 1987) similar to the rosy locus of Drosophila melanogaster(e.g., Hilliker et al., 1991). So far, only two similar examples of an age-related restoration of wild-type phenotype have been shown, i.e., in the analbuminemic rat (Esumi et al., 1983, 1985) and the mdx mouse, a model for human Duchenne muscular dystrophy (Hoffman et al., 1990). In the rat mutant a deletion of seven bases occurs in an intron of the albumin gene. In the course of life of the analbuminemic rat within the liver mRNA and albumin is again synthesized in minute quantities (i.e., from approximately one cell in 105 in young rats to one cell in lo2 at 24 months: Esumi et al., 1985; Saber et a1.,1990). The frequency can be increased by treatment with a specific hepatomutagen. In the mdx mouse an age-dependent reversion of dystrophin expression has been reported (Hoffman et al., 1990).Also, in human patients a similar reversion has been reported (Gold et al., 1990; Burrow et al., 1991). No molecular biological data have so far been obtained to explain these particular phenomena. Whether such phenomena also occur at a larger scale (i.e., in humans) has yet to be established. However, the di/di rat model has shown that alterations at the level of DNA of post-mitotic neurons cannot be ruled out a priori. Furthermore, it has provided us with fundamental data on the synthesis of peptide precursors (Van Leeuwen et al., 1991;Gabreelsetal., 1992)anditisamodelnot only for osmoregulatory disturbances but also for many more physiological processes (Sokol and Valtin, 1982). It has been shown in two different human families that a single nucleotide alteration leads to a single amino acid exchange within the highly conserved VP-neurophysin domain encoding domain. So far, such a substitution has been observed at two different sites in the exon B encoding for neurophysin, i.e., a Gly-Val (neurophysin-17; Schmale et al., 1991; Bahnsen et al., 1992) and a Gly-Ser (neurophysin-57, Ito et al., 1991)substitution. Interestingly, the latter nucleotide substitution is not far from the area (21 bases upstream) where in the di/di rat and ostrich respectivelya deletion and an amino acid insertion have been reported (Schmale and Richter,

280

1984; Lazure et al., 1987; Evans et al., 1991). Subsequently, for reasons as yet unknown, a V P deficiency occurs, resulting in di. Since both substitutions concern highly conserved amino acids, it may be that the three-dimensional configuration of the VP precursor has changed (Chen et al., 1991), possibly resulting in a different VP binding pocket of the neurophysin and a subsequent disturbance of intracytoplasmic routing of the wild-type VP precursor. The reason why the unaffected expressed allele is not able to provide sufficient VP precursor is not clear at the moment. Bahnsen et a]. (1992) speculated that the presence of the mutant VP precursor interferes with the self-aggregation process of the VP-neurophysin precursor in the transGolgi, the site at which the targeting to secretory granules is determined. In contrast, the expression of the wild-type VP precursor in the heterozygous Brattleboro rat is not affected, which may be explained by the suppression of mutant allele expression (Sherman and Watson, 1988). We were indeed unable to show immunoreactive mutant VP precursor in young and middle-aged heterozygous Brattleboro rats, even after osmotic challenge (Van Leeuwen and Van der Beek, 1991; Van Leeuwen, unpublished results; Fig. 3). Once again the use of transgenic animals (Young et al., 1990) may help to detect the site at which the targeting of the VP precursor is disturbed in human di. Other changes in the areaof the neurophysin gene are likely to occur in two different families, as shown by restriction fragment length polymorphism studies (cf., Repaske et al., 1990, this volume), and indicate a heterogeneous genetic basis for familial neurogenic di. This heterogenicity is similar to the mutations of the human androgen receptor gene causing the androgen insensitivity syndrome and resulting in a wide spectrum of abnormalities in male sexual development (e.g., testicular feminization; Brinkman and Trapman, 1992).

Summary and conclusions For the various types of di in humans, animal models are available. However, their value for explain-

ing human di is for the major part an indirect one; by studying cellular mechanisms in these animal models, fundamental aspects of the cellular processes become available, which will help to understand similar processes in human di and subsequently lead to the molecular cause(s) of the various types of human di. Finally, it is to be expected that in the very near future transgenic animals will be raised in which very specific genetic information is overexpressed (or knocked out by homologous recombination; McMahon and Bradley, 1990). Recently hypervasopressinemia could be shown in transgenic mice, providing an animal model for the syndrome of the inappropriate VP secretion (Bartter and Schwartz, 1967), which is often observed in patients with lung cancers that ectopically express the V P gene (Habener et al., 1989). Furthermore it will be possible to study the exact cause(s) of human di by performing in vitro mutagenesis and to express the RNA constructs within a cell-free translation system and in oocytes (e.g., Schmale et al., 1989)and subsequently study the pattern of precursor synthesis, packaging and processing. References Bahnsen, U., Oosting, P., Swaab, D.F., Nahke, P., Richter, D. and Schmale, H. (1992) A missense mutation in the vasopressin-neurophysin precursor gene cosegregates with human autosomal dominant neurohypophyseal diabetes insipidus. EMBO J., 11: 19-23. Bartter, F.C. and Schwartz, W.B. (1967) The syndrome of inappropriate secretion of antidiuretic hormone. Am. J. Med., 42: 790 - 806. Bergeron, C., Kovacs, K., Ezrin, C. and Mizzen, C. (1991) Hereditary diabetes insipidus: an immunohistochemical study of the hypothalamus and pituitary gland. Actu Neuropathol. (Bed.), 81: 345 - 348. Bichet, D.G., Arthus, M.F. and Lonergen, M. (1991) Cellular defect in hereditary nephrogenic diabetes insipidus. In: S. Jard and R. Jamison (Eds.), Vasopressin - Colloque INSERM, Vol. 208, John Libbey Eurotext Ltd., London, pp. 557 - 564. Blackett, P.R., Seif, S.M., Altmiller D.H. and Robinson, A.G. (1983) Case report. Familial central diabetes insipidus: vasopressin and nicotine stimulated neurophysin deficiency with subnormal oxytocin and estrogen stimulated neurophysin. Am. J. Med. Sci., 286: 42 - 46, Blotner, H. (1958) Primary of idiopathic diabetes insipidus: a

28 1 system disease. Metab. Clin. Exp., 7: 191 -200. Braverman, L.E., Mancini, J.P. and McGoldrick, D.M. (1965) Hereditary idiopathic diabetes insipidus. A case report with autopsy findings. Ann. Intern. Med., 63: 503 -508. Brinkman, A.O. and Trapman, J. (1992) Androgen receptor mutants that affect normal growth and development. Cancer Surveys, 14, in press. Bruguier, A., Poisson, D., Lestradet, H. and Labrune, B. (1981) Diabkte insipide familial d’origine centrale. Nouvelle Presse Medicale, 10: 897 - 899. Burrow, K.L., Coovert, D.D., Klein, C.J., Bulman, D.E., Kissel, J.T., Rammonen, K.W., Burghes, A.H.M. and Mendell, J.R. (1991)Dystrophin expressionand somatic reversion in prednisone-treated and untreated Duchenne dystrophy. Neurology, 41: 661 - 666; Chen, L., Rose, J.P., Breslow, E., Yang, D., Chang, W.-R., Furey, Jr., W.F., Sax, M. and Wang, B.-C. (1991) Crystal structure of a bovine neurophysin I1 dipeptide complex at 2.8 A determined from the single wave length anomalous scattering signal of an incorporated iodine atom. Proc. Nutl. Acad. Sci. U.S.A., 88: 4240-4244. Cunningham, Jr., E.T. and Sawchenko, P.E. (1991) Reflex control of magnocellular vasopressin and oxytocin secretion. Trends Neurosci., 14: 406 - 41 1 . Esumi, H., Takahashi, Y., Sato, S., Nagase, S. and Sugimura, T. (1983) A seven-base-pair deletion in an intron of the albumin gene of analbuminemic rats. Proc. Natl. Acad. Sci. U.S.A., 80: 95-99. Esumi, H., Takahashi, Y ., Makino, R., Sato, S. and Sugimura, T. (1985) Appearance of albumin-producing cells in the liver of analbuminemic rats on aging and administration of mutagens. Adv. Exp. Med. Biol., 190: 637-650. Evans, A.P., lvell, R., Van Leeuwen, F.W., Corner, M. and Burbach, J.P.H. (1991) Sequence analysis of vasopressin mRNAs in the homozygous Brattleboro rat which code for glycopeptide immunoreactivity. Abstracts of the 21st Meeting of the Society for Neurosciences, New Orleans, LA, 17: 380. Gabreels, B.A.Th.F., Sonnemans, M.A.F., Seidah, N.G., ChrCtien, M. andVanLeeuwen,F.W. (1992)Dynamicsof7B2and galanin expression on solitary magnocellular hypothalamic vasopressin neurons of the homozygous Brattleboro rat. Brain Res., 582: in press. Gold, R., Meurers, B., Reichmann, H., Kress, W. and Miiller, C.R. (1990) Duchenne muscular dystrophy: evidence for somatic reversion of the mutation in man. J. Neurol., 237: 494 - 498. Goudsmit, E., Hofman, M.A., Fliers, E. and Swaab, D.F. (1990) The supraoptic and paraventricular nuclei of the human hypothalamus in relation to sex, age and Alzheimer’s disease. Neurobiol. Aging, 1 1 : 529 - 536. Green, J.R., Buchan, G.C., Alvord, Jr., E.C. and Swanson, A.G. (1967) Hereditary and idiopathic types of diabetes insipidus. Brain J. Neurol., 90:707 - 714. Habener, J.F., Cwikel, B.J., Hermann, H., Hammer, R.E., Palmiter, R.D. and Brinster, R.L. (1989) Metallothionein-

vasopressin fusion gene expression in transgenic mice. J. Biol. Chem., 264: 18844- 18852. Hattori, Y. and Koizumi, K. (1990) Sensitivity to angiotensin I1 of neurons in the circumventricular organs of polydipsic inbred mice. Brain Rex, 524: 181 - 186. Hattori, Y., Katafuchi, T. and Koizumi, K. (1991) Characterization of opioid-sensitive neurons in the anteroventral third ventricle region of polydipsic inbred micein vitro. Brain Res., 538: 283 - 288. Hilliker, A.J., Clark, S.H. and Chovnick, A. (1991) The effect of DNA sequence polymorphisms in the rosy locus of Drosophila melanogaster. Genetics, 129: 779 - 781. Hoffman, E.P., Morgan, J.E., Watkins, S.C. and Partridge, T.A. (1990) Somatic reversion/suppression of the mouse mdx phenotype in vivo. J. Neurol. Sci., 99: 9 - 25. Ito, M., Mori, Y., Oiso, Y. and Saito, H. (1991) A single base substitution in the coding region for neurophysin I1 associated with familial central diabetes insipidus. J. Clin. Invest., 87: 725 - 728. Ivell, R., Burbach, J.P.H. and Van Leeuwen, F.W. (1990) The molecular biology of the Brattleboro rat. Front. Neuroendocrinol., 1 1 : 313 - 338. Johnson, A.K. and Buggy, J. (1987) Periventricular preoptichypothalamus is vital for thirst and normal water economy. Am. J. Physiol., 234: 122- 129. Johnson, A.K. and Cunningham, J.T. (1987) Brain mechanisms and drinking: the role of lamina terminalis-associated systems in extracellular thirst. Kidney Znt., 32: 35 -42. Katafuchi, T., Hattori, Y., Nagatomo, I., Koizumi, K. and Silverstein, E. (1991) Involvement of angiotensin I1 in water intake of genetically polydipsic mice. A m . J. Physiol., 260: 1152- 1158. Lazure, C., Saayman, H.S., NaudC, R.J., Oelofsen, W. and Chretien, M. (1987) Complete amino acid sequence of a VLDV-type neurophysin from ostrich differs markedly from known mammalian neurophysins. Znt. J. Pept. Prot. Res., 30: 634 - 645. Levinger, E.L. and Escamilla, R.F. (1955) Hereditary diabetes insipidus. J. Clin. Endocrinol. Metab., 15: 542 - 552. McMahon, A.P. and Bradley, A. (1990)The Wnt-1 (int-1) protooncogene is required for development of a large region of the mouse brain. Cell, 62: 1073- 1085. Miselis, R.R., Shapiro, R.E. and Hand, P.J. (1979) Subfornical organ efferents to neural systems for control of body water. Science, 205: 1022- 1025. Nagai, I., Li, C.H., Hsieh, S.M., Kizaki, T. and Urano, Y. (1984) Two cases of hereditary diabetes insipidus, with an autopsy finding in one. Acta Endocrinol., 105: 318- 323. Ohkubo, H., Kageyama, R., Ujihara, M., Hirose, T., Inayama, S. and Nakanishi, S. (1983) Cloning and sequence analysis of cDNA for rat angiotensinogen. Proc. Natl. Acad. Sci. U.S.A., 80: 2196-2200. Repaske, D.R., Phillips 111, J.A.,Kirby, L.T., Tze, W.Y., D’ErCole, J.D. and Battey, J. (1990) Molecular analysis of autosomal dominant neurohypophyseal diabetes insipidus. J.

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Clin. Endocrinol. Metabol., 70: 752 - 757. Rhodes, C.H., Morrell, J.I. and Pfaff, D.W. (1981) Immunohistochemical analysis of magnocellular elements in rat hypothalamus: distribution and number of cells containing neurophysin, oxytocin and vasopressin. J. Comp. Neurol., 198: 45 - 64. Robertson, G.L. (1988) Differential diagnosis of polyuria. Annu. Rev. Med., 39: 425 - 442. Saber, M.A., Novikoff, P.M. and Shafritz, D.A. (1990) Albumin and collagen mRNA expression in normal and analbuminemic rodent liver: analysis by in situ hybridization using biotinylated probes. J. Histochem. Cytochem., 38: 199- 207. Schmale, H. and Richter, D. (1984) Single base deletion in the vasopressin gene is the cause of diabetes insipidus in Brattleboro rats. Nature, 308: 705 - 709. Schmale, H., Borowiak, B., Holtgreve-Grez, H. and Richter, D. (1989) Impact of altered protein structures on the intracellular traffic of a mutated vasopressin precursor from Brattleboro rats. Eur. J. Biochem., 182: 621 -627. Schmale, H., Bahnsen, U., Fehr, S., Nahke, P. and Richter, D. (1991) Hereditarydiabetesinsipidus inmanandrat. In: S. Jard and R. Jamison (Eds.), Vasopressin - Collogue INSERM, Vol. 208, John Libbey Eurotext Ltd., London, pp. 57 - 62. Sherman, T.G. and Watson, S.J. (1988) Differential expression of vasopressin alleles in the Brattleboro heterozygote. J. Neurosci., 8: 3799 - 381 1. Silverstein, E., Sokoloff, L., Mickelsen, 0. and Jay, Jr., G.E. (1961) Primary polydipsia and hydronephrosis in an inbred strain of mice. A m . J. Pathol., 38: 143 - 159. Sokol, H.W. and Valtin, H. (1982) The Brattleboro rat. Ann. N . Y. Acad. Sci., 394: 1 - 828. Swaab, D.F., Boer, G.J. and Nolten, J.W.L. (1973) The hypothalamo-neurohypophyseal system (HNS) of the Brattleboro rat. Acta Endocrinol. (Suppl.), 177: 80. Takeda, S., Lin, C.-T., Morgano, P.G., McIntyre, S.J. and Dousa, T.P. (1991) High activity of low-Michaelis-Menten constant 3 ' 5 ' -cyclic adenosine monophosphate-phosphodiesterase isozymesin renal inner medulla of mice with hereditary nephrogenic diabetes insipidus. Endocrinology, 129: 287 294. Uhlich, E., Eigler, J., Trzyna, G., Finke, K., Winkelmann, W. and Buchborn, E. (1971) Zum Wirkungsmechanismus von Chlorpropamid bei Diabetes insipidus. Klin. Wochenschr., 49: 314-322. Valtin, H., Sawyer, W.H. and Sokol, H.W. (1965) Neurohypophyseal principles in rats homozygous and heterozygous for hypothalamic diabetes insipidus (Brattleboro strain). Endocrinology, 77: 101 - 706. Valtin, H. (1992) Genetic models of diabetes insipidus. In: E.E. Windhagen (Ed.), Handbook of Physiology, Section 8. Renal Physiologiy, Vol. 11, Oxford University Press, New York, pp. 1281 - 1316. Van Leeuwen, F.W. and Van der Beek, E.M. (1991) The amount of mutant vasopressin precursor in the supraoptic and paraventricular nucleus of Brattleboro rats increases with age.

Brain Res., 542: 163 - 166. Van Leeuwen, F.W., Caffe, R., Van der Sluis, P.J., Sluiter, A.A., Van der Woude, T.P., Seidah, N.G. and Chretien, M. (1986) Propressophysin is present in neurones at multiple sites in Wistar and homozygous Brattleboro rat brain. Brain Res., 379: 171-175. Van Leeuwen, F.W., Van der Beek, E.M., Seger, M., Burbach, P. and Ivell, R. (1989) Age-related development of a heterozygous phenotype in solitary neurons of homozygous Brattleboro rats. Proc. Natl. Acad. Sci. U.S.A., 86: 6417- 6420. Van Leeuwen, F.W., Van der Beek, E.M., Van Heerikhuize, J.J., Sluiter, A.A., Felix, D. and Imboden, H. (1991) Vasopressin and angiotensin I1 are absent but spontaneously reappear in solitary hypothalamic neurons of the homozygous Brattleboro rat. Neurosci. Lett., 127: 207 -211. Whitnall, M.H., Castel, M., Key, S. and Gainer, H. (1985) Immunocytochemical identification of dynorphin-containing vesicles in Brattleboro rats. Peptides, 6: 241 - 247. Young, 111, W.S., Reynolds, K., Shepard, E.A., Gainer, H. and Caste, M. (1990) Cell-specific expression of the rat oxytocin gene in transgenic mice. J. Neuroendocrinol., 2: 917- 925.

Discussion D.R. Repaske: Brattleboro rat do have normal amounts of VP in the adrenals and ovaries. If the translation of the poly A-tail is not what disrupts VP production in the hypothalamus, can you speculate on how these rats are able to make VP elaborate in their body. F.W. van Leeuwen: So far, no explanation is available for this difference in assayable VP between adrenals and ovaries on the one hand and the hypothalamus on the other hand (see e.g., Ivell etal., 1990).Thepoly A-tailofVPmRNAisindeed much shorter than the one of the hypothalamus which was a reasonable explanation for this phenomenon until the results of Schmale et al. (1989) excluded the poly A-tail (translated into polylysine and thus highly charged and possibly sticking to the membranes of the endoplasmic reticulum). The results of Schmale et al. (1989) indicated that the folding of thevasopressin precursor is essential for passage from the endoplasmic reticulum to the Golgi apparatus. It might be speculated that in the peripheral tissue the folding is less important than in the hypothalamus. In this respect it is of interest to realize that VP within adrenal cells does not have to be axonally transported and thus may be packaged differently from the hypothalamic cells.

References Ivell, R., Burbach, J.P.H. and Van Leeuwen, F.W. (1990) The molecular biology of the Brattleboro rat. Front. Neuroendocrinol., 11: 313-338. Schmale, H., Borowiak, B., Holtgrave-Grez, H. and Richter, D. (1989) Impact of altered protein-structures on the intracellular traffic of a mutated vasopressin precursor from Brattleboro rats. Eur. .I. Biochem., 182: 621 -627.

D.F. Swaab, M.A. Hofman, M. Mirmiran, R. Ravid and F.W. van Leeuwen (Eds.) Progress in Brain Research, Vol. 93 @ 1992 Elsevier Science Publishers B.V. All rights reserved.

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CHAPTER 20

Autoimmune hypothalamic diabetes insipidus (‘ ‘autoimmune hypothalamitis’ ’) W .A. Scherbaum Department of Internal Medicine, University Hospital, Ulm, Germany

Introduction Diabetes insipidus (DI) is a syndrome characterized by hypotonic polyuria and polydipsia. In hypothalamic DI, also known as central, neurogenic or hypothalamic DI, the disorder results from decreased secretion of osmoregulated arginine vasopressin (AVP) hormone which is synthesized in the perikarya of large neurosecretory cells located mainly in the supraoptic and paraventricular nuclei of the hypothalamus. Hypothalamic DI is a rare disorder which may be inherited as either a dominant (Repaske et al., 1990) or recessive (Forssmann, 1955) trait or it may be acquired at any age. Once the diagnosis of hypothalamic DI has been established, every effort should be undertaken to find out its underlying cause which may require specific therapy. Table I gives a list of possible causes of hypothalamic DI. In many cases no underlying disease is found, and for lack of better insight this form of the disorder is called idiopathic. Idiopathic hypothalamic DI comprised 30 - 45% of all spontaneously acquired cases, even if unrecognized diseases were excluded at long-term follow-up investigations (Blotner, 1958; Moses, 1985). By definition, this form had been ill-defined due to a lack of any specific markers. This matter was fortunately changed by the observation of the case of a patient with a polyendocrine autoimmune syndjome and central DI which led us to the search for and the detection

of circulating autoantibodies to hypothalamic vasopressin cells in cases of idiopathic DI (Scherbaum and Bottazzo, 1983). Our findings suggest that idiopathic hypothalamic DI consists of pathogenetically heterogeneous entities. I shall here try to provide an update of the data on autoimmune hypothalamic DI and to give an outlook for possible further research into autoimmune hypothalamitis. General features of autoimmune diseases are listed in Table 11. Autoimmune diseases are characterized by lympho-plasmacellular infiltration of the affected organ and by the detection of humoral and cellular autoimmune reactions directed towards the respective autoantigen(s). Ideally, one should be able to experimentally induce the diseasein a healthy animal by the transfer of cells or antibodies from an animal with the disease (Milgrom and Witebsky, 1972). In most endocrine autoimmune diseases of peripheral glands, production of antibodies, specific cellular infiltration as well as secondary atrophy of the organs have been experimentally induced by immunization with extracts from these glands together with complete Freund’s adjuvant (Rose and Witebsky, 1956). Immunization with the respective hormones appears to be less harmful to the animals. An animal model for experimental hypothalamitis has not been established so far. When antibodies to vasopressin were raised in rabbits by injecting the hormone together with complete Freund’s adjuvant, Swaab and colleagues (Swaab et al., 1975)

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observed transient diabetes insipidus. However, the hypothalamo-neurohypophyseal system was intact on histology with signs of increased hormone synthesis. Only a few cases of hypothalamic DI have been described where the histopathology of the hypothalamus was studied (Gaupp, 1941; Blotner, 1958; Green et al., 1958; Braverman et al., 1965). In both, idiopathic as well as in familial hypothalamic DI there was a marked loss of neurons as well as gliosis in the supraoptic nuclei. Similar, but less pronounced changes were also seen in the paraventricular nuclei. This is in parallel with the findings in atrophic autoimmune thyroiditis (primary myxoe-

TABLE I Causes of acquired hypothalamic diabetes insipidus Trauma Hypophysectomy, radiation therapy, skull fracture Tumour Craniopharyngioma, germinoma, pinealoma, suprasellar cysts, leukaemic infiltrates, metastases (carcinoma of breast, lung) Granulomatous diseases Histiocytosis X, sarcoidosis, tuberculosis, syphillis Encephalitis, meningitis Vascular lesions Cerebral thrombosis, haemorrhagia, aneurysm, post-partum necrosis Autoimmune Idiopathic

TABLE I1 General features of autoimmune diseases No evident cause for precipitation of diseases Associated autoimmune disease Familial trait Improve during pregnancy Local infiltration with mononuclear cells Specifically sensitized lymphocytes Specific autoantibodies in the serum

dema), autoimmune Addison’s disease and premature ovarian failure with Addison’s disease where a loss of specific hormone-producing cells and replacement by fibrous tissue are seen at histopathology (Doniach et al., 1982). Although the brain is traditionally considered to be “immunologically privileged”, immune and autoimmune responses can occur. The lack of conventional lymphatic drainage of the brain impedes transport of neural antigens to the lymphoid organs (Bradbury, 1981). However, antigen can be carried from the brain via the CSF. Under normal conditions, the brain is protected from a variety of circulating cellular and humoral components of the blood by the blood-brain barrier. This barrier consists of a capillary and endothelial cell layer with tight junctions, a dense basement membrane and an almost complete layer of surrounding astrocytes with their protruding foot processes (Reese and Karnovsky, 1967; Brightman and Reese, 1969), so that passage of molecules and cells is only possible after penetration of the luminal and anteluminal cell membrane. This prevents the passive entry of immunoglobulins, large immunomodulators, and immunocompetent cells. However, in the circumventricular organs, such as the organum vasculosum laminae terminalis of the hypothalamus as well as at the median eminence, such a barrier does not exist (Weindl, 1973), so that an exchange can take place between the peripheral immune system and hypothalamic structures. It has also been shown that pathological clinical conditions such as encephalitis and primary or secondary brain tumors (Hiranoetal., 1971;Long, 1979)aswellasanumber of experimental conditions such as drug-induced seizures (Lorenzo et al., 1972), intra-arterial infusions of hypertonic solutions or intracerebral injection of arachidonic acid (Westergaard, 1980; Chan and Fishman, 1984) may cause shrinking of endothelia, disconnection of tight junctions and reversible opening of the blood-brain barrier. It has also been shown that leukocytes may leave the blood vesseIs in the brain in response to specific homing molecules or chemotactic factors and then secrete degradative enzymes to cleave a passage

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through tissue (Naparstek et al., 1984). Thus, the normal absence of leukocytes from the brain, and their occurrence under pathological conditions such as multiple sclerosis and viral encephalitis (Hafler and Weiner, 1987)as well as in experimental models (Matsumoto et al., 1986) may represent changes in the expression of homing molecules as much as in the state of the physical blood-brain barrier (Lampson, 1987). In addition to the immunological isolation of the brain, both neurons and glia normally lack major histocompatibility class I and class I1 antigens on their surface, which are essential for the ability of cells to interact with T lymphocytes. Thus, normal neural tissues cannot respond to immunocompetent T lymphocytes even if they have entered the brain (Lampson and Hickey, 1986). However, HLA class I expression can be induced in neuronal cells after exposure to, e.g., interferon (Wong et al., 1985). Cultured glial, but not neuronal cells are able to express HLA class I1 molecules which can present antigens in vitro (Fontana et al., 1984). A further immunological protection of the brain , is provided by the fact that lymphocytes are much less adhesive for brain microvessel endothelium than extracerebral endothelium, but adhesion can be increased by stimulation of endothelial cells with interferon gamma or tumor necrosis factor-alpha (Male et al., 1990a). Mitogen-activated lymphocytes or T cells activated by their specific antigen bind more effectively than resting lymphocytes (Male et al., 1990b). Detection of cytoplasmic vasopressin cell antibodies Like other cell-type specific autoantibodies within complex organs hypothalamic vasopressin cell antibodies are detected by indirect immunofluorescence testing on organ sections. Unlike the well-preserved neurotransmitters and peptide hormones such as vasopressin (Emson et al., 1982; Rossor et al., 1982), the autoantigen of these vasopressin cell antibodies is very susceptible to post-mortem decay (Scherbaum and Bottazzo, 1983), so that fresh, shock-frozen tissue has to be used in the assay. Post-

fixation of cryostat sections with acetone does not influence the antibody reactivity whereas fixatives like Bouin’s or formaldehyde lead to unpredictable results, often giving false-positive or false-negative reactions (own unpublished observation). Human fetal hypothalamus has been applied as a source of antigen in all previous investigations. It should be mentioned, however, that the suitability of such specimens from prostaglandin-induced abortions is uncertain, and it should now be recommended to limit the use of hypothalamic tissues to those specimens obtained within 8 h after induction of abortion when hypothalamic cells still respond to physiological stimuli in perifusion systems (Rasmussen et al., 1986). In our obstetrical unit, however, the abortions are induced by gradually increasing doses of prostaglandins which cause a minimum of side effects to the women, but lead to delayed abortions, so that the time of fetal death in utero cannot be clearly denominated. Therefore, vasopressin cell antibody positive sera have always to serve as controls to test the suitability of a given tissue. Human adult post-mortem tissue would be a preferable source of antigen, but post-mortem delay of more than 2 h renders this tissue unsuitable for the test (Scherbaum, 1987). Also tissues from older donors are unsuitable since the autofluorescence of lipofuscin granules within neurosecretory cells of such individuals impair the reading of specific reactions in the indirect immunofluorescence test (Scherbaum et al., 1985a). By definition, human tissue has to be applied in the first place when human autoantibodies are to be demonstrated. Some autoantibodies such as antimitochondria1 antibodies, anti-nuclear antibodies or gastric parietal cell antibodies show a wide crossreactivity with non-human species. Others, such as thyroid or adrenal antibodies give a more restricted reactivity with mammalian tissues (Scherbaum et al., 1986a). However, heterophilic antibodies may still give false-positive reactions. This also applies to non-human primate tissue which has been shown to be unsuitable for the detection of pituitary autoantibodies (Gluck and Scherbaum, 1990). The suitability of baboon tissue as a source of antigen to test for

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vasopressin cell antibodies has not been extensively studied. In the limited number of comparative tests we have performed so far, the number of falsenegative reactions has not been evaluated. However, eight out of ten cases which had been positive for vasopressin cell antibodies on human tissue, were also positive when baboon hypothalamus was used (Scherbaum et al., 1985a). Vasopressin cell antibodies give a coarse cytoplasmic staining pattern of vasopressin cells. The titers are very low, so that undiluted sera have to be applied in the first place. The antibodies may be of the IgG and/or IgA and/or IgM class so that the serum reaction has to be visualized with a polyvalent anti-IgG/IgA/IgM anti-human immunoglobulin conjugate. Vasopressin cell antibodies are equally frequent of the IgG and IgA class, but IgM antibodies rarely occur. Some of the vasopressincontaining sera also react with oxytocin cells, but others are strictly cell type-specific (Scherbaum and Bottazzo, 1983). About half the vasopressin cell antibodies fix complement. The complement-fixing ability of antibodies detected in the sera of healthy individuals has been shown to indicate high risk for rapid progression to clinical disease in islet cell antibody-positive non-diabetic individuals (Tarn et al., 1988) as well as in adrenal antibody-positive individuals without Addison’s disease (Scherbaum and Berg, 1982). Healthy individuals with vasopressin cell antibodies in their sera have not been detected so far so that the impact of complementfixing vasopressin cell antibodies cannot be deter-

mined. However, no correlation has been found between the complement-fixing ability of vasopressin cell antibodies and a distinct subgroup of patients with hypothalamic DI, nor with the severity of disease (Scherbaum, 1987). Reactivity of the specific vasopressin cell autoantigen While the autoantigens corresponding to a number of autoantibodies to peripheral endocrine glands have been characterized, the biochemical nature of the autoantigen reacting with vasopressin cell antibodies is still unknown. It is evident from absorption experiments that the autoantigen is distinct from the hormones arginine vasopressin, oxytocin, or their corresponding neurophysins NPII and NPI. Preincubation with an excess of the above mentioned substances does not affect the vasopressin cell reactivity of positive sera. Also all vasopressin cell antibody positive and negative patients’ sera tested so far were negative for circulating vasopressin or neurophysin antibodies when tested by a sensitive direct radiobinding assay with iodinated vasopressin and neurophysin (Scherbaum et al., 1985b). This is in line with other cytoplasmic autoantibodies to endocrine glands detected by immunofluorescence testing, such as thyroid microsoma1 antibodies which are not directed to the hormones, but to cytoplasmic enzymes associated with the function of the respective cells (Czarnocka et al., 1985).

Fig. 1. a. Autoantibodies to hypothalamic vasopressin cells. An unfixed 7 pm cryostat section of human hypothalamus at the level of the supraoptic nucleus (SON) was incubated with native serum from a patient with idiopathic hypothalamic diabetes insipidus and stained with FITC-labeled anti-human-IgG. Note that the cytoplasm of large cells is stained. It could be shown by the four-layer double-fluorochrome immunofluorescence test with anti-vasopressin in the second sandwich that vasopressin cells were stained (magnification: x 250). b. The same area of the SON as in Fig. la incubated with normal human serum and FITC-labeled polyvalent anti-human immunoglobulin. Note that the background is brighter than the dark neurosecretory cell bodies (magnification: x 400). c. The same area of the SON as in Fig. la incubated with the serum of a patient with systemic lupus erythematosus containing the rare anti-ribosomal antibodies visualized by FITC-labeled anti-human IgG which may in a very rare case disturb the detection of vasopressin cell antibodies. Note the coarsely granulated cytoplasmic staining of the two large cell bodies (magnification: x 400). d. 7 pm cryostat section of human hypothalamus at the level of the SON. The specimen was obtained from a donor aged 50. The section was incubated with normal human serum and FITC-labeled polyvalent anti-human immunoglobulin. The autofluorescent lipofuscin deposits in the cell bodies of large neurosecretory cells hamper the evaluation of test results (magnification: x 250).

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and sensitive pituitary function tests may now reveal, e.g., germinomas, cranial sarcoidosis or tumour metastases long before clinical symptoms become apparent. In a more recent series by Moses (1985) 30% of the cases of spontaneously acquired hypothalamic DI were classified as idiopathic. The detection of vasopressin cell antibodies now helps to distinguish patients with possible autoimmune hypothalamic DI from other forms of idiopathic DI. One third of patients with idiopathic hypothalamic DI are positive for these antibodies. The percentage is about the same in adults (Scherbaum and Bottazzo, 1983) and in children (Scherbaum et al., 1985b) with this diagnosis. The sera from mixed hospital controls, cases of familial DI or from patients with nephrogenic DI were all negative. So were the sera from patients with DIDMOAD, a syndrome including diabetes insipidus, diabetes mellitus, optic atrophy and deafness. As shown in Table 111, there was one important group of patients with hypothalamic DI who were frequently positive for vasopressin cell antibodies, but there were only individual positive cases in other forms of symptomatic DI. Vasopressin cell antibodies have been detected in about half the cases of hypothalamic DI, secondary to histiocytosis X (Scherbaum et al., 1986b). Thymic abnormalities and antibodies to autologous erythrocytes have been observed in histiocytosis X

In order to account for a possible functional role, vasopressin cell antibodies should theoretically bind to structures represented on the surface of the cells, so that they can react with their respective autoantigens in vivo. Such a cell surface reactivity has been detected with the thyroid microsomal (Khoury et al;, 1981), the adrenocortical (Khoury et al., 1980) and the gastric parietal cell antigen (Masala et al., 1980)as well as with the 64-kDa islet cell autoantigen glutamate decarboxylase GAD (Christie et al., 1990). Similar to the above observations we have shown in selected cases that vasopressin cell antibodies react with the surface membrane of cultured human fetal hypothalamic vasopressin cells in vitro (Scherbaum et al., 1985~). Vasopressin cell antibodies in different forms of diabetes insipidus The symptoms of hypothalamic DI can now easily be cured by the substitution with vasopressin analogs. However, it is clinically essential to search for a specific disease underlying hypothalamic DI. The detection of such diseases often depends on the sensitivity of the diagnostic procedures and on the duration of follow-up investigations. Among 124 patients with hypothalamic DI published by Blotner (1958), 45% were classified as idiopathic in origin. Computed tomography, nuclear magnetic imaging

TABLE 111 Prevalence of vasopressin cell antibodies in different forms of diabetes insipidus (DI) Form of diabetes insipidus

Number tested

Number positive AVP cell antibodies

Idiopathic hypothalamic DI Histiocytosis X with DI Craniopharyngioma Other forms of symptomatic hypothalamic DI* Familial hypothalamic DI Nephrogenic DI

52 11 11 55

20 (38%) 6 (54%) 1 ( 9To) 0 ( 0%) 0 ( 0%) 0 ( 0%)

6 15

*Other forms of symptomatic hypothalamic D1 included: pituitary adenomas with post-operative DI (20), germinomas (7), DID MOAD (diabetes insipidus, diabetes mellitus, optic atrophy and deafness, lo), metastatic tumours (9,traumatic DI (4), sarcoidosi! with DI (3), post-irradiation (2), acute myeloid leukaemia (2), malformation of the hypothalamus (2).

289

(Nesbit et al., 1981). Clinical observations with spontaneous remissions also suggest that autoimmunity is involved at least in a subgroup of such patients (Broadbent et al., 1984). Histiocytosis X cells may invade the hypothalamus (Cline and Golde, 1973) and break the blood-brain barrier through a local inflammatory reaction. Like antigen presenting cells of the macrophage type, histiocytosis X cells bear HLA class I1 antigens on their surface (Murphy et al., 1981) which render them readily active in presenting locally disrupted antigen to preexisting lymphocytes. Non-processed antigen is well recognized by B lymphocytes (Lanzavecchia, 1985) which may be stimulated to proliferate and produce a specific immune response to hypothalamic tissues. A similar phenomenon has also been described in the case of a patient with histiocytosis X invading the thyroid gland, where high titers of thyroid autoantibodies were observed (Sinisi et al., 1986). Determination of vasopressin cell antibodies in cases of histiocytosis X may allow to recognize hypothalamic infiltration before the onset of clinical symptoms of hypothalamic DI which may prove to be helpful in the therapeutic management of patients with histiocytosis X. Polyendocrine autoimmunity and hypothalamic diabetes insipidus

The coexistence of established autoimmune diseases with an idiopathic endocrine disorder suggests the possibility of an autoimmune pathogenesis of the latter. Patients with polyendocrine autoimmune diseases have provided the best source for the detection of new autoantibody specificities in endocrine diseases of unknown origin, and such a case was also the basis of our original hypothesis to suggest an autoimmune variant of hypothalamic diabetes insipidus. Two years later, an interesting further case was reported with hypothalamic diabetes insipidus associated with hypopituitarism, type I (insulindependent) diabetes mellitus, pernicious anemia and circulating antibodies to the thyroid gland, adrenal cortex, gastric parietal cells and pancreatic islet cells (Bhan and O’Brien, 1982).

Polyendocrine autoimmune syndromes are classified into three groups (Neufeld et al., 1980): the rare type 1 with primary hypoparathyroidism and recurrent mucocutaneous candidiasis which appears during the first years of life and where Addison’s disease mostly develops later in childhood; type 2 polyendocrine autoimmunity centered around autoimmune Addison’s disease which is associated with autoimmune thyroid diseases and/or type 1 diabetes mellitus; and type 3 polyendocrine autoimmunity characterized by either autoimmune thyroid disease or/and type 1 diabetes mellitus which is associated with another autoimmune disorder such as vitiligo, pernicious anemia or autoimmune myasthenia gravis. Out of 39 cases of idiopathic hypothalamic DI studied, one or more autoimmune diseases were associated in 11 cases (28%). In two additional cases, autoantibodies to thyroid microsomes or to thyroglobulin were detected in the serum, suggesting subclinical autoimmunity. The list of autoimmune diseases and autoantibodies associated with idiopathic hypothalamic DI in our series is given in Tables IV and V. Thyroid autoimmunity is the main association, but it was not uncommon to observe more than one endocrine disease occurring together with DI. When a series of children with TABLE IV Associated organ-specific autoimmune diseases in 39 patients with idiopathic hypothalamic diabetes insipidus ~~

M 22 Primary hypoparathyroidism, alopecia totalis, mucocutaneous candidiasis, pernicious anemia F 13 Addison’s disease, primary myxoedema, pernicious anemia M 53 Addison’s disease, Hashimoto’s thyroiditis F 62 Type 1 diabetes mellitus, Hashimoto’s thyroiditis M 61 Primary myxoedema, Sjogren’s syndrome M 21 Type 1 diabetes mellitus (and family history of type 1 diabetes) F 24 Hashimoto’s thyroiditis F 39 Hashimoto’s thyroiditis F 52 Graves’ thyrotoxicosis F 31 Graves’ thyrotoxicosis M 60 Alopecia totalis, myasthenia gravis

290 TABLE V Prevalence of endocrine autoantibodies in patients with idiopathic and secondary diabetes insipidus

Form of diabetes insipidus Idiopathic (n = 39) Autoantibodies to: Thyroid microsomes Thyroglobulin Gastric parietal cells Intrinsic factor Adrenal cortex Gonadal steroid cells Pancreatic islet cells Pituitary prolactin cells Number of cases with organ-specific antibodies

6 3 2 1 2 2 2 3 11 (28%)

Secondary (n = 81)

4 1

2 -

-

6 (7%)

idiopathic hypothalamic DI was investigated in a separate study (Scherbaum et al., 1985b), polyendocrine cases were rare. This may reflect a distinct time course of polyendocrine autoimmunity where associated endocrine autoimmune diseases may develop later in life. Conclusions and outlook After the description of an autoimmune form of hypothalamic diabetes insipidus the spectrum of endocrine autoimmune diseases now extends to the, hypothalamus. Our findings suggest that autoimmunity may also play a role in other hypothalamic endocrine defects which have hitherto been regarded asidiopathic. It is theoretically possible that some cases of corticotropin deficiency, hypothyroidism, hypogonadism or growth hormone deficiency are related to cell type-specific hypothalamic autoimmunity. The inaccessability of hypothalamic tissue in vivo and the poor availability of fresh human post-mortem hypothalamus, however, remain a major obstacle in further investigations. It would be interesting to assess the functional role of

vasopressin cell antibodies by applying them in perifusion studies of human post-mortem hypothalamus explants. It is also unclear if vasopressin cell surface antibodies detected in the here described binding studies exert a cytotoxic effect which may be releated to the specific cell loss in the supraoptic and paraventricular nuclei. This matter as well as the question whether complement fixing vasopressin cell antibodies participate in the destructive process may be answered by cell culture studies. Will it be possible to establish an animal model of experimentally induced or even of spontaneous autoimmune hypothalamitis? Animal models like the spontaneously diabetic non-obese (NOD) mouse have provided new insights into the pathogenesis of autoimmune diseases and have allowed to develop new therapeutic strategies. What is the role of T lymphocytes in autoimmune DI? Is it possible to detect specifically sensitized T lymphocytes in the peripheral blood of patients with autoimmune DI? To answer these questions, one should get hold of the responsible autoantigen@.) in the hypothalamus. The availability of new techniques such as the screening of a human cDNA library with the autoantibodies may now allow to proceed in this field. References Bhan, G.L. and O’Brien, T.D. (1982) Autoimmune endocrinopathy associated with diabetes insipidus. Postgrad. Med. J., 58: 165- 166. Blotner, H. (1958) Primary or idiopathic diabetes insipidus: a system disease. Metabolism, 7: 191 - 200. Bradbury, M. (1981) Lymphatics and the central nervous system. Trends Neurosci., 4: 100- 101. Braverman, L.E., Mancini, J.P. and McGoldrick, D.M. (1965) Hereditary idiopathic diabetes insipidus. A case report with autopsy findings. Ann. Intern. Med., 63: 503 - 508. Brightman, M.W. and Reese, T.S. (1969) Junctions between intimately apposed cell membranes in the vertebrate brain. J. Cell Biol., 40: 648 - 677. Broadbent, V., Davies, E.G., Heaf, D., Pincott, J.R., Pritchard, J., Levinsky, R.J., Atherton, D.J. and Tucker, S. (1984) Spontaneous remission of multi-system histiocytosis X. Lancet, i: 253 -254. Chan, M.J. and Fishman, R.A. (1984) The role of arachidonic acid in vasogenic brain edema. Fed. Proc., 43: 210-213. Christie, M.R., Pipeleers, D.G., Lernmark, A. and Baekkeskov,

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analysis of MHC expression in human brain biopsies: tissue ranging from “histologically” normal to that showing different levels of glial tumor involvement. J. Immunol., 136: 4054 - 4062. Lanzavecchia, A. (1985) Antigen-specific interaction between T and B cells. Nature, 314: 537-542. Long, D.M. (1979) Capillary ultrastructure in human metastatic brain tumors. J. Neurosurg., 51: 53 - 58. Lorenzo, A.V., Shirahige, J., Liang, M. and Barlow, C.F. (1972) Temporary alteration of cerebrovascular permeability to plasma proteinduring drug-induced seizures. Am. J. Physiol., 223: 268 - 277. Male, D.K., Pryce, G., Hughes, C.C.W. and Lantos, P.L. (1990a) Lymphocyte migration into brain modelled in vitro: control by lymphocyte activation, cytokines and antigen. Cell. Immunol., 127: 1 - 11. Male, D.K., Pryce, G. and Rahman, J. (1990b) Comparison of the immunological properties of rat cerebral and aortic endothelium. J. Neuroimmunol., 30: 161 - 168. Masala, C., Smurra, G., Di Prima, M.A., Amendolea, M.A., Celestino, D. and Salsano, F. (1980) Gastric parietal cell antibodies: demonstration by immunofluorescence of their reactivity with the surface of the gastric parietal cells. Clin. Exp. Immunol., 41: 271 - 280. Matsumoto, Y., Naoyaki, H., Tanaki, R. and Fujiwara, M. (1986) Immunochemical analysis of the rat central nervous system during experimental allergic encephalomyolitis with special reference to Ia-positive cells with dendritic morphology. J. Immunol., 136: 3668 - 3676. Milgrom, F. and Witebsky, E. (1972) Autoantibodies and autoimmune diseases. JAMA, 181: 706-710. Moses, A.M. (1985) Clinical and laboratory observations in the adult with diabetes insipidus and related syndromes. In: P. Czernichow and A.G. Robinson (Eds.), Digbetes Insipidus in Man - Frontiers in Hormone Research, Vol. 13, Karger, Basel, pp. 156- 175. Murphy, G.F., Bhan, A.K., Sato, S., Harrist, T.J. and Mihm, M. (1981) Characterization of Langerhans cells by the use of monoclonal antibodies. Lab. Invest., 45: 465 - 468. Naparstek, Y., Cohen, J.R., Fuks, Z. and Vlodavsky, J. (1984) Activated T lymphocytes produce a matrix-degrading heparan sulfate endoglycosidase. Nature, 310: 241 -244. Nesbit, M.E., O’Leary, M., Dehner, L.H. and Ramsay, N.K.C. (1981) The immune system and the histiocytosis syndromes. Am. J. Pediatr. Hematol. Oncol., 3: 141 - 149. Neufeld, M., MacLaren, N. and Blizzard, R. (1980) Autoimmune polyglandular syndromes. Pediatr. Ann., 9: 43 - 53. Rasmussen, D.D., Liu, J.H., Swartz, W.H., Tueros, V.S., Wolf, P.L. and Yen, S.S.C. (1986) Human fetal hypothalamic GnRH neurosecretion: dopaminergic regulation in vitro. Clin. Endocrinol., 25: 127 - 132. Reese, T.S. andKarnovsky, M.J. (1967) Fine structural localization of a blood-brain barrier to exogenous peroxidase. J. Cell Biol., 34: 207-217.

292 Repaske, D.R., Phillips, J.A., Kirby, L.T., Tze, W.J., d’Ercole, A.J. and Battey, J. (1990) Molecular analysis of autosomal dominant neurohypophyseal diabetes insipidus. J. Clin. Endocrinol. Metab., 10: 152 - 157. Rose, N.R. and Witebsky, E. (1956) Studies on organ specificity: production of specific rabbit thyroid antibodies in the rabbit. J. Immunol., 76: 408 - 416. Rossor, M.N., Hunt, S.P., Iversen, L.L., Bannister, R., Hawthorn, J., Ang, V.T.Y. and Jenkins, J.S. (1982) Extrahypothalamic vasopressin is unchanged in Parkinson’s disease and Huntington’s disease. Brain Res., 252: 341 - 343. Scherbaum, W.A. (1987) Role of autoimmunity in hypothalamic disorders. Bailtiire’s Clin. Immunol. Allergy, 1: 231 - 245. Scherbaum, W.A. and Berg, P.Ar (1982) Development of adrenocortical failure in non-addisonian patients with antibodies to adrenal cortex. Clin. Endocrinol., 16: 345 - 352. Scherbaum, W.A. and Bottazzo, G.F. (1983) Autoantibodies to vasopressin cells in idiopathic diabetes insipidus: evidence for an autoimmune variant. Lancet, i: 897 - 901. Scherbaum, W.A., Bottazzo, G.F., Czernichow, P., Wass, J.A.H. and Doniach, D. (1985a) Role of autoimmunity in central dihbetes insipidus. In: P. Czernichow and A.G. Robinson (Eds.), Diabetes Insipidus in Man - Frontiers in Hormone Research, Vol. 13, Karger, Basel, pp. 156- 177. Scherbaum, W.A., Czernichow, P., Bottazzo, G.F. and Doniach, D. (1985b) Diabetes insipidus in children. IV. A possible autoimmune type with vasopressin cell antibodies. J. Pediatr., 107: 922- 925. Scherbaum, W.A., Hauner, H. and Pfeiffer, E.F. (1985~) Vasopressin cell surface antibodies in central diabetes insipidus detected on cultured human foetal hypothalamus. Horm. Metab. Res., 11: 623. Scherbaum, W.A., Mirakian, R., Pujol-Borrell, R., Dean, B.M. and Bottazzo, G.F. (1986a) Immunocytochemistry in the study and diagnosis of organ-specific autoimmune disease. In: J.M. Polak and S. Van Noorden (Eds.), Immunocyfochemistry. Modern Methods andApplications, Wright, Bristol, pp. 456 - 476. Scherbaum, W.A., Wass, J.A.H., Besser,G.M.,Bottazzo,G.F. and Doniach, D. (1986b) Autoimmune cranial diabetes insipidus: its association with other endocrine diseases and with hystiocytosis X. Clin. Endocrinol., 25: 41 1 -420. Sinisi, A.A., Criscuolo, T., Palombini, L., Bellastella, A. and Faggiano, M. (1986) Thyroid localization inadult histiocytosis X. J. Endocrinol. Invest., 9: 411 - 420. Swaab, D.F., Pool, C.W. and Nijveldt, F. (1975) Immunofluorescence of vasopressin and oxytocin in the rat hypothalamo-neurohypophyseal system. J. Neural Transm., 36: 195-215. Tarn, A.C., Thomas, J.M., Dean, B.M., Ingram, D., Schwarz, G . , Bottazzo, G.F. and Gale, E.A.M. (1988) Predicting insulin-dependent diabetes. Lancet, i: 845 - 850. Weindl, A. (1973) Neuroendocrine aspects of circumventricular organs. In: W.F. Ganong and L. Martini (Eds.), Frontiers in

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Discussion D.F. Swaab: One of the observations in other autoimmune diseases is that they might improve during pregnancy. What has been reported on such changes during pregnancy for idiopathic diabetes insipidus (DI) in literature and what is your experience? Secondly, do you think that the vasopressin cell autoantibodies are responsible for the destruction of the vasopressin neurons in autoimmune hypothalamic diabetes insipidus? W.A. Scherbaurn:This is a very important point which is true for Graves’ thyrotoxicosis, autoimmune myasthenia gravis, systemic lupus erythematosus, and probably also for autoimmune hypothalamic DI. We have observed a patient with hypothalamic DI due to histiocytosis X who was also positive for vasopressin cell antibodies. In this patient the severe histiocytic skin lesions and lymph node swellings as well as DI remitted at about the 28th week of gestation and recurred after delivery. This is very remarkable in light of the fact that vasopressinase activity increases during pregnancy so that patients with symptomatic hypothalamic DI usually require larger doses of DDAVP during pregnancy. Although this matter has not been studied in hypothalamic DI, I would not suspect that the vasopressin cell antibodies cause the disease. There are just a few autoimmune diseases, such as autoimmune myasthenia gravis or Graves’ thyrotoxicosis, where specific receptor antibodies may directly interfere with the function of the corresponding organ. In other autoimmune diseases, such as experimentally induced autoimmune encephalomyelitis (EAE) or insulin-dependent diabetes mellitus in the non-obese diabetic (NOD) mouse, this matter could be studied by transfer experiments of serum or lymphocytes from diseased animals to unaffected individuals. There it could be shown that the antibodies apparently do not cause functional defects or cellular lesions. Such lesions, however, are caused by the transfer of (antigen-specific, cytotoxic) T-lymphocytes which thus appear to be more directly involved in the effector mechanisms. G.J. Boer: It is very interesting to see that the autoantibodies you described are so specific for the vasopressin cells. 1 have two questions in this respect: (1) have you obtained evidence that human serum containing the autoantibodies can stain the HNS of other species? Using a sample of one of the sera we obtained from you a couple of years ago, Fred van Leeuwen and I failed to find staining in rat brain.

293 (2) Have you checked whether the nerve terminals of the VP cells also stain, in order to see if the staining is only at the level of the perikarya? W.A. Scherbaum: (1) Vasopressin cell autoantibodies are higly species-specific so that I am not surprised you did not obtain a positive reaction when you applied our positive control serum on rat hypothalamus. (2) We lookedat the perikarya because we expected the autoantigen to be located there. We have not looked at nerve terminals. H.M. Charlton: Why don’t you use a rat vasopressin cell line to induce experimental hypothalamic DI in rats? W. A. Scherbaum: To my knowledge nobody has raised such cell lines so far. D.F. Swaab: Tixier-Vidal and De Vitry (1979) have described a vasopressin-containing cell line. V. Ceenen: (1) Did you characterize auto-antigen(s) in autoimmune DI? (2)Have you completely eliminated the hypothesis that the autoantigens could be members of the neurohypophyseal peptide family? W.A. Scherbaum: (1) This is a difficult task since we would need a large amount of such human vasopressin cells on the one hand, and B-lymphocyte clones producing vasopressin cell antibodies, isolated from patients with idiopathic cranial DI to immunoprecipitate the autoantigens. Screening of a human brain-specific library would be another way. (2) There are arguments supporting the idea that the vasopressin cell antigen reacting in our own assay is different from the hormones vasopressin and oxytocin or their corresponding neurophysins: (a) Patients with hypothalamic DI who have been treated with vasopressin for a long time are usually negative in our assay. (b) We have done absorption studies with the above substances and the reactivity of our autoantibodies remained unchanged. Also there were no such antibodies detected ina direct radiobinding assay to elucidate this issue. (c) In no system - islet cell antibodies/insulin, thyroid microsomal antibodiedthyroid hormones, and others - was the method of indirect immunofluorescence sensitive enough to detect the respective anti-hormone antibodies which are detected by other assays. E. Braak: Have you been able to demonstrate components of the complement system with immunocytochemistry in the hypothalamus of patients with DI? W.A. Scherbaum: What we have done is to incubate patients’ sera with normal human hypothalamus, and we looked for the complement-fixing ability of autoantibodies by anti-complement conjugates. Here, about half the vasopressin cell antibodies were able to fix complement. What one wants to know, of course, is if there are immune complex deposits in the hypothalamus of such patients. Such deposits which are shown by direct im-

munofluorescence and staining, e.g., with anti-IgG or anticomplement antibodies, have indeed been shown in the pancreatic islets of patients who died with newly diagnosed type 1 diabetes mellitus and on the basement membranes of goitres removed from patients with Hashimoto’s thyroiditis. Patients with idiopathic hypothalamic DI live a long lifeand do not usually die of their disease, so that no such immunocytochemical studies are available from patients with idiopathic DI. H.P.H. Kremer: Were you able to demonstrate CSF abnormalities, i.e., antibodies in the CSF or oligoclonal bands in CSF or an increase in mononuclear cells? W.A. Scherbaum: We have not tested CSF from these people since in most of our cases the diagnosis of DI had been already established, so that collection of CSF was not justifiable. Therefore we just looked at serum. Nevertheless, investigation of CSF in cases of DI would be very interesting indeed. E. Fliers: (1) In type.1 diabetes mellitus (IDDM) it has been shown that asymptomatic NOD mice have antibodies against Langerhans islets long before they develop diabetes. Do you have data on the prevalence of anti-VP cell antibodies in subjects without DI? (2) Might autoimmune hypothalamitis be treated with corticosteroids? W.A. Scherbaum: (1) It would certainly be very interesting to investigate sera from normal individuals for vasopressin cell antibodies and thus for preclinical autoimmune hypothalamitis. However, hypothalamic DI and its autoimmune variant are very rare, so that I guess one would have to test thousands of individuals to pick up a positive not yet affected individual. As you can imagine, this would be impossible with the time-consuming vasopressin cell antibody test and the shortage of appropriate tissues. (2) Theoretically, the autoimmune reaction should be ameliorated by immunosuppresive drugs. However, this is not justifiable from the clinical standpoint, since replacement therapy of vasopressin with its analogs is very easy and safe, without known side effects when appropriately applied. On the other hand you have to consider that at the time when complete hypothalamic DI is diagnosed, it is likely that already 90% of the vasopressin neurons are destroyed. Since the repair capacity of the adult brain is limited, you will just deal with the remaining 10% of cells. From type 1 diabetes mellitus we know that this is - at least in the long run - a point of no return, and all individuals will eventually. require full hormone replacement therapy even when they are treated with potent immunosuppressive drugs, such as cyclosporin A.

References Tixier-Vidal, A. and De Vitry, F. (1979) Hypothalamic neurons in cell culture. Int. Rev. Cytol., 58: 291 -331.

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D.F. Swaab, M.A. Hofman, M. Mirmiran, R. Ravid and F.W. van Leeuwen (Eds.) Progress in Brain Research, Vol. 93 0 1992 Elsevier Science Publishers B.V. All rights reserved.

295

CHA$TER 21

The molecular biology of human hereditary central diabetes insipidus David R. Repaske and John A. Phillips, 111' Division of Pediatric Endocrinology, Department of Pediatrics, University of North Carolina at Chapel Hill, Chapel Hill, NC 27599-7220, U.S.A.; and I Division of Genetics, Department of Pediatrics, Vanderbilt University, Nashville, TN 37232, U.S.A.

Introduction Diabetes insipidus (DI) is a disease characterized by excessive excretion of water in the urine. In this disorder hypothalamic sensing of plasma osmolality is uncoupled from renal mechanisms for conserving water. The hypothalamus normally communicates fluid status to the kidney via regulation of release of a posterior pituitary hormone, arginine vasopressin (AVP), otherwise known as antidiuretic hormone. AVP is a nine amino acid peptide hormone with one intramolecular disulfide bond. It is synthesized in magnocellular neurons of the supraoptic and paraventricular nuclei of the hypothalamus, and axonally transported in neurosecretory granules with its carrier protein, neurophysin 11, and a glycoprotein to nerve terminals in the posterior pituitary (Brownstein, 1983). AVP is normally secreted into the circulation as the free peptide in response to plasma hyperosmolality sensed directly in the hypothalamus or in response to peripheral stimuli communicated to the hypothalamus by afferent neurons (Sawchenko and Swanson, 1981). Decreased extracellular fluid volume is sensed primarily by atrial stretch receptors, and hypotension by aortic baroreceptors. There are also humoral influences that potentiate AVP secretion, including angiotensin I1 (Iovino and Steardo, 1984), 0-adrenergic hormones (Schrier et al., 1979), and possibly others (Zerbe and Robertson, 1987). AVP acts on epithelial cells of the collecting

tubules of the kidney to increase their water permeability, allowing reabsorption of water from the renal ultrafiltrate. This action is mediated by a membrane-bound AVP receptor that stimulates an intracellular adenylate cyclase to increase the intracellular cyclic AMP (CAMP)concentration (Jard et al., 1975). CAMP-dependent kinases then catalyze phosphorylations that promote insertion of preformed intracellular stores of water channels into the cell membrane. The increase in the water permeability of the collecting tubule epithelium allows the resorption of water from the dilute ultrafiltrate to the hypertonic renal medullary interstitium (Dousa et al., 1977; Brown and Orci, 1983). In addition, AVP, also by a CAMP-dependent mechanism, acts in the medullary thick ascending limb of Henle to enhance Na+ and C1- transport from the ultrafiltrate to the renal medulla, thereby further increasing the medullary hypertonicity and the ability of the kidney to resorb water (Herbert and Andreoli, 1984). DI can result from one of two distinct types of defects. It can be central, that is, due to insufficient release of AVP from the posterior pituitary, or nephrogenic, that is, due to inadequate response of the kidney to circulating AVP. Both types have multiple etiologies, but they can usually be distinguished by assay of circulating AVP when the plasma has a relatively high osmolality and/or by testing the effect of exogenous AVP or AVP analog on the ability of the kidney to produce a concentrated urine. In central

296

DI, circulating AVP is inappropriately low, and the kidney will concentrate urine in response to exogenous AVP. In nephrogenic DI, the circulating AVP concentration is appropriate for plasma osmolality, and therefore additional exogenous AVP has no additional effect on the urine. Nephrogenic DI can be inherited as an X-linked recessive disorder (Bode and Crawford, 1969), but is usually sporadic or drug-induced (Reeves and Andreoli, 1989). Clinically, the majority of cases of central DI result from damage to the hypothalamus, pituitary stalk, or posterior pituitary. Most cases are secondary to head trauma, surgical procedures, or inflammatory or neoplastic disease. A small minority of cases of human central DI are familial. X-linked inheritance has been described in a limited number of families (Forssman, 1955; Green et al., 1967). Usually, inheritance follows an autosomal dominant pattern (Pender and Fraser, 1953; Levinger and Escamilla, 1955; Moehlig and Schultz, 1955; Martin, 1959; Braverman et al., 1965; Meinders and Bijlsma, 1970; Kaplowitz et al., 1982; Blackett et al., 1983; Toth et al., 1984; Pedersen et al., 1985). Patients with autosomal dominant central DI typically have polyuria and polydipsia with variable clinical severity in different affected members of a single family and even variable severity in an individual over time (Pender and Fraser, 1953; Martin, 1959; Green et al., 1967; Kaplowitz et al., 1982). Onset of clinical symptoms occurs from infancy to several years of age, and some patients report amelioration of symptoms in the third to fifth decades. Biochemical, histological and genetic investigations

The first measurements of plasma AVP concentration in human autosomal dominant central DI were reported by Kaplowitz et al. (1982) who found marked deficiencies in two affected brothers. The severely affected brother had no detectable circulating AVP, even after fluid restriction that raised his plasma osmolality well above normal to 306 mOsm/kg. The less severely affected brother was able to produce a small amount of AVP in both water deprivation and hypertonic saline infusion

tests. This paper documented a relationship between the severity of disease in individual family members and the degree of vasopressin deficiency in these individuals. A limited number of autopsy studies (Braverman et al., 1965; Green et al., 1967; Nagai et al., 1984; Bergeron et al., 1991)have examined the hypothalamus and pituitary of individuals affected with autosomal dominant central DI. Braverman et al. (1965) reported an autopsy study of a 49-year-old man who had two sisters with confirmed central DI and an extended family history very suggestive of autosomal dominant central DI. This man was consuming 4- 17 1 of fluid per day (with a similar amount of urine output) as an adult and reported having had polydipsia, polyuria and nocturia since childhood. An autopsy performed after death from a myocardial infarction revealed a small posterior pituitary that was “histologically intact”. His brain was not abnormal except for the hypothalamus. The supraoptic nucleus showed ‘‘a chronic degenerative process consisting of a marked loss of nerve cells, a mild gliosis, and varying degrees of karyorrhexis, chromatolysis, and loss of Nissl substance in the few remaining nerve cells” and the paraventricular nucleus showed similar, but less striking, changes. Green et al. (1967) reported an autopsy study of a 37-year-old woman with a very extensive family history of DI who had had central DI since the age of three. After her death from widely metastatic breast carcinoma, she was found to have a posterior pituitary that was “normal in appearance, but no neurosecretory material was identifiable”. The hypothalamus showed a paucity (“less than 5 % ” ) of the large neuron bodies that normally make up the supraoptic nucleus and an even more complete loss of the large neurosecretory cell bodies, but not the smaller neurons, in the paraventricular nucleus. There was a mild to moderate gliosis of these hypothalamic nuclei. Neurosecretory material was demonstrated in the few remaining large neurons by an aldehyde-fuchsin staining technique. Recently, Bergeron et al. (1991) reported an immunohistochemical study of the hypothalamus of a 72-yearold man with biochemically documented central DI

297

who had three clinically affected sons and one affected grandson. After death from heart failure, examination of the supraoptic nucleus showed virtual absence of magnocellular neurons, but those remaining were non-immunostaining for AVP. A fourth autopsy study (Nagai et al., 1984) did hot demonstrate any histological abnormality in the hypothalamus of a 44-year-old man with polyuria and polydipsia and a family history of DI who died of a myocardial infarction. However, this patient was able to concentrate his urine, produce some AVP, and maintain a nearly normal serum osmolality after a 14-h fluid restriction. Furthermore, the family history of DI included two brothers, a maternal aunt, and the maternal grandfather, but neither his mother nor any of his children or children of his affected siblings were affected. Thus this family had atypical familial polydipsia and polyuria that may represent an extremely mild variant of autosomal dominant central DI, or possibly a distinct disorder. These few histological studies in humans suggested that autosomal dominant central DI results from a dysgenesis or degeneration of the neurons that produce AVP in the supraoptic and paraventricular nuclei of the hypothalamus. The variation in severity of illness over time and between various members of a family may be related to the degree of anatomic and functional abnormality in the hypothalamus. While these findings did not shed light on the genetic basis for the disorder, they suggested a developmental abnormality or possibly a hereditary autoimmune disorder or an abnormal apoptosis (programmed cell death). Initial attempts to define the genetic locus of autosomal dominant central DI were unsuccessful. Fraser (1955) and Pedersen et al. (1985) examined large affected families for linkage (co-inheritance) of this form of DI with various biochemical, phenotypical, or cytogenetic markers such as red blood cell antigens, hair and eye color, HLA type, polymorphic red blood cell enzymes and chromosomal centromeric heteromorphisms. Neither investigator was able to identify significant linkage between DI and any of these markers that are distributed across the human genome.

Molecular biology of the AVP gene

The rat (Schmale et al., 1983) and human (Sausville et al., 1985) genes that encode AVP have been sequenced and have a similar organization (Fig. 1). Riddell et al. (1985) demonstrated that the human gene is located on chromosome 20. Both the rat and human genes are small (2.5 kilobases (kb)) composite genes comprised of three exons that encode three peptides downstream from a signal peptide: AVP, neurophysin I1 (NP 11), and a small glycoprotein (see Van Leeuwen, this volume). The first exon encodes the signal peptide, AVP, and the first nine amino acids of NP 11. The second exon encodes the mid-portion of NP I1 and the third exon encodes the last sixteen amino acids of NP I1 and the small glycoprotein of unknown function. A single mRNA is transcribed from the gene and translated to produce a single precursor protein. Post-translational cleavage yields the component peptides (Gainer et al., 1977). Molecular biology of hereditary central DI in the rat

The mutation that causes hereditary central DI in an animal model, the Brattleboro rat, falls within the AVP-NP I1 gene. This model system has recently been reviewed (Ivell et al., 1990). In the Brattleboro rat, DI is autosomal recessive, in sharp contrast to the human disease. Homozygous Brattleboro rats exhibit poly-dipsia and polyuria due to absence of detectable circulating AVP (Valtin et al., 1974; Pickering and North, 1982) and these rats are also unable to produce NP 11. They can concentrate their urine in response to exogenous AVP, as expected, and they apparently have normal oxytocin production and release from the posterior pituitary (Land

AVP-NP I1

1

OT-NP I

c32 1

2 3

w Fig. 1. Organization of AVP/OT region of human chromosome 20. Arrows indicate orientation of each gene from 5 ' to 3 ' . Numerals and wide line segments indicate exons.

298

et al., 1983; Ivell and Richter, 1984). Sequencing of the Brattleboro rat AVP-NP I1 gene (Schmale and Richter, 1984) revealed a single base pair deletion in the distal portion of the second exon, resulting in a frameshift mutation within the region of the AVPNP I1 gene encoding NP 11. The mutation does not affect transcription of the messenger RNA (mRNA) for AVP-NP 11; however, translation of this mutant mRNA, if it did occur, would produce avery abnormal NP 11. The mutant mRNA encodes a NP I1 with multiple amino acid substitutions in the C-terminus including alteration of an arginine that is probably required to direct proteolytic cleavage of the glycoprotein from the NP I1 and destruction of a glycosylation site. Furthermore, the termination codon is deleted, potentially allowing protein translation to continue abnormally into the mRNA's poly-A tail and thus adding a poly-lysine tail to the protein (Schmale and Richter, 1984; hell et al., 1986). In spite of the completely normal AVPcoding region in this mutant AVP-NP I1 gene, the downstream disruption within NP I1 prevents the successful production of adequate amounts of AVP (Majzoub et al., 1984). The mutant gene does remain capable of being transcribed and translated and the precursor protein appears in the endoplasmic reticulum (ER) but not in the Golgi apparatus or neurosecretory granules (Guldenaar et aI., 1986; Ivell et al., 1986; Krisch et al., 1986). Elegant in vitro experiments by Schmaleet al. (1989) suggest that the N-terminus of the abnormal precursor protein enters the ER but the C-terminus is unable to follow, leaving the precursor anchored in the ER membrane. Neither introducing a stop codon, a glycosylation site, nor a shortened or deleted poly-lysine tail into the mutant mRNA by genetic' engineering techniques allowed normal internalization and processing of the precursor protein in the ER. This suggested that another feature of the mutant NP I1 sequence was responsible for failure of protein processing. Surprisingly, the Brattleboro rats have been found to have a normal amount of AVP of unknown function in their adrenals and ovaries (Lim et al., 1984; Nussey et al., 1984). The only known difference between the

1

1

I

'

-

I I

I I Fig. 2. Pedigrees of autosomal dominant central DI families. Solid circles and squares indicate affected females and males, respectively. Open circles and squares indicate unaffected individuals. Slashes indicate deceased individuals. * Signifies affected persons studied in Fig. 3.

AVP-NP I1 mRNA in these tissues and in the hypothalamus is the addition of a relatively short poly-A tail in both the adrenals and ovaries (Ivell et al., 1986). Thus these tissues would produce a mutant precursor with a shorter poly-lysine tail, but the experiments of Schmale et al. (1989) suggest that this alone does not account for the ability of these tissues to produce AVP. In contrast to humans with familial DI, Brattleboro rats have hypertrophied supraoptic and paraventricular nuclei (Sokol and Valtin, 1965).

Molecular biology of human AVP gene in central DI We investigated the possibility that human autosoma1 dominant central DI might be encoded by a mutation in the AVP-NP I1 gene. Our studies involved thirteen affected and eight unaffected members of three multigenerational families (Fig. 2). Affected individuals had otherwise unexplained polyuria and polydipsia since childhood with a urine concentrating response to exogenous AVP or AVP analog and a family history of DI consistent with autosomal dominant inheritance. First, the AVP-NP I1 genes of one affected member of each family were examined for a large

299

Bgl I 1

Pvu I I

kb

-

7.5 6.7

=

5.3 5.0

-

3.8

-

2.3

- 0.8 Fig. 3. Autoradiogram of Southern blot after digestion of genomic DNA with Bgl I1 (left) or Pvu I1 (right) and hybridization with the AVP-NP I1 probe. Lanes I and 2 in each group are unaffected individuals. Lanes3 ,l a n d 5 are affected individuals from families 2, 1, and 3, respectively, as indicated by an asterisk in Fig. 2. Lane 3 in each group contained less DNA than the others, but showed identical patterns upon longer exposure. (From Repaske et al., 1990, with permission from the publisher.)

molecular disruption such as a deletion, insertion or rearrangement (Repaske et al., 1990). Genomic DNA was isolated from peripheral leukocytes (Kunkel et al., 1977) and analyzed by restriction endonuclease digestion followed by Southern blotting (Southern, 1975) and hybridization with a genomic AVP-NP I1 probe. In general, this approach involves digestion of genomic DNA with a restriction enzyme that produces multiple specific DNA fragments of varying sizes. The ends of these fragments are determined by the location of the specific DNA sequence at which that enzyme cuts double-stranded DNA. The resultant collection of DNA fragments is

separated on the basis of size by agarose gel electrophoresis, and the DNA is transferred to a nylon membrane to immobilize the fragments. Fragments that contain some or all of a particular DNA sequence can then be detected by hybridization with a radioactively labeled probe composed of that DNA sequence and by visualization using autoradiography (see Summar, this volume). In these experiments, 5 pg of genomic DNA from three unrelated affected individuals and two controls were digested to completion with the restriction endonuclease Bgl 11, and additional 5 pg aliquots were digested with Pvu 11. After electrophoresis and Southern blotting, the fragments containing portions of the AVP-NP I1 gene were detected by hybridization with a probe containing this entire gene. As shown in Fig. 3, the sizes of the genomic fragments of the AVP-NP I1 gene from affected and control individuals were identical. A large deletion involving the AVP-NP I1 gene would have produced a smaller AVP-NP 11-hybridizing fragment, or, if the deletion included a restriction enzyme recognition site, the total number of visualized fragments would have been decreased. Likewise, an insertion that disrupts the gene would be likely to have increased the size of at least one visualized fragment, and a rearrangement would be likely to have altered the pattern of visualized fragments. The results shown in Fig. 3 indicate that a major structural alteration of the AVP-NP I1 gene was not responsible for the disease in these families. Of course, a small gene disruption, such as the single base deletion of the Brattleboro rat or a base substitution, can have major genetic consequences without being detectable by this methodology (see Van Leeuwen, this volume). Linkage strategy We subsequently studied the AVP-NP I1 locus in autosomal dominant central DI using a genetic linkage strategy. This approach does not depend upon directly identifying or detecting the mutation responsible for the disease. Instead, it is an analysis of co-inheritance of individual AVP-NP I1 alleles

300

and the disease phenotype in affected family members. The goal is to demonstrate that one particular AVP-NP I1 allele, that is, the putative mutant gene, is inherited by all affected family members and not by any unaffected family members. If such an allele can be identified, it provides strong evidence that that allele, or the associated allele of another tightly linked gene, is responsible for the disease. The crucial prerequisite for this type of study is to develop a marker to identify and trace individual alleles through the pedigree. Earlier studies (Fraser, 1955; Pedersen et al., 1985) used panels of convenient cytogenetic or biochemical markers scattered across the genome for their linkage studies of autosoma1 dominant central DI. These investigators hoped that the disease locus would fall close to the locus of one of their markers and that they would therefore detect co-inheritance of that marker and the disease phenotype. However, they were not fortunate enough to have one of their markers close enough to the autosomal dominant central DI locus to detect significant linkage. On the other hand, we were able to develop linkage markers specific for a particular candidate locus, the AVP-NP I1 locus, and thus were able to demonstrate linkage in our families between the AVP region of chromosome 20 and autosomal dominant central DI. We used restriction fragment length polymorphisms (RFLPs) as our markers for the AVP-NP I1 alleles. Scattered throughout the genome, frequently in introns or between genes, are single base pair variations that have no effect on phenotype. Such variations - polymorphisms - are usually undetected, but can easily be detected if they fall within a restriction endonuclease recognition sequence as they then produce a polymorphic restriction site (Botsteinet al., 1980; Antonarakis et al., 1982). The presence of a polymorphic restriction site creates an RFLP, a variation in the size of fragments visualized after a restriction digest. Genomic DNA digested with the appropriate restriction enzyme will yield one large fragment (usually designated the - RFLP allele) if the polymorphic restriction siteis absent, or two smaller DNA fragments if the restriction site is present. Usually only one of the smaller fragments

hybridizes with the radiolabeled probe and is visualized. This smaller fragment is usually designated the + RFLP allele. Occasionally, two different polymorphic sites can occur within one large restriction fragment, producing three different restriction fragment lengths. These RFLP alleles are then named by their fragment sizes. The presence of different polymorphic restriction sites, detected as different RFLP alleles, on each of a person’s two homologous chromosomes can serve as a marker to distinguish the .two chromosomes. Identifying a set of RFLPs in a given region of a chromosome allows construction of an RFLP haplotype associated with that region of each chromosome. An RFLP haplotype frequently allows informative analysis of an element of a pedigree where the information from a single RFLP allele might be ambiguous. We first attempted to find RFLPs associated with the AVP-NP I1 gene that could be detected with the AVP-NP I1 DNA probe. Genomic DNA from nine control individuals (representing 18 AVP-NP I1 alleles) was digested with each of 33 restriction endonucleases and analyzed by Southern blotting using the AVP-NP 11 probe. All of the resulting fragments from each enzyme were identical in size in all of the individuals examined (Repaske et al., 1990). In other words, no RFLPs were detected using these restriction enzymes and the AVP-NP I1 probe. We then used a DNA probe from an adjacent gene to identify RFLPs that are very tightly linked to, but not in, the AVP-NP I1 gene. The gene that encodes human oxytocin (OT) and its carrier protein, neurophysin I (NP I), is located adjacent (approximately 10 kb away) to the AVP-NP I1 gene (Fig. 1). At this distance, the likelihood that these two loci would recombine during meiosis is less than 1 in 10000. Thus the AVP-NP I1 allele and the OT-NP I allele on each chromosome 20 are inherited together 9999 times out of 10000.The OT-NP I gene is highly homologous to the AVP-NP I1 gene but is oriented in the opposite direction. It has been cloned by Sausvilleet al. (1985). We used an OT-NP I genomic clone, radioactively labeled, to rescreen the restriction enzyme digested DNA from the nine control in-

301 Xba I

Apa I

21/9 21/3 9/3

3/3

-/-

- / + +/+

Dde I

kb

--/+

+I+

21

9

5

3

kb 1.1

0.7 0.6

0.9

Fig. 4. RFLPs in the AVP/OT region of human chromosome 20. Autoradiograms of Southern blots of control DNA after digestion with indicated restriction endonuclease and hybridization with the OT-NP I probe. RFLP alleles present in each sample are indicated above each lane. Apa I alleles are labeled by approximate fragment size in kb. Xba I and Dde I alleles are labeled (for absence of the polymorphic restriction site) for the larger fragment and + (for presence of the polymorphic restriction site) for the smaller fragment. The Dde I digest reveals a non-polymorphic fragment of 1.1 kb in association with the RFLP. (From Repaske et al., 1990, with permission from the publisher.)

TABLE 1 OT-NP I RFLP allele sizes and frequencies Enzyme

Allele desig- Size (kb) Frequency Chromosomes nation examined

Apa I

21 9 3

21.4 9.4 3.1

0.21 0.40 0.39

62

XbaI

-

9.8 5.4

0.92 0.08

100

DdeI

-

0.66 0.60

0.07 0.93

58

sea I

-

0.90 0.10

18

+ + +

17.5 15.5

dividuals (Repaske et al., 1990). Four different restriction enzymes (Apa I, Xba I , Dde I, and Sca I) revealed RFLPs with the OT-NP I probe (Fig. 4, Table I ) . For example, there is a polymorphic XbaI site adjacent to the OT-NP I gene that falls between two constant Xba I sites, 9.8 kb apart, that flank this gene. Thus, the OT-NP I probe detects a 9.8 kb fragment if the polymorphic Xba I site is absent but detects a 5.4 kb fragment in the presence of the site. Evaluation of additional control individuals for these RFLPs allowed determination of the frequency of occurrence of each fragment length in the control population (Table I). Five microgram aliquots of genomic DNA from each available member of the three affected families were then digested separately with Xba I (Fig. 5), Apa I (Fig. 6 ) , and Dde I. In a few cases, not all digestions could be performed because of limited DNA quantity. The pattern of OT-NP I RFLPs in each individual allowed construction of RFLP haplotypes that demonstrate the inheritance pattern of individual OT-NP I (and therefore the tightly linked AVP-NP 11) alleles for families 1 and 2 (Fig. 7). In family 1, the haplotype (Apa I/Dde I/Xba I ) 3/ + / + occurs in all affected persons and not in unaffected persons. Likewise, in family 2, the disease is associated with the 3/ - / - haplotype. Unfortunately, all members of family 3 were homozygous for all three of these RFLPs as well as the Sca I RFLP. Thus these RFLPs were unable to distinguish the various OT-NP I alleles in this family. The statistical significance of co-inheritance of the RFLP haplotype and the DI phenotype, taking into account such variables as the RFLP allele frequencies, was calculated as a LOD (log odds) score (Morton, 1955). This analysis allows data from different families with the same disease to be combined in order to achieve greater statistical significance than with any one family alone. The maximum LOD score for family 1 alone was 2.40 which means that there is only a 1 in 102.4,or 1 in 250, probability that this association between the disease and the RFLP haplotype in the AVP/OT region of chromosome 20 occurred by chance alone. Combining the data from the families 1 and 2 raises the LOD score to 2.7, or

302

kb

9

w

9.89

5.4Fig. 5 . Autoradiograms of genomic DNA from members of families 1 (left) and 2 (right) after digestion with Xba I, Southern blotting, and hybridization with the OT-NP I probe. (From Repaske et al., 1990, with permission from the publisher.)

-2 1 - 9

Fig. 6. Autoradiograms of genomic DNA from members of families 1 (left) and 2 (right) after digestion with Apa I, Southern blotting, and hybridization with the OT-NP I probe, (From Repaske et al., 1990, with permission from the publisher.)

c

303

Apa I

Dde I Xba I

Fig. 7. RFLP allele haplotypes of individuals from families 1 (top) and 2 (bottom Symbols representing the haplotypes of RFLPs on each chromosome are displayed vertically. The RFLP alleles were detected by hybridization to the OT-NP I probe after digestion with (from top to bottom) Apa I, Dde I, and Xba I. DNA was not available from the deceased members of family 1 or from the members of family 2 lacking haplotype symbols. Insufficient quantities of DNA prevented determination of complete haplotypes in all cases. (From Repaske et al. 1990, with permission from the publisher.)

a 1 in 500 probability that these results occurred by chance alone. Recently we have studied DNA from a fourth unrelated family that contains 11 affected individuals. Preliminary results in five informative matings substantiate the findings that the AVP/OT locus cosegregates with the DI phenotype. Sequence analysis of AVP gene

These results very strongly suggest that the genetic locus for autosomal dominant central DI is in or near the AVP-NP I1 gene as in the Brattleboro rat. A recent study by Ito et al. (1991) examined the nucleic acid sequence of the AVP-NP I1 genes of two affected sisters from a Japanese family with this disease. They identified a single base pair alteration in one allele of both of these patients compared with three Japanese and two Caucasian controls and with the previously published gene sequence (Sausville et al., 1985). This sequence difference is a G to A transition located in the NP I1 coding sequence in exon 2, just 21 base pairs 5 ’ to the corresponding site of the Brattleboro rat deletion. As a result of this alteration a glycine (position 57) in the NP I1 is

changed to a serine. Unfortunately, this study could not include any unaffected family members to demonstrate that this alteration is present only in affected individuals. Thus it remains possible that this alteration is a phenotypically silent polymorphism in this family and that the disease is encoded by a mutation at a different locus. The G to A transition in this Japanese family destroys an Msp I restriction endonuclease recognition site (CCGG to CCAG). This allowed relatively rapid screening of individuals for the alteration. Ito et al. designed specific polymerase chain reaction (PCR) primers that flank exon 2 of the AVP-NP I1 gene and allow amplification of a 304 base pair (bp) fragment including all of exon 2. There are three Msp I sites within this fragment in control individuals, and therefore digestion of this PCR product with Msp I produces four fragments of 32 bp, 56 bp, 93 bp and 123 bp. One of the two AVP-NP I1 alleles of each of the affected individuals in the Japanese family was missing the Msp I site between the 123 and 93 bp fragments, and thus these individuals have a 216 bp fragment in addition to the fragments from their normal allele. No 216 bp frag-

304

ments were detected in a screening of 20 unrelated, unaffected Japanese individuals, providing additional evidence that this alteration is not simply a polymorphism and may be responsible for autosoma1 dominant central DI in this family. We have recently examined two unaffected and four affected members of our family 1 by a similar PCR amplification of AVP-NP I1 exon 2 and did not find that this Msp I site is destroyed in any of these individuals, suggesting heterogeneity in the molecular basis of autosomal dominant central DI. Further studies of the AVP-NP I1 gene in this and other affected families are in progress. An additional unique single base substitution (G T at nucleotide position 1740causing a Glu --* Val substitution at position 29) in the coding region for neurophysin I1 has recently been shown to be associated with a family affected by autosomal dominant central DI (Schmale et al., 1991). Further studies have suggested that this mutation is the cause of autosomal dominant central DI in this family (see Van Leeuwen, this volume). +

Summary and conclusions Molecular biology techniques have begun to shed light on the genetic basis of autosomal dominant central DI, but several very basic questions remain to be answered. The disorder was initially presumed to have a developmental, degenerative, or autoimmune basis based on the autopsy findings in the hypothalamus of a limited number of patients. The molecular cloning of the AVP-NP I1 gene and the clue from the Brattleboro rat that at least this one form of hereditary DI involved an AVP-NP I1 gene mutation allowed us to focus on this gene in our study of human hereditary DI. Our initial experiments did not show this gene to have a major structural alteration such as a deletion, insertion, or rearrangement, but the approach was not capable of detecting more subtle defects. The linkage studies provided substantial evidence that one particular OT-NP I haplotype was linked to the disease phenotype in each family, and thus, a mutation in the AVP/OT region of chromosome 20 is responsible

for this disease. Ito et al. (1991) then identified a single base change in the AVP-NP I1 gene in affected members of one Japanese family. This change was not detected in unrelated, unaffected persons and thus is a good candidate for the mutation causing the disease in this family. However, there appears to be diversity in the molecular basis of autosomal dominant central DI as affected members of one of our families did not have this particular base change in either AVP-NP I1 allele and recently another distinct AVP-NP I1 gene base change has been associated with this disorder. One interesting question still to be addressed is how a mutation in the NP-I1 coding region of this gene prevents AVP release from the posterior pituitary in the rat or the human disease. Does the disrupted AVP-NP I1 coding sequence prevent normal processing of the mRNA so that it can not be properly translated into protein? Does the mutated AVP-NP I1 glycoprotein precursor protein interfere with normal post-translational processing to prevent release of AVP? Is an altered NP I1 protein not able to protect the AVP from proteolysis within the magnocellular neuron? An even more puzzling question is how a mutation in the gene encoding a hormone is inherited in an autosomal dominant pattern. The Brattleboro rat model follows the a priori expectation of autosomal recessive inheritance: the animal only exhibits a defect in hormone function if both genes encoding the hormone are defective. In the human disease, the dysfunction of one gene somehow inhibits the normal functioning of the other AVP-NP I1 gene. The mutant gene product may irreversibly bind the ribosome blocking further AVP synthesis, even that encoded by the normal gene. The degenerative changes of the cells that produce AVP in autosomal dominant central DI could be the result of a gene product that is somehow toxic, although this would not easily explain the variable expression of the disease over time and the variability from one affected family member to another. Thus while the molecular genetic approach has allowed us to begin to elucidate the molecular basis of autosomal dominant central DI, many unanswer-

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ed questions still exist about the pathophysiology of this disorder. Sequencing of the mutant gene in additional families and then study of mRNA synthesis, protein synthesis, and subsequent protein processing from these mutant genes should allow a definitive answer to the questions raised above and contribute to a fuller understanding of the molecular basis of autosomal dominant central DI.

References Antonarakis, S.E., Phillips, J.A., 111and Kazazian, H.H. (1982) Genetic diseases: diagnosis by restriction endonuclease analysis. J. Pediatr., 100: 845 - 856. Bergeron, C., Kovacs, K., Ezrin, C. and Mizzen, C. (1991) Hereditary diabetes insipidus: an immunohistochemical study of the hypothalamus and pituitary gland. Acta Neuropathol. (Bed.), 81: 345 - 348. Blackett, P.R., Seif, S.M., Altmiller, D.H. and Robinson, A.G. (1983) Familial central diabetes insipidus: vasopressin and nicotine stimulated neurophysin deficiency with subnormal oxytocin and estrogen stimulated neurophysin. Am. J. Med. Sci., 286: 42-46. Bode, H.H. andcrawford, J.D. (1969) Nephrogenic diabetes insipidus in North America - The Hopewell hypothesis. N . Engl. J. Med., 280: 750- 754. Botstein, D., White, R.L., Skolnick, M. andDavis, R.W. (1980) Construction of a genetic linkage map in man using restriction fragment length polymorphisms. A m . J. Hum. Genet., 32: 314-331. Braverman, L.E., Mancini, J.P. and McGoldrick, D.M. (1965) Hereditary idiopathic diabetes insipidus. A case report with autopsy findings. Ann. Int. Med., 63: 503 -508. Brown, D. and Orci, L. (1983) Vasopressin stimulates formation of coated pits in rat kidney collecting ducts. Nature, 302: 253 - 255. Brownstein, M. J. (1983) Biosynthesis of vasopressin and oxytocin. Annu. Rev. Physiol., 45: 129- 135. Dousa, T.P., Barnes, L.D. and Kim, J.K. (1977) The role of cyclic AMP-dependent protein phosphorylations and microtubules in the cellular action of vasopressin in mammalian kidney. In: A.M. Moses and L. Share (Eds.), Neurohypophysis, Karger, Basel, pp. 220- 235. Forssman, H. (1955) Two different mutations of the Xchromosomecausing diabetes insipidus. Am. J. Hum. Genet., 7: 21 -27. Fraser, F.C. (1955) Hereditk dominante d’un diabete insipide du a une dkficience en pitressine. J. Gener. Hum., 4: 193 - 203. Gainer, H., Same, Y. and Brownstein, M.J. (1977) Biosynthesis and axonal transport of rat neurohypophyseal proteins and peptides. J. Cell Biol., 13: 366-381.

Green, J.R., Buchan, G.C., Alvord, E.C. and Swanson, A.G. (1967) Hereditary and idiopathic types of diabetes insipidus. Brain, 90: 707-714. Guldenaar, S.E.F., Nahke, P. and Pickering, B.T. (1986) Immunocytochemical evidence for the presence of a mutant vasopressin precursor in the supraoptic nucleus of the homozygous Brattleboro rat. Cell TissueRes., 244: 431 - 436. Herbert, S.C. and Andreoli, T.E. (1984) Effects of antidiuretic hormone on cellular conductive pathways in mouse medullary thick ascending limb of Henle. 11. Determinants of the ADHmediated increases in transepithelial voltage in net C1- absorption. J. Membr. Biol., 80: 221 -233. Iovino, M. and Steardo, L. (1984) Vasopressin release to central and peripheral angiotensin I1 in rats with lesions of the subfornical organ. Brain Res., 322: 365 - 368. Ito, M., Mori, Y., Oiso, Y. and Saito, H. (1991) A single base substitution in the coding region for neurophysin I1 associated with familial central diabetes insipidus. J. Clin. Invest., 87: 725 - 728. Ivell, R. and Richter, D. (1984) Structure and comparison of the oxytocin and vasopressin genes from rat. Proc. Natl. Acad. Sci. U.S.A., 81: 2006-2010. Ivell, R., Schmale, H., Krisch, B., Nahke, P. and Richter, D. (1986) Expression of a mutant vasopressin gene: differential polyadenylation and read-through of the mRNA 3’ end in a frameshift mutant. EMBO J., 5: 971 - 977. Ivell, R., Burbach, J.P.H. and Van Leeuwen, F.W. (1990) The molecular biology of the Brattleboro rat. Front. Neuroendocrinol., 11: 313 - 338. Jard, S., Roy, C., Barth, T., Rajerison, R. and Bockaert, J. (1975) Antidiuretic hormone-sensitive kidney adenylate cyclase. Adv. Cyclic Nucleotide Res., 5: 31 - 52. Kaplowitz, P.B., D’Ercole, A.J. and Robertson, G.L. (1982) Radioimmunoassay of vasopressin in familial central diabetes insipidus. J. Pediatr., 100: 76 - 81. Krisch, B., Nahke, P. and Richter, D. (1986) Immunocytochemical staining of supraoptic neurons from homozygous Brattleboro rats by use of antibodies against two domains of the mutated vasopressin precursor. Cell Tissue Res., 244: 351 - 358. Kunkel, L.M., Smith, K.D., Boyer, S.H., Borgaonkar, D.S. and Wachtel, S.S. (1977) Analysis of human Y chromosome specific reiterated DNA in chromosome variants. Proc. Natl. Acad. Sci. lJ,S.A., 14: 1245- 1249. Land, H., Grez, M., Ruppert, S., Schmale, H., Rehbein, M., Richter, D. and Schutz, G. (1983) Deduced amino acid sequence from the bovine oxytocin-neurophysin I precursor cDNA. Nature, 302: 342 - 344. Levinger, E.L. and Escamilfa, R.F. (1955) Hereditary diabetes insipidus: report of 20 cases in seven generations. J. Clin. Endocrinol. Metab., 15: 547- 552. Lim, A.T.W., Lolait, S. J., Barlow, J.W., Autelitano, D.J., Toh, B.H., Boublik, J., Abraham, J., Johnston, C.I. and

306 Funder, J.W. (1984) Immunoreactive arginine-vasopressin in Brattleboro rat ovary. Nature, 310: 61 -64. Majzoub, J.A., Pappey, A., Burg, R. and Habener, J.F. (1984) Vasopressin gene is expressed at low levels in the hypothalamus of theBrattleboro rat. Proc. Natl. Acad. Sci. U.S.A.,81: 5296- 5299. Martin, F.I.R. (1959) Familial diabetes insipidus. J. Med., 112: 573 - 582. Meinders, A.E. and Bijlsma, J.B. (1970) A family with congenital hypothalamic neurohypophyseal diabetes insipidus. Folia Med. Neerl., 13: 68 - 72. Moehlig, R.C. and Schultz, R.C. (1955) Familial diabetes insipidus. Report of one of fourteen cases in four generations. JAMA, 158: 725-727. Morton, N.E. (1955) Sequential tests for the detection of linkage. A m . J. Hum. Genet., 7: 277-318. Nagai, I., Li, C.H., Hsieh, S.M., Kizaki, T. and Urano, Y. (1984) Two cases of hereditary diabetes insipidus, with an autopsy finding in one. Acta Endocrinol., 105: 318 - 323. Nussey, S.S., Ang, V.T.Y., Jenkins, J.S., Chowdrey, K.S. and Bisset, G.W. (1984) Brattleboro rat adrenal contains vasopressin. Nature, 310: 64 -66. Pedersen, E.B., Lamm, L.U., Albertsen, K., Madsen, G., Bruun-Petersen, G., Henningsen, K., Friedrich, U. and Magnusson, K. (1985) Familial cranial diabetes insipidus: a report of five families. Genetic, diagnostic and therapeutic aspects. Q. J. Med., 224: 883 - 896. Pender, C.B. and Fraser, F.C. (1953) Dominant inheritance of diabetes insipidus. A family study. Pediatrics, 11: 246- 254. Pickering, B.T. and North, W.G. (1982) Biochemical and functional aspects of magnocellular neurons and hypothalamic diabetes insipidus. Ann. N . Y . Acad. Sci., 394: 72-81. Repaske, D.R., Phillips, J.A., 111, Kirby, L.T.,Tze, W.J., D’ErCole, A.J. and Battey, J . (1990) Molecular analysis of autosomal dominant neurohypophyseal diabetes insipidus. J. Clin. Endocrinol. Metab., 70: 752 - 757. Reeves, W.B. and Andreoli, T.E. (1989) Nephrogenic diabetes insipidus. In: C.R. Scriver, A.L. Beaudet, W.S. Sly and D. Valle (Eds.), Metabolic Basis of Inherited Disease, 6th edn., McGraw-Hill, New York, pp. 1985 - 201 1. Riddell, D.C., Mallonee, R., Phillips, J.A., 111, Parks, J.S., Sexton, L.A. and Hamerton, J.L. (1985) Chromosomal assignment of human sequences encoding arginine vasopressinneurophysin I1 and growth hormone releasing factor. Somat. Cell, Mol. Genet., 11: 189- 195. Sausville, E., Carney, D. and Battey, J . (1985) The human vasopressin gene is linked to the oxytocin gene and is selectively expressed in a cultured lung cancer cell line. J. Biol. Chem., 260: 10236- 10241. Sawchenko, P.E. and Swanson, L.W. (1981) Central noradrenergic pathways for the integration of hypothalamic neuroendocrine and autonomic responses. Science, 214: 685 - 687. Schmale, H. and Richter, D. (1984) Single base deletion in the

vasopressin gene is the cause of diabetes insipidus in Brattleboro rats. Nature, 308: 705 -709. Schmale, H., Heinsohn, S. and Richter, D. (1983) Structural organization of the rat gene for the arginine vasopressinneurophysin precursor. EMBO J., 2: 763 - 767. Schmale, H., Borowiak, B., Holtgreve-Grez, H. and Richter, D. (1989) Impact of altered protein structures on the intracellular traffic of a mutated vasopressin precursor from Brattleboro rats. Eur. J. Biochem., 182: 621 -627. Schmale, H., Bahnsen, U., Fehr, S., Nahke, P. and Richter, D. (1991) Hereditary diabetes insipidus in man and rat. In: S. Jard and R. Jamison (Eds.), Vasopressin - Colloque INSERM, Vol. 208, John Libbey Eurotext Ltd., pp. 57 - 62. Schrier, R.W., Berl, T. and Anderson, R.J. (1979) Osmotic and non-osmotic control of vasopressin release. Am. J. Physiol., 236: F321- F332. Sokol, H.W. and Valtin, H. (1965) Morphology of the neurosecretory system in rats homozygous and heterozygous for hypothalamic diabetes insipidus (Brattleboro strain). Endocrinology, 77: 692 - 700. Southern, E.M. (1975) Detection of specific sequences among DNA fragments separated by gel electrophoresis. J. Mol. Biol., 98: 503 - 517. Toth, E.L., Bowen, P.A. and Crockford, P.M. (1984) Hereditary central diabetes insipidus: plasma levels of antidiuretic hormone in a family with a possible osmoregulator defect. Can. Med. Assoc. J . , 131: 1237- 1241. Valtin, H., Stewart, J. and Sokol, H.W. (1974) Genetic control of the production of posterior pituitary principles. Handbk. Physiol., 7: 131- 171. Zerbe, R.L. and Robertson, G.L. (1987) Osmotic and nonosmotic regulation of thirst and vasopressin secretion. In: M.H. Maxwell, C.R. Kleeman and R.G. Narins (Eds.), Clinical Disorders of Fluid and Electrolyte Metabolism, 4th edn., McGraw-Hill, New York, pp. 61 - 78.

Discussion D.F. Swaab: How can we explain that in human hereditary diabetes insipidus (DI) the vasopressin cells in the supraoptic and paraventricular nuclei (but probably not in the suprachiasmatic nucleus) die off (Bergeron et al., 1991)but not in the Brattleboro rat? D.R. Repaske: This is a good question and the answer is not yet clear. The Brattleboro rat has hypertrophied supraoptic and paraventricular nuclei and posterior pituitary (Sokol and Valtin, 1965). All of these structures are relatively atrophic in the human disease (Braverman et al., 1965; Green et al., 1967; Bergeron et al., 1991). This difference must reflect different pathophysiological mechanisms which cause the disease in the rat and the human. The mechanisms must be very different as the heterozygous state in the rat produces a nearly normal phenotype while

307 in the human the disease is manifest (see also, Van Leeuwen, this volume). Perhaps the mutant precursor accumulates in neurons in the human disease in a manner that is toxic to these cells or in some manner that leaves them susceptible to autoimmune attack. The selective atrophy of the AVP-containing neurons in the supraoptic and paraventricular nuclei is reminiscent of the changes seen after high stalk section (MacCubbin and Van Buren, 1963; Morton, 1969) and may reflect a primary lesion (such as autoimmune attack or toxic degeneration) which takes place in the neurohypophyseal nerve terminals. F.W. van Leeuwen: With respect to the point of the explanation of cell loss in the supraoptic and paraventricular nucleus (SON and PVN) in human hypothalamic DI, it might be that superactivation of vasopressin (VP) cells leads to cell death whereas hyperactivity results in a prolonged life as compared to hypoactivity. D.F. Swaab: I agree that even if the “use it or lose it” hypothesis will be proven to be correct, it will only hold within certain physiological limits. Extreme stimulation by excitoxin administration for instance has shown to lead to cell death (Swaab, 1991a,b). For the hypothalamic diabetes insipidus (DI) Brattleboro rat it has been repeatedly shown that the SON and PVN are hyperactive(Soko1 and Valtin, 1965; Swaab et al., 1973; Van Tol, 1987; Sherman et al., 1988). Although nobody, t o my knowledge, has counted the SON and PVN cells in the homozygous DI rat, we have no reason to assume that in these nuclei cell loss in any considerable degree occurred. Nor is there any reason to assume that in the human DI the SON and PVN cells should be much more stimulated than in the DI rat (what you called superstimulation). Therefore I prefer the idea that some compound produced or accumulated by the disease process in the human DI neurons is causing their degeneration rather than their hyperactivity. J.B. Martin: Do you have any explanation for how a point mutation in one allele can cause an autosomal dominant disorder? Why does not the normal allele generate sufficient AVP t o give rise to a partial deficiency? Is it possible that there is adequate vasopressin secretion early in life and that the secretory capacity fails with advancing age? D.R. Repaske: We do not yet know the pathophysiological mechanism of the human disease. Expression of the human mutant allele, in contrast to the Brattleboro rat mutant allele, must create a significant block to expression of the normal allele. The mutant allele may cause the death of AVP-producing neurons by toxic or autoimmune mechanisms or it may otherwise interfere with a critical step in the production of the normal hormone. Possibilities include an inability of the mutant precursor protein to release from the ribosome, from a transport system in the ER/Golgi apparatus, or from a critical post-translational processing enzyme. Reply to J.B. Martin by F.W. Van Leeuwen: Most probably a very low amount of AVP is present in the plasma of human hypothalamic DI persons since chlorpropamide only acts in the presence of AVP (Miller and Moses, 1970). This makes it likely

that some AVP is released from the hypothalamo-neurohypophyseal system. This indicates that the normal AVP allele is expressed and that some is translated, packaged, transported and released. This is different from the DI + / + rat in which no AVP is detectable in the plasma and chlorpropamide has no effect (Berndt et al., 1970). Furthermore it has been shown by Schmale et al. (1989) that in the homozygous Brattleboro rat the cause of the arrested transport from the endoplasmic reticulum to the Golgi apparatus of the altered AVP precursor is most probably a change in the internal domain of the precursor. In the human situation a similar phenomenon may occur where the slightly different AVP precursor may disturb the intracellular transport or packaging of the wild-type precursor (see Van Leeuwen, this volume). D.R. Repaske: Kaplowitz et al. (1982) also reported that AVP was detectable in low amounts in an individual with central DI, while his affected brother had essentially no detectable AVP. Also, case reports indicate that the degree of phenotypic expression of DI varies with age. This is likely due to variations in the amount of AVP production during life. Thus, while the mutation creates sufficient dysfunction in AVP production to cause the clinical disease, the capacity to make some AVP is retained. D.R. Repaske: Failure of DNA repair mechanisms in the AVPNP I1 gene is an unlikely explanation for autosomal dominant central DI as: (1) some affected individuals express the disease from birth; (2) some affected individuals clinically improve in later life; (3) there appears to be a single consistent AVP-NP 11 gene mutation within each family where a mutation has been identified; and (4) failure of a repair mechanism might be expected to affect either AVP-NP I1 allele with equal frequency; however, our linkage analysis demonstrates that the disease is consistently associated with one particular AVP-NP I1 allele in each family.

References Bergeron, C., Kovacs, K., Ezrin, C. and Mizzen, C. (1991) Hereditary diabetes insipidus: an immunohistochemical study of the hypothalamus and pituitary gland. Acta Neuropathol. (Bed.), 81: 345 - 348. Berndt, W.O., Miller, M., Kettyle, W.M. and Valtin, H. (1970) Potentiation of the antidiuretic effect of vasopressin by chlorpropamide. Endocrinology, 86: 1028- 1032. Braverman, L.E., Mancini, J.P. and McGoldrick, D.M. (1965) Hereditary idiopathic diabetes insipidus. A case report with autopsy findings. Ann. Int. Med., 63: 503 - 508. Green, J.R., Buchan, G.C., Alvord, E.C. and Swanson, A.G. (1967) Hereditary and idiopathic types of diabetes insipidus. Brain, 90: 707 - 714. Kaplowitz, P.B., D’Ercole, A.J. and Robertson, G.L. (1982) Radioimmunoassay of vasopressin in familial central diabetes insipidus. J. Pediatr., 100: 76-81. MacCubbin, D.A. and Van Buren, J.M. (1963) A quantitative

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evaluation of hypothalamic degeneration and its relation to diabetes insipidus following interruption of human hypophyseal stalk. Brain, 86: 443 - 464. Miller, M. and Moses, A.M. (1970) Mechanism of chlorpropamide action in diabetes insipidus. J. Clin. Endocrinol. Mefab., 30: 488 - 496. Morton, A. (1969) A quantitative analysis of the normal neuron population of the hypothalamic magnocellular nuclei in man and of their projections to the neurohypophysis. J. Comp. Neurol., 136: 143- 158. Schmale, H., Bahnsen, U., Fehr, S., Nahke, P. and Richter, D. (1991) Hereditarydiabetes insipidusin manand rat. In: S. Jard and R. Jamison (Eds.), Vasopressin, John Libbey, London, pp. 57 - 62. Sherman, T.G., Day, R., Civielli, O., Douglas, J . , Herbert, E., Akil, H. and Watson, S.J. (1988) Regulation of hypothalamic magnocellular neuropeptides and their mRNAs in the Brattleboro rat: coordinate responses to further osmotic challenge.

J. Neurosci., 8: 3785 - 3796. Sokol, H.W. and Valtin, H. (1965) Morphology of the neurosecretory system in rats homozygous and heterozygous for hypothalamic diabetes insipidus (Brattleboro strain). Endocrinology, 77: 692 - 700. Swaab, D.F. (1991a) Brainagingand Alzheimer’s disease, “wear and tear” versus “use it or lose it”. Neurobiol. Aging, 12: 317-324. Swaab, D.F. (1991b) Author’s response to commentaries. Neurobiol. Aging, 12: 352- 355. Swaab, D.F., Boer, G.J. and Nolten, J.W.L. (1973) The hypothalamo-neurohypophyseal system (HNS) of the Brattleboro rat. Acta Endocrinoi., Suppl., 177: 80. Van Tol, H.H.M. (1987) Regulation of Vasopressin and Oxytocin GeneExpression in the Hypofhalamo-Neurohypophyseal System of the Rat. Dissertation, State University of Utrecht, Utrecht, The Netherlands, p. 129.

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0 1992 Elsevier Science Publishers B.V. All rights reserved.

CHAPTER 22

The use of linkage analysis and the Centre d’Etude Polymorphisme Humain (CEPH) panel of DNA in the study of the arginine vasopressin, oxytocin and prodynorphin gene loci Marshall L. Summar Division of Genetics, Department of Pediatrics, Vanderbilt University Medical Center, Nashville, TN, U.S.A.

Introduction

The hormones arginine vasopressin (AVP) and oxytocin (OT), which are released from the human posterior pituitary gland, play important roles in fluid homeostasis, parturition and lactation (Cunningham and Sawchenko, 1991). Prepro-AVP-NPII consists of a single polypeptide chain containing a secretory signal sequence, the nonapeptide AVP, the hormone carrier protein neurophysin I1 of unknown function, and a glycopeptide also of unknown function (Land et al., 1982). Prepro-OTNPI consists of the same configuration, except that the AVP moiety is replaced by OT, the glycopeptide portion is absent, and the neurophysin moiety is NPI instead of NPII (Land et al., 1983). While the AVP and OTgenes have been shown to be physically very close since they are separated by only about 12 kb of DNA. Their transcription occurs on opposite DNA strands and does not seem to be coupled to the same stimuli. Somatic cell hybridization with AVP probes has shown AVP to reside on chromosome 20 without assignment to a specific region (Riddell et al., 1985). Previous genetic linkage studies of AVP have been difficult to date because of the lack of known restriction fragment length polymorphisms (RFLPs) within or close to the AVP locus. Recently we have identified an RFLP within 15 kb of AVP.

The polymorphism is detected using genomic sequences from the flanking regions of the adjacent OT-NPI locus and the restriction endonuclease Xba I (Repaske et al., 1990). Because of the close physical proximity of the AVP and the OT loci, the RFLP detected by OT provides a reliable genetic marker for both loci. Another hormone locus on chromosome 20 is prodynorphin (PDYN), which is one of the three distinct precursor genes for opioid peptides (Douglas et al., 1984). The PDYN gene has been mapped to chromosome 20~12-pter by in situ hybridization (Litt et al., 1988). Interestingly, AVP and PDYN, which share no obvious biochemical or physiological homology, have been colocalized in the rat within the same neurosecretory granules of posterior pituitary terminals whose cell bodies are located in the supraoptic and paraventricular nuclei of the hypothalamus (Whitnall et al., 1983; Sherman et al., 1986). While AVP and PDYN are both found to be present in these neurosecretory granules they are separate in other cells (Whitnall et al., 1983). In addition to AVP, OT and PDYN, we studied additional anonymous DNA loci, D20S5, D20S4, and D20S6, that have been previously mapped to chromosome 20 (Goodfellow et al., 1987; Kidd et al., 1989). To study the causality or association of clinical

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disorders to a particular gene locus such as AVP, it is first necessary to establish a statistical link between the disorder and the gene in question. One way to do this is through family linkage studies in which restriction fragment length polymorphism (RFLP) data for the candidate locus are compared to the disease status of family members. These studies rely upon the presence of either DNA polymorphisms, detected by the gene in question, or closely linked RFLPs, whose genetic distance from the gene is well established. If no RFLPs can be detected using the AVP-NPII gene, alternative markers may be established and used in linkage studies. We have used such an approach to determine that an autosomal dominant form of neurohypophyseal diabetes insipidus exhibits apparent cosegregation with the AVP gene (Repaske et al., 1990) However, studies of this an other neuroendocrine disorders have proven difficult due to the lack of known RFLPs for loci such as adenosine deaminase (ADA) and growth hormone-releasing hormone (GHRH) both located on chromosome 20, as well as the low degree of informativeness of RFLPs detected by other loci. By constructing a linkage map of chromosome 20, more RFLPs of known reliability are made available for linkage studies of various hormones. Constructing a map of chromosome 20 also facilitates the linkage placement of additional hormone loci such as GHRH, and loci such as the locus for benign neonatal convulsions, and ADA, as well as additional new candidate loci (Riddell et al., 1985; Sykes et al., 1986; Willard et al., 1986).

History of linkage analysis In the human genome there are an estimated 3 x lo9 base pairs of DNA per haploid copy (McKusick, 1988). Within this vast array of base pairs reside about 50- 100 000 genes ranging from less than 1000 bases to more than 200 000 bases of DNA in length (McKusick, 1988). In the efforts to uncover some of the underlying order and relationships of these genes, linkage analysis has developed as one of the most powerful tools. In the field of brain research, numerous genetic loci come under scrutiny as candidate genes for

pathologic disorders. Since readily apparent mutations or defects in these genes often cannot be found and an exhaustive search for specific defects consumes considerable time and materials, it is preferable and cost-effective to use an indirect method such as linkage analysis to test the association between disease locus and gene locus. The origins of linkage analysis began in the early 1900s with the work of Dr. Thomas Hunt Morgan in Drosophila melanogaster. Classic genetic theory held with the postulates of Gregor Mendel concerning the assortment and inheritance of genes. Mendel’s third postulate states that, “members of different gene pairs assort to the gametes independently of one another”. Morgan’s experiments showed that Mendel’s law of independent assortment was violated by some phenotypic characteristics in Drosophila. Morgan and his coworkers found four gene groups which each segregated with the others in that group but independently of others. These four groups were later found to be the four chromosomes of the fruit fly. Further work found that the coassortment of alleles directly reflected their physical distance. The concept of recombination was developed to explain the exchange of chromosomal material between homologous chromosomes in prophase of meiosis I (Fig. 1). The extent to which recombination occurs between two loci reflects the actual physical distance between the two. The estimate of the degree of recombination between two loci can thus be used to give an idea of

Recombination Between Sister Chromatids Fig. 1. Recombination event during meiosis I in which sister chromatids exchange equivalent segments. Note that the recombinant chromatids have rearranged the combinations of the A(a) and B(b) alleles.

31 1

their intervening distance. This distance is measured in centimorgans with 1 centimorgan equalling 1 To recombination which approximates 1 x lo6 base pairs of DNA (Conneally and Rivas, 1980). Random assortment is reflected by a recombination fraction of 0.5 or 50% which indicates that the loci in question assort independently (see below).

system and compare it to the markers for which data have already been generated. There are currently data on over 1000 markers in the CEPH database, and the probability of demonstrating linkage with any of these is high and improves as more markers are added. Basics of linkage analysis

Current use of linkage Linkage analysis is currently used for numerous purposes. One of the most popular uses of linkage analysis is locating and testing candidate gene loci in the study of genetic disorders. Linkage analysis has proved essential in isolating the genes for disorders such as cystic fibrosis and neurofibromatosis. Linkage analysis can also be of great use in testing candidate genes for association with a given disorder such as the arginine vasopressin gene (AVP) locus and its involvement in the familial form of diabetes insipidus. Linkage analysis can furthermore be of use in diseases where the gene locus is unknown such as Huntington’s disease and adult polycystic kidney disease. Linkage analysis is also being used to explore such questions as the heritable psychiatric disorders and Alzheimer’s disease. To identify and study new genes and loci as they become available requires an international reference system. The construction of the human linkage map is a project with this goal in mind. By providing linked markers at known intervals over the human genome, new genes can rapidly be placed within a known framework and informative markers can be identified to aid in diagnostic linkage studies. To accomplish this, a standard set of family pedigrees is needed on which all markers can be tested. One such system exists. CEPH (Centre d’Etude du Polymorphisme Humain) consists of DNA isolated from all members of 42 families with three generations each, usually with more than eight children per family. All the individuals in the CEPH families have had lymphoblastoid cell lines created to produce a virtually unlimited supply of DNA. Using this system, a researcher studying a gene need not test many different markers looking for linkage with the gene, but instead may test his marker against the CEPH

For many single gene disorders, there is either a polymorphic locus with few alleles hence a low polymorphism information content (PIC), or no restriction fragment length polymorphism (RFLP) at all. In this case one must rely upon closely linked markers. The relationships between close markers are established through the study of statistical linkage, which evaluates the probability that two syntenic loci (loci on the same chromosome) will cosegregate. Mathematically, linkage is an expression of the percentage of recombination between two loci that occurs during meiosis I at the chiasmata of sister chromatids (Ott, 1974; Conneally and Rivas, 1980). This recombination frequency is measured in centimorgans (1 cM = 1070 recombination) and studies indicate that the human genome comprises approximately 3000 centimorgans, thereby yielding about 1 x lo6 bp/cM for small recombination distances (3 x lo9 bp/3 x lo3 cM) in the human genome. The linkage relationships between syntenic loci can be determined by studying transmission patterns in families over several generations and noting the frequency of recombinations that occur between the loci. Thecloser genes are physically, the less likely they are to recombine and assort to separate gametes. The first linkage relationships were detected at the chromosome level when it was noted that genes on the same chromosome tend to travel together (Ott, 1974; Conneally and Rivas, 1980). This was first described by T.H. Morgan and his colleagues at the beginning of this century in the fruit fly (Drosophila melanogaster) (Conneally and Rivas, 1980). Morgan observed that in some cases, genes an the same chromosome did not cosegregate and that the closer the physical proximity of the

312

genes, the less likely they were to recombine and assort independently (Ott, 1974; Conneally and Rivas, 1980). The frequency of recombination, 8 is an expression of the percentage of all meiosis in which recombination occurs. Therefore, a 8 of 0.5 (50% recombination) would indicate independent assortment, whereas a 8 of 0.0 would indicate that the two loci are very closely linked together such that recombination occurs only very rarely. LOD scores The probability of a given 8 is stated by means of the LOD or Zscore. The LOD score, log of the odds, is a comparison of the probability of a particular 8 against the probability that the loci assort independently (Le., 8 = 0.5) (Ott, 1974; Conneally and Rivas, 1980). The probability of a given recombination frequency is given by the equation:

where Le is the likelihood of a given recombination frequency, 0 is the given recombination frequency, R is the number of observed recombinants in a pedigree, and S is the number of observed non-recombinants. The LOD (Z)score is then determined by the equation (Ott, 1974; Conneally and Rivas, 1980): LOD (Z)= Le/LB = '3 A LOD score of 3 (odds ratio of 1000/1) is generally accepted as proof of linkage. The peak LOD score calculated for a range of 8s is used to select the most likely recombination frequency (method of maximum likelihood; Ott, 1974; Conneally and Rivas, 1980). A LOD score of 3, however, does not correspond to a P value of 0.001, but rather to a P value of about 0.04 because of a prior probability of linkage of 1/46, since there are 46 chromosomes. The error range for the most likely 8 is determined by the two 8s with LOD scores corresponding to Zm" - 1 , which provides a reliable confidence interval. The calculation of LOD scores rests on several assumptions: first, that the chiasmata at

which recombination takes place in meiosis I are evenly distributed; second, that there is no interference of recombination between genes; and finally, that position on the chromosome does not affect recombination. In fact, the frequency of recombination decreases as one approaches the centromere, and there are known areas of certain chromosomes where recombination is more likely to occur. Also, in males and females there is some variable degree of interference with recombination, as well as sex-specific differences in recombination rates (Ott, 1974; Conneally and Rivas, 1980). To calculate the probability for a wide range of 8s for several families by hand or using tables would be prohibitively time consuming. Fortunately, computer programs have been developed which perform these tasks quickly. One such program package, LINKAGE, calculates two-point and multi-point linkage and tests loci order and differentiates male and female recombination rates (Lathrop and Lalouel, 1984; Lathrop et al., 1985). With the current capacity of microcomputers, one can easily run these programs and calculate linkage using standard desktop units. Linkage maps By combining and comparing 8s for several overlapping loci, one can construct a linkage map of a given region or linkage group of a chromosome. These linkage maps are useful for determining the relationships of markers in clinical studies, and for placing new markers in their proper relationship to established markers. When constructing linkage maps and performing linkage studies, several things must be considered. First, if the loci are close to the centromere, recombination events may be suppressed resulting in 8 values, obtained for pericentromeric loci, representing greater physical distances than those derived for telomeric loci (Conneally and Rivas, 1980). Second, recombination frequencies can vary greatly between male and female meiosis, with the female frequencies usually being greater (Ott, 1974; Conneally and Rivas, 1980; Donis-Keller and Botstein, 1988). This sex difference varies depending upon the chromosome and chromosome

313

region being studied (Conneally and Rivas, 1980). For routine linkage studies, however, the sex specific recombination frequencies are treated as equal, and the positional differences in 8 do not affect the statistical reliability of family studies. Restriction fragment length polymorphisms Classical genetic markers consist of observable or measurable traits in humans. These comprise physical findings such as the Nail-patella syndrome, protein variants such as hemoglobin, and immunologic distinctions such as the A 3 0 blood group or MHC antigens. In the last decade, a new generation of markers has been developed which consist of DNA sequence polymorphisms, referred to as restriction fragment length polymorphisms or RFLPs, which can be detected by probes for specific genes or for anonymous or uncharacterized fragments of DNA (Southern, 1975; Feinberg and Vogelstein, 1983).TheDNAprobes used to detect these RFLPs are derived from numerous sources such as cloned DNA libraries or cDNA sequences, obtained from messenger RNA isolated from a variety of tissues. In some cases these clones contain inserts that encode known protein products while in most cases their sequences are unknown. The use of these probes and the RFLPs they identify as markers has revolutionized the way in which we look at inherited diseases, as well as certain acquired diseases, such as neoplasias which are associated with chromosome abnormalities. The use of DNA polymorphisms allows direct or indirect detection of a rapidly growing list of familial and acquired disorders that perturb the human genome. It is estimated that variations in intergenic genomic DNA sequences occur about every 250 500 bp (Cooper et al., 1985; Gusella, 1986). This variation leads in some cases to differences in the number and location of sites recognized by various restriction endonucleases. These RFLPs yield fragments of different sizes which, when separated by gel electrophoresis and hybridized to labeled probes, generate patterns which are co-dominant traits that can be used to trace transmission of alleles. The size of the specific fragments, referred to as alleles,

depends upon the presence or absence of a polymorphic endonuclease cleavage site. These polymorphic sites are usually located within the introns or the flanking regions of genes rather than in the coding sequences, and they are usually unrelated to the functional qualities of the gene. Using RFLPs, one can trace the transmission of a particular gene or DNA segment through a family tree by the pattern of polymorphic alleles linked to it. The feasibility of such studies depends upon the parents being heterozygous (i.e., informative) for the RFLP which in turn depends upon the frequency and distribution of the various RFLP alleles in the population under study. The informativeness of an allele, referred to as the polymorphism information content (PIC), within a population can be expressed mathematically by the equation: PIC

=

1 - A2 - B2 - N 2 - 2A2B2N2

where A is the frequency of the first allele, B is the frequency of the second allele, and N of other alleles (McKusick, 1988). The closer the PIC is to 1, the more informative the probe becomes. There are some probe enzyme systems with over 20 distinct alleles available and PICs approaching 1; however, as the number of fragments increases, it becomes very difficult to distinguish one band from another. Southern blots and restriction enzyme analysis

Materials and methods Southern blotting. One of the most commonly used techniques in molecular genetics is Southern blotting combined with restriction endonuclease digestion of DNA (Southern, 1975). The use of sequencespecific restriction endonuclease derived from eukaryotic organisms has greatly expanded our ability to track copies of a gene through several generations (Gusella, 1986). These enzymes cleave specific DNA sequences, called recognition sites, which are most often palindromic. In the human genome, there are approximately 3 x lo9 bp of

314 Blotting paper

I ,

Restriction Endonuclease Digestion

Electroohoretic Gel Separation

I I ..--.1

Tr

c

+Hybridization with Probe And Washing

1

Autoradiogram

Fig. 2. Schematic representation of Southern blotting.

DNA, throughout which the recognition sequences for restriction endonucleases are widely and, in most cases, evenly distributed (Cooper et al., 1985). In the technique of Southern blotting (Fig. 2) aliquots of genomic DNA, isolated from peripheral leukocytes or tissue, are incubated with restriction endonucleases to obtain fragments of a reproducible and manageable size which can then be separated by subjecting them to gel electrophoresis. These fragments, after denaturing to render them singlestranded, are then transferred to a membrane filter (usually composed of nylon, nitrocellulose or a combination of the two), by either capillary-, electro-, or vacuum-blotting (Southern, 1975). The DNA fragments are then fixed to the membrane by either drying and/or exposure to ultraviolet light. These filters are then hybridized to labeled DNA probes which are specific for certain DNA sequence(s). The label incorporated into these probes can either be radiolabeled or coupled to an antigen type marker, which is then utilized in an enzymatic reaction. After the hybridization, the excess probe is removed by washing the filter which is then either placed on film to detect the radioactive label or subjected to an enzymatically driven colorimetric reaction. The fragments which contain the complimentary sequences for the probe then appear as bands whose size is determined by the relative location of restriction endonuclease sites limiting their ends.

Probes. The DNA probes used in this study include those encoding known genes as well as anonymous DNA segments. The prepro-OT-NPI

probe is pHuOT, which is a 1.6 kb fragment inserted into the pJB327 vector using the BamH I and EcoR I restriction sites (Sausville et al., 1985). The genomic insert contains exons 1, 2 and 3 of the prepro-OT-NPI gene. Other DNA probes used include: the PDYN clone (PDYN), regionally assigned between p12.21 and pter; the clone pRI12.21, which recognizes the D20S5 locus at band 12.21 of the short arm of chromosome 20; the clone pMS1-27 which recognizes the D20S4 locus mapped to the 13.2 band of the long arm of chromosome 20; and the probe D3H12, assigned to the D20S6 locus on the short arm of chromosome 20 (Riddell et al., 1985; Sausville et al., 1985; Litt et al., 1988, 1989; Kidd et al., 1989). Table I lists the restriction endonucleases used to detect these various RFLPs, as well as the sizes and the frequencies of their alleles.

Polymorphism typing. For this study both the Vanderbilt and Yale laboratories used human genomic DNA supplied by CEPH, isolated from transformed lymphoblastoid cell lines. In the Vanderbilt laboratory high-molecular weight DNA was digested with Xba I, separated by agarose gel electrophoresis, transferred to nylon filters, and hybridized according to standard protocols with the pHuOT clone (Sykes et al., 1986; Willard et al., 1986). The filters were then exposed to film and the TABLE I Frequencies and sizes of probe alleles with PIC value Probe

Locus

Enzyme

PIC

Fragments

pHuOT

AVP/OT XbaI

0.27

PDYN

PDYN

Taq1

0.38

pR12.21

D20S5

Msp I

0.32

pMS127

D20S4

Msp I

0.36

pD3H12

D20S6

TuqI

0.38

9.0 5.0 2.1 2.0 3.0 3.8 6.5 1.5 13.1 8.6

Frequencies ~

Source of data: CEPH, release 3.

0.80 0.20 0.65 0.34 0.74 0.25 0.39 0.61 0.56 0.44

315

autoradiograms interpreted by two different individuals. Similar procedures were used in the Yale laboratory to type the PDYN and D20S6 loci on Taq I digested samples, and the D20SS loci on Msp I digested samples. Data on the D20S4 locus were provided by the Collaborative Research Inc. (CRI) laboratory. Linkage analysis. Linkage analysis was used to determine the statistical relationships of the loci in question by comparing the meiotic recombination rates between loci. The frequency of this recombination (6) and it’s associated odds ratio are determined by use of the method of maximum likelihood (Conneally and Rivas, 1980). These calculations were performed using version 4.7 of the LINKAGE programs MLINK, CILINK, LINKMAP, and CLODSCORE (Lathrop and Lalouel, 1984). A LOD score of 3 (odds of 1000/1) was used as the point at which significant evidence of linkage was achieved. Equal recombination rates were assumed in males and females (6, = 6,) for the two-point scores, and the map function option was not used. The 6s which correspond to LOD(6,, - 1) are used as the confidence interval for this study (Conneally et al., 1985). The two-point linkage scores were calculated using MLINK, and multi-locus mapping was performed using the program LINKMAP. The order of the loci was determined by assigning probabilities to the various orders using the program LINKMAP and selecting the most probable configuration. Using the map distance of 0.15 cM between AVP/OT/PDYN and D3H12, the other loci were tested against this group to determine the most

probable order based on the recombination frequencies. Results Examination of the recombination frequencies between loci demonstrated significant linkage between six different loci (see Table 11). The PDYN and AVP/OT loci showed no recombination in the pedigrees studied, with a LOD score of 5.2 corresponding to an odds ratio of 158 50011 that these results were due to linkage rather than chance. By adding the 6s between the loci, an approximation of the size of the region mapped can be reached. The combination of the 6s for the loci studied spans 30 cM of chromosome 20 encompassing regions on both the short and long arms. The probabilities of the loci order were determined by LINKMAP and their 8s were: AVP/OT -0- PDYN - 0.15 (D20S6 0.03 D20S5) - 0.15 - D20S4. The regional assignments of loci by hybridization studies as well as their linkage placements and the probabilities of the order of the loci are shown in Fig. 3. The relatively precise assignments of PDYN, D20S5, and D20S4 enable us to determine that the AVP/OT loci map to the telomeric side of the p12.21 band. This map adds to those already established for chromosome 20 by placing additional loci on the map.

- -

Conclusions Using the six genomic DNA probes described in studies of DNA samples constituting the CEPH panel, we have been able to establish a multipoint linkage map of chromosome 20 which extends from

TABLE I1 Two-point linkage data from C E P H families Loci AVP/OT

AVP/OT

PDYN D20S6

-

D20S5

-

D20S6

PDYN 0.0/5.2

NI

-

0.15/3.8 -

-

-

-

NI, Non-informative; UL, unlinked (peak L O D at 8 = 0.5).

-

D20S5 0.30/0.54 0.15/2.7 0.03/7.8 -

D20S4 0.2/0.4 UL

0.15/2.7 0.30/0.3

316

13 12 11.2 11.1

U

11.2 12 13.1 13.2

.I:

, D20S4

13.3

U Fig. 3. A schematic of the two-point recombination distances (0) between the loci and the probabilities of the order of loci. The probabilities represent the odds of the two loci for an interval being inverted. The linkage relationships are compared to the regional chromosomal assignments of several of the probes.

the telomeric portion of the short arm to the band q 13.2 on the long arm. However, the actual physical distance encompassed may vary greatly in different portions of the linkage map because of the positional effects on recombination which are known to occur near the centromere (decreased rate of recombination) and near the telomere (increased rate of recombination) (Conneally and Rivas, 1980; Lathrop et al., 1985). The greatest improvement to the map is in the short arm where D20S5 and D20S6 were previously reported to show about 7% recombination (Goodfellow et al., 1987). Our data strengthen the close linkage of those two loci, suggesting they are even closer, but still fail to resolve their order with respect to the centromere. Since PDYN was previously mapped to the short arm but is clearly not on the long arm side of D20S5 and D20S6, it is now mapped more telomeric, as are both OT and AVP which had not previously been regionally localized on chromosome 20. The distance from D20S4, at the tip of 20q, to these short arm loci is not estimated well in this study because it shows such weak evidence of linkage.

The fact that AVP and PDYN, although dissimilar in function, are closely linked genetically and physically, and are coexpressed in response to osmotic challenge (Sherman et al., 1986) suggests that the two genes could possibly share the same transcriptional activation system. Thus, further studies might be considered to determine if the coexpression of these two genes is due to their sharing the same promoter region or if a homologous promoter region has been duplicated regionally. Comparison of sequence data between human AVP and PDYN was not possible due to a lack of sequence data on human PDYN. While preliminary comparison between human AVP and rodent PDYN cDNA sequences shows no areas of significant homology, this needs to be confirmed once the human PDYN sequence is available. Although no recombinants were seen, the physical distance between the two loci could be quite large, since the confidence interval (LOD,, - 1) for this study is 0 - 9% recombination. In addition to providing information on the genes and markers studied, the placement of markers spanning at least 30 cM of chromosome 20 should assist in the mapping of additional loci on this chromosome. As more markers are placed on the map linkage analysis of new loci should become quicker and easier. Therefore, once RFLPs for genes such as GHRH become available, their location on the linkage map shown in Fig. 3 should be easily determined by linkage studies. This, in turn, could provide additional RFLPs to facilitate linkage studies in families with putative derangements of the GHRH or hormonal genes. References Conneally, P.M. and Rivas, M.L. (1980) Linkage analysis in man. In: H. Harris and K . Hirschhorn (Eds.), Advances in Human Genetics, Vol. 10, Plenum, New York, pp. 209- 266. Conneally, P.M., Edwards, J.H., Kidd, K.K., Lalouel, J.-M., Morton,N.E.,Ott, J.and White, R. (1985)Reportofthecommittee on methods of linkage analysis and reporting. HGM8: 8th International Workshop on Human Gene Mapping Cytogenet. Cell Genet., 40: 356 - 359. Niemann, S. and Cooper, D.N., Smith, B.A., Cooke, H.J., Schmidtke, J. (1985) An estimate of unique DNA sequence

317 heterozygosity in the human genome. Hum. Genet., 69: 201 - 205. Donis-Keller, H. and Botstein, D. (1988) Recombinant DNA methods: applications to human genetics. In: B. Childs, N.A. Holtzman, H.H., Kazazian, Jr. and D.L. Valle (Eds.), Molecular Genetics in Medicine, Elsevier, New York, pp. 17-42. Douglas, J . , Civelli, 0. and Herbert, E. (1984) Polyprotein gene expression: generation of diversity of neuroendocrine peptides. Annu. Rev. Biochem., 53: 665-715. Feinberg, A.P. and Vogelstein, B. (1983) A technique for radiolabeling DNA restriction endonuclease fragments to high specific activity. Anal. Biochem., 132: 6- 13. Goodfellow, P. J ., Duncan, A.M.V., Farrer, L.A., Holden, J.J.A., White, B.N., Kidd, J .R., Kidd, K.K. and Simpson, N.E. (1987) Localization and linkage of three polymorphic DNA sequences on human chromosome 20. Cytogenet. Cell Genet., 44: 62-67. Gusella, J.F. (1986) Recombinant DNA techniques in the diagnosis of inherited disorders. J. Clin. Invest., 77: 1723 - 1726. Kidd, K.K., Bowcock, A.M., Schmidtke, J . , Track, R.K., Ricciuti, F., Hutchings, G., Bale, A., Pearson, P. and Willard, H.F. (1989) Report of the DNA committee and catalogs of cloned and mapped genes and DNA polymorphisms. Cytogenet. Cell Genet., 51: 622 - 947. Land, H., Schutz, G . , Schmale, H. and Richter, D. (1982) Nucleotide sequence of cloned cDNA encoding bovine arginine vasopressin-neurophysin I1 precursor. Nature, 295: 299 - 303. Land, H., Grez, M., Ruppert, S., Schmale, H., Rehbein, M., Richter, D. and Schutz, G. (1983) Deduced amino acid sequence from the bovine oxytocin-neurophysin I precursor cDNA. Nature, 302: 342 - 344. Lathrop, G.M. and Lalouel, J.M.(1984) Easy calculations of Lod scores and genetic risks on small computers. A m . J. Hum. Genet., 36: 40-465. Lathrop, G.M., Lalouel, J.M., Julier, C. and Ott, J. (1985) Multilocus linkage analysis in humans: detection of linkage and estimation of recombination. A m . J. Hum. Genet., 37: 482 - 498. Litt, M., Buroker, N.E., Kondoleon, S., Douglas, J., Liston, D., Sheehy, R. and Magenis, R.E. (1988) Chromosomal localization of the human proenkephalin and prodynorphin genes. Am. J. Hum. Genet., 42: 327 - 334. McKusick, V.A. (Ed.) (1988) The Morbid Anatomy of the Human Genome, Williams and Wilkins, Baltimore, MD. Ott, J . (1974) Estimation of the recombination fraction in human pedigrees: efficient computation of the likelihood for human linkage studies. Am. J. Hum. Genet., 26: 588 - 597. Repaske, R., Phillips 111, J.A., Kirby, L.T., Tze, W.J., D’Er-

Cole, A.J. and Battey, J. (1990) Molecular analysis of autosomal dominant neurohyphyseal diabetes insipidus. J. Clin. Endocrinol. Metab., 70: 752 - 757. Riddell, D.C., Mallonee, R., Phillips 111, J.A., Parks, J.S., Sexton, L.A. and Hamerton, J.L. (1985) Chromosomal assignment of human sequences encoding arginine vasopressinneurophysin I1 and growth hormone releasing factor. In: B. Sykes et al. (Eds.), Somatic Cell and Molecular Genetics, p. 189. Sausville, E.A., Carney, D. and Battey, J.F. (1985) The human vasopressin gene is linked to the oxytocin gene and is selectively expressed in a cultured lung cancer cell line. J. Biol. Chem., 260: 10236- 10241. Sherman, T.G., Olivier, C., Douglas, J., Herbert, E. and Watson, S.J. (1986) Coordinate expression of hypothalamic prodynorphin and pro-vasopressin mRNAs with osmotic stimulation. Neuroendocrinology, 44: 222 - 228. Southern, E.M. (1975) Detection of specific sequences among DNA fragments separated by gel electrophoresis. J. Mol. Biol., 98: 503-517. Sykes, B., Ogilvie, D., Wordsworth, P., Anderson, J . and Jones, N. (1986) Osteogenesis imperfecta is linked to both type I collagen structural genes. Lancet, ii: 69 - 72. Whitnall, M.H., Gainer, H., Cox, B.M. and Molineaux, J. (1983) Dynorphin-A-(1 - 8) is contained within vasopressin neurosecretory vesicles in rat pituitary. Science, 222: 37 - 39. Willard, H.F., Wayne, J.S., Skolnick, M.H., Schwartz, C.E., Powers, V.E. and England, S.B. (1986) Detection of restriction fragment length polymorphisms at the centromeres of human chromosomes by using chromosome specific a satellite DNA probes: implications for development of centromerebasedgeneticlinkagemaps. Proc. Nail. Acad. Sci. U.S.A., 83: 561 1 - 5615.

Discussion J.P.H. Burbach: Is the fact that the dynorphin and vasopressin loci are both present on chromosome 20 just a matter of coincidence? M.L. Summar: Yes, this is very well possible. Although the close physical association and colocalization in neurosecretory granules would be against it. F.W. Van Leeuwen: With reference to the point raised by Burbach of a coincidence of the small distance between dynorphin (DYN) and vasopressin (AVP) loci on chromosome 20, it might be worthwhile to study the loci of other co-expressed peptides such as galanin, neuropeptide Y and F8f amide. M.L. Summar: I agree. In fact that is how we came to study these loci initially.

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SECTION VII

Hypothalamus and Reproduction

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D.F. Swaab, M.A. Hofman, M. Mirmiran, R. Ravid and F.W. van Leeuwen (Eds.) Progress in Brain Research, Vol. 93

32 1

0 1992 Elsevier Science Publishers B.V. All rights reserved.

CHAPTER 23

Animal models for brain and pituitary gonadal disturbances H.M. Charlton and M.J. Wood Department of Human Anatomy, University of Oxford, Oxford OX1 3QX, U.K.

Introduction The hypothesis that the brain controls, in large part, reproductive processes via the hypothalamohypophyseal system depended originally upon an analysis of such natural phenomena as seasonal breeding cycles and reflex ovulation. It seems selfevident that the effects of day length depend upon retinal stimulation and the transduction of that stimulus into a neuroendocrine response. Similarly if ovulation follows coitus, rather than occurring spontaneously, then there must be a pathway, or pathways, relaying the mating stimuli to centres controlling the luteinising hormone (LH) surge upon which ovulation depends. Indeed, even in spontaneously ovulating species the length of the estrus cycle is usually dependent upon the lighting regime in which the animals are housed. These natural phenomena led to experimentation and the development of laboratory model systems aimed initially at determining a critical role for the pituitary gland in gonadal stimulation and then for the hypothalamus in the control of the pituitary itself. At the present moment much research effort has moved one point higher and is concerned with determining how other areas of the central nervous system inhibit or stimulate peptide and amine release from the median eminence into the portal vessels draining to the pituitary gland. Much of the early work involved the use of electrolytic lesioning or stimulation of the brain and can be read in detail

in the Dale lecture delivered by Geoffrey Harris (1972) and also in the recent monograph published by Everett (1989). The development of deafferentation of the hypothalamus by Halasz and his colleagues provided another model system. The evidence became overwhelming that the brain must control reproductive processes by releasing factors into the vessels leading to the pituitary gland. Crude though the experimentation was, the end result was the isolation and characterisation of the gonadotrophic hormone-releasing hormone (GnRH), and the demonstration that it was a decapeptide that provided the final biochemical link between the brain and the control of gonadotrophin hormone synthesis and secretion. The gonads subserve both endocrine and gametogenic functions and their activity begins in utero and extends into middle and old age. The role of the testes in determining sexual differentiation of the internal and external genitalia is well documented. In male rodents androgen production by the testes in the perinatal period is critical for sexual differentiation of the brain and the behaviour of the male rodent in adulthood. At puberty the secretions of the pituitary coordinate spermatogenic functions in males and follicular development and ovulation in females. Feedback control between the gonads and brain/ pituitary has also been the subject of a large volume of research. In determining the potential role of GnRH in the above phenomena we need to be able to prevent its

322

production, for example by CNS lesions, or to counteract its actions by the use of antagonist drugs or by immunoneutralisation with antibodies. New experimental model systems are being developed all the time, but occasionally nature performs a precise lesion for us in the form of a genetic mutation. One such mutant is the hypogonadal (hpg) mouse discovered and initially characterised by Cattenach et al. (1977). In this mutant the gonads fail to develop post-natally with a paired testis weight, when adult, of approximately 6 - 8 mg compared with 200 mg for normal littermate males. The vagina fails to open in the majority of females, the uterus is threadlike and the ovaries are minute with little or no antral follicle development. Pituitary luteinising hormone (LH) and follicle stimulating hormone (FSH) content is a fraction of normal in both sexes. The hypogonadal mouse

The first analysis of the defect in these mice depended upon radioimmunoassay of hypothalamic extracts and immunohistochemical staining of brain sections. Both approaches confirmed a lack of GnRH within the brain and we now know that there is a massive deletion removing half of the GnRH gene and rendering it impossible for hpg mice to transcribe and translate the gene and to produce the mature GnRH protein (Mason et al., 1986a). Thus we have a very precise lesion within the hypothalamo-hypophyseal-gonadalsystems. Perhaps one of the earliest functions of the testes is to influence sexual differentiation of the internal and external genitalia. The hpg mouse testes might be expected to produce the peptide Mullerian duct inhibitory substance and thus fail to maintain structures giving rise to the uterus, and this is the case. On the other hand differentiation of the epididymis, vas deferens, seminal vesicles, prostate and external genitalia all depend upon testicular androgens. If androgen production in utero itself depends upon normal hypothalamic/pituitary interactions, then we might expect that hpg males would exhibit abnormalities in sexual differentiation; they do not. Despite the fact that the testes fail to develop post-

natally, hpg males are fully sexually differentiated anatomically both internally and externally. This observation indicates that intra-uterine sexual differentiation in male mice may be independent of pituitary gonadotrophic hormones, and therefore also of hypothalamic influences. Moving to the perinatal events involved in sexual differentiation of the brain and we find a different story. Treatment of hpg males with testosterone when adult stimulates spermatogenesis, growth of seminal vesicles and prostate but with no activation of male sexual behaviour. However, priming mutants with testosterone or estrogen at birth and then with testosterone when adult results in the stimulation of all aspects of male behaviour. The indeterminate state of the hpg male brain has been demonstrated by the fact that treating adult mutant males with estrogen and progesterone results in female sexual behaviour with lordosis (Ward, 1981). Hpg females when treated with estrogen and progesterone as adults also exhibit the full spectrum of female behaviour (Ward and Charlton, 1981). GnRH has also been implicated as a neuromodulator of normal adult sexual behaviour , one argument being that the GnRH surge before ovulation may also enhance female sexual behaviour on the night of estrus (Moss and Dudley, 1988). Whilst this is entirely possible, the presence of GnRH is not an absolute prerequisite for normal sexual behaviour patterns because of the experiments upon female and male hpg mice mentioned above. In both sexes therefore apparently normal sexual behaviour can be elicited in a mutant lacking GnRH. Before leaving normal embryogenesis and development the hpg mouse also provides some insight into the events involved in migration of primordial GnRH cells from the olfactory placode, into the brain and eventually to the preoptic area (POA). Schwanzel-Fukuda and Pfaff (1989) have demonstrated that GnRH neurones are actually born within the olfactory placode and migrate from there to the medial basal forebrain. Even at the embryonic stage within the olfactory placode of normal mice these neurones stain positively for GnRH and il

323

might be considered that expression of the peptide could be a prerequisite for correct migration. Although GnRH cannot be detected immunologically nor indeed physiologically within hpg mice, Seeburg and his colleagues have shown, by in situ hybridisation, that cells within the POA actually attempt to transcribe the GnRH gene (Mason et al., 1986a). This indicates that GnRH cell bodies are capable of normal migration, even in the hpg mouse, and that the decapeptide is not a prerequisite for correct migration and guidance. This aspect of GnRH cell migration has also been approached by Radovick et al. (1991) and Mellon et al. (1990). These two groups have produced hypogonadal mice by introducing the simian virus big T antigen driven by the GnRH promoter into mice. In the experiments of Radovick et al. (1991) the human GnRH promoter was used and resulted in the disruption of migration of GnRH neurones from the olfactory placode into the preoptic area. Mellon et al. (1990) used the rat GnRH promoter, and in their experiments GnRH cell bodies reached the preoptic area, but failed to extend axons to the median eminence. In both cases the mice suffered from hypogonadotrophic hypogonadism. The relevance of GnRH cell migration to Kallmann’s syndrome in man (clinically identified by hypogonadotrophic hypogonadism and anosmia) has been dramatically demonstrated by Schwanzel-Fukuda et al. (1989). The brain of a foetus affected by Kallmann’s syndrome showed an arrest in the migration of GnRH neurones into the central nervous system, and this migration was suggested to depend upon a neural cell adhesion molecule. Very recently Franco et al. (1991) have demonstrated that a gene deleted in Kallmann’s syndrome in fact shares homologies with neural cell adhesion molecules. Our knowledge of the embryological derivation, migration and function of the GnRH neurones is a triumph of the interplay between the basic and medical sciences. The ability of individual cells specifically to transcribe and translate unique gene products depends upon the expression of transcription factors and enhancers which bind to the 5 ’ and 3 ’ DNA sequences associated with the gene in question. The

group at Genentech undertook the experiment of replacing the GnRH gene with several kilobases of 5 ’ and 3 ’ DNA back into hpg mice. They found expression of the gene was correctly encoded both temporally and geographically thus demonstrating that the DNA sequences associated with the injected GnRH gene were sufficient to target GnRH expression. In these transgenic hpg mice gonadal growth was normal. The females demonstrated normal estrus cycles and the males were capable of full masculine behaviour patterns. A small amount of activity was found within the paraventricular nucleus of the thalamus which is not normally associated with GnRH expression. As there were multiple copies of the gene introduced into the transgenic line it is difficult to determine any physiological significance for this final observation (Mason et al., 1986b; Charlton, 1987). Within the pituitary itself it has been suggested that GnRH is essential for the embryonic development of gonadotrophs. However, the fact that both LH and FSH can be assayed in the hpg mouse, and that LH immunoreactive cells can be stained within the pituitary argues against this. Nevertheless there is a marked reduction in gonadotroph numbers which is normalised within 2 - 3 weeks by GnRH injection (McDowell et al., 1982).

Neural transplantation One of the most fertile areas of research in which hpg mice have been used is that of neural transplantation. The demonstration that the restoration of CNS function by the intracerebral transplantation of normal tissue depends, as does most in vivo experimental work, upon adequate model systems. The restoration of motor function to 6-hydroxydopamine-lesioned rats by transplants of foetal substantia nigra into the denervated caudate/putamen is one of the classical experiments of modern time. On the neuroendocrine front the first experiments involved the vasopressin-deficient Brattleboro rat (Gash et al., 1980)and then Krieger et al., (1982) undertook similar experiments in hpg mice. The results were unequivocal, transplantation of

3 24

A EFFECT

B

OF POA IMPLANTS IN MALE hpg MICE

EFFECT

OF POA IMPLANTS IN FEMALE hpg MICE

I

d a

3

,d

0 LH CONTENT

e

T

T

NORMAL hpg

hpg

lODay

26DW 4 0 D a y

NORMAL

I

hpg

hpg+POA

CORTEX 25Day -hpg*POA-

Fig. 1. A . Pituitary LH and FSH content, serum FSH levels and testicular weight in hpg male mice given either cortical or POA grafts into the third ventricle. B. Pituitary LH and FSH content, ovarian and uterine weights in hpg female mice given P O A grafts into the third ventricle. ND. Not detectable.

normal/early neonatal POA tissue gave rise to increased pituitary LH and FSH synthesis and secretion with the stimulation of full spermatogenesis and seminal vesicle growth in males and follicular development, uterine growth and vaginal opening in females (see Fig. 1). The excellent physiological response of hpg mice to POA grafts could be explained on the basis that the GnRH cells were merely acting as a form of mini

pump with the decapeptide reaching the median eminence by diffusion. However, histological evidence has demonstrated that GnRH axons leave the grafts with over 95% of them specifically innervating the median eminence and with their nerve endings abutting onto portal vessels supplying the pituitary gland (Silverman et al., 1985). This must mean that, even in the adult, this region of the brain maintains the capacity to attract such axons. W,hen

325

grafts were placed within the lateral ventricle they did not grow towards the median eminence but rather left the ventricle to enter large fibre tracts such as the fornix and stria terminalis (Kokoris et al., 1987) and there was no stimulation of the pituitarylgonadal axis. If the grafts were specificallyinnervating the host, was there any evidence for host influences upon the grafts? This has proved a difficult area to interpret, The first observation that grafts may not be functioning normally was that female hpg mice bearing POA grafts never ovulated (i.e., possessed corpora lutea in their ovaries) and that vaginal smears from these animals demonstrated extended periods of estrus with no evidence of 4 - 5 day estrus cycles. In other words the grafts could not transduce day length. With excellent folliculogenesis, uterine growth and vaginal opening it was obvious that the internal steroid environment of these females might be conducive to female sexual behaviour. Indeed Gibson et al. (1984) demonstrated that many POAgrafted female hpg mice would mate with male mice and this was not surprising. What was surprising, however, was that 70% of mated females became pregnant. Dogma would expect that ovulation must be preceded by an LH surge and, in rodents, such a surge of LH must be preceded by a GnRH surge. The only source of GnRH in these experimental animals must be the GnRH neurones within the graft. It would appear that POA-grafted hpg female mice had become reflex ovulators and this was conclusively demonstrated by Gibson et al. (1987) who measured an LH peak in the serum of females within minutes of mating. One interpretation of this experiment could be that host axons transducing the mating stimulus had a direct input into the GnRH neurone. However, there is very little evidence either histologically or electrophysiologically of host axons entering graft tissue. It should also be pointed out that the lack of estrus cycles in virgin grafted females indicates that pathways transducing day length do not appear to be able to influence spontaneous GnRH surges. Donor and host axons cannot help but intermingle within the host median eminence and it is per-

haps pertinent to mention that although ovulation is spontaneous in rats and mice, the activation of corpora lutea depends upon a mating-induced release of prolactin. There is abundant evidence that releasing factors axons can be activated by the products of their near neighbours. For example corticotropinreleasing hormone (CRH) can inhibit the release of GnRH by median eminence nerve terminals (Frias et al., 1990) and prolactin can enhance release (Azad et al., 1990). Galanin has also been implicated in the control of GnRH secretion from the median eminence (Lopez and Negro-War, 1990) and NegroW a r (1982) has previously argued for local control of neural peptides within the median eminence itself. At the moment it does not seem necessary to invoke specific innervation of graft neurones to explain the ovulatory response of hpg females. One of the earliest experiments in which hpg mice were used to investigate the actions of GnRH demonstrated that multiple daily injections of the decapeptide were most efficacious in stimulating pituitary and gonadal function (Charlton et al., 1983). The excellent physiological response to POA grafts of both male and female mutants could indicate that the mice were releasing LH in a pulsatile manner. This has been demonstrated by Gibson et al. (1991) and it is tempting to suggest that pulsatile LH reflects pulsatile GnRH secretion. Again interpretation of these results is difficult. Is the pulse generator within the GnRH neurones themselves? Does it depend upon co-grafted interneurones? Could it be that, again, episodic release may be activated at the level of axon terminals within the median eminence? At the beginning of this chapter mention was made of the advances in research aimed at investigating factors controlling hypothalamicreleasing hormone synthesis and release and the review of Kalra and Kalra (1983) specifically covered the field of feedback control of gonadotrophic hormones. The hpg mutant represents an excellent model for investigating these phenomena, for example, if testosterone in the male has its negative feedback at the level of the pituitary gland, then subcutaneous silastic capsules of testosterone

326

should prevent the synthesis and secretion of LH and FSH which follows injections of GnRH - they do not, however (Charlton et al., 1987), which suggests that feedback may reside at a higher level than the pituitary, perhaps within the GnRH neurone itself, or at an even higher level within the CNS. The POA-grafted hpg male mouse has proved of use in tackling these questions. In hpg male mice bearing POA grafts within the third ventricle we have GnRH neurones removed from their normal anatomical site and disconnected from their normal neuronal input. Such neurones could still be susceptible to testosterone negative feedback but when mutant males with surviving and functional grafts were given androgen pellets there was no evidence of the normal negative feedback suppression of pituitary LH levels. The work has recently been extended to measure LHP mRNA levels within the pituitary. In normal males testosterone treatment reduced LHP mRNA levels whilst castration caused a dramatic increase. However, in POA-grafted hpg males neither castration nor testosterone treatment had any effect upon LHP mRNA (Charlton et al., 1988). These experiments indicate that, in mice, a major component of the negative feedback of testosterone upon pituitary LH synthesis and secretion probably depends upon androgen-sensitive neurones which normally project to GnRH cell bodies within the preoptic area. In our laboratory negative estrogen feedback appears to have a major site of feedback at the level of the pituitary gland in that steroid treatment prevents

increased gonadotrophic hormone synthesis in response to GnRH injections in female hpg mice. In POA-grafted mutants we have looked at the effects of ovariectomy or estradiol injections upon pituitary LH content and LHP mRNA levels. In all cases the steroid treatment depressed both parameters measured whilst ovariectomy elicited a significant increase (Scott et al., 1991). This is in contrast to the work of Gibson and Silverman (1989) who measured serum LH levels with apparently no significant effects of the two treatments. The above experiments cannot rule out an effect of estradiol within the CNS but the evidence that GnRH cell bodies do not contain estrogen receptors rules out a direct action on these neurones themselves. However, there are many estrogen-sensitive cells within POA grafts (Gibson et al., 1989) and it may be that these can influence GnRH output from their neighbours. It is also possible that estrogen-sensitive cells within the host brain may influence GnRH release at nerve terminals within the median eminence itself. What factors might influence the apparent specific outgrowth of GnRH axons from the grafts? In many cases fibres have been seen to leave third ventricular grafts laterally to course through the arcuate nucleus before entering the median eminence. In order to see if interactions with arcuate nucleus neurones played a part in any guidance mechanism, hpg mice were injected neonatally with monosodium glutamate (MSG) which destroys the majority of these neurones. When such animals were given POA grafts as adults over 95% of GnRH-positive

Fig. 2. Histological sections in the coronal plane through the mediobasal hypothalamus of the mouse. A . Section through the median eminence of a normal mouse demonstrating immunoreactive GnRH fibres. Note the concentration of GnRH fibres at the lateral sulcus and the arching fibres through the arcuate nucleus (an). B. Section through the median eminence of a hypogonadal (hpg) mouse immunostained as in A . Note the complete absence of immunostaining in the mutant. C. Section through the median eminence of an hpg mouse 30 days after receiving an intraventricular graft of foetal mouse POA. The graft fills the third ventricle and fibres can be seen to innervate the median eminence with the greatest concentration in the lateral sulcus. D.Section through the median eminence of an hpg mouse 30 days after receiving an intraventricular xenograft of foetal rat POA with low-dose anti-CD4 monoclonal antibody treatment. The graft fills the third ventricle, as in C, but fewer GnRH immunoreactive fibres are present. Again these fibres are seen specifically to innervate the median eminence. E. Section through the median eminence of an hpg mouse 60 days after receiving an intraventricular foetal rat POA xenograft with low-dose anti-CD4 treatment. The graft has undergone rejection and only a few GnRH immunoreactive fibres remain (arrow). F. Section through the median eminence of an hpg mouse 60 days after receiving an intraventricular foetal rat POA xenograft with high-dose anti-CD4 antibody treatment. GnRH immunoreactive fibres can be seen to survive and to specifically innervate the median eminence.

327

328

axons again projected to the median eminence indicating that the neuronal population of the arcuate nucleus is not directly involved in attracting axon terminals to the median eminence (Silverman et al., 1990). This still leaves glial cells as a possible scaffold. Exactly what is the guidance mechanism? In vitro cultured experiments suggest that pituitary tissue itself can act as a target for GnRH axons (Wray et al., 1988). In co-grafted experiments with POA and pituitary tissue placed within the lateral ventricle Silverman and Gibson (1990) have provided tentative evidence that axons from the neural graft grow towards the pituitary tissue. With suitable controls and an increase in experimental numbers this promises to be a fruitful area of research. In situ hybridisation studies have been quoted above as evidence that cell bodies leave the olfactory placode and reach the POA in hpg mice indicating that this migration is not dependent upon expression of the GnRH gene. Whatever the guidance cues are for the journey from the olfactory placode to the final neuroanatomical site during embryogenesis, would such cells from normal foetuses grafted into adult mice be capable of migrating into the hpg POA? It might be expected that there could be some temporal program for foetal GnRH cell bodies which first dictates the migration pattern of the cells to the POA and later controls axonal guidance to the median eminence. By grafting El3 placodal tissue directly into the adult third ventricle it should be possible to see if these cells are immediately capable of innervating the host median eminence to stimulate pituitary and gonadal growth. An interesting extension of the transplant work in the field of guidance mechanisms has come from experiments in which monoclonal antibody blockade of Tlymphocytes has allowed us to graft rat POA into hpg mouse with graft survival of beyond 60 days. It has long been recognised that graft rejection is especially dependent upon thymus-derived T-cells (Billingham et al., 1954). More recently two major subsets of T lymphocytes have been defined according to function and surface antigen characteristics, namely helper or CD4+ T-cells and cytotoxic T-

cells which bear the CD8 antigen. The brain may be considered an immunologically privileged site, but this privilege is not absolute and xenografts are rejected (Sloan et al., 1991). Use of monoclonal antibodies against the CD4 and CD8 antigens (Cobbold et al., 1984) has allowed us to obtain specific subset depletion and thereby to investigate their respective roles in the rejection of neural xenografts. Early results (Honey et al., 1991) suggested that CD4 T-cell depletion could prolong the survival of rat POA xenografts in hpg male mice for 30 days after which rejection occurred, GnRH immunoreactivity decreased and paired testicular weight declined to approach hpg values (Table I and Fig. 2D,E). Subsequent work involving altering antibody administration regimes and utilising combinations of anti-CD4 and anti-CD8 antibodies has allowed rat xenografts to survive (Fig. 2F)and function, maintaining testicular weight (Table I), for more than 60 days. In these studies the rat GnRH axons specifically innervate the median eminence of the mouse so, whatever the guidance cues are, they are shared by the two species. With long-term survival of xenografts we are now in a position to ask questions about feedback mechanisms between the graft, pituitary and gonads in this system. +

Sex differences in neurotransmitters There are many instances of brain differences between the sexes other than those most obviously involved in sexual behaviour, and this field has recently been reviewed in depth by De Vries (1990). A number of these sex differences are influenced by gonadal steroids and the hpg mouse represents a useful model in this field of research because neither the male nor the female brain have been subjected to significant gonadal steroid input post-natally. In rats and mice there is a vasopressinergic innervation of the lateral habenula nuclei in both sexes. This innervation is reduced or absent in gonadectomised animals and cannot be visualised immunocytochemically in the hpg mouse, male or female. However, treatment of the mutants with testosterone or estradiol results in an abundant vasopres-

329 TABLE I Paired testicular weights (in mg) of male hpg mice 30 and 60 days after receiving foetal mouse or rat POA grafts Experiment

Mouse POA implant into hpg mouse, no treatment Mouse POA implant into hpg mouse, anti-CD4 treatment (low dose) Rat POA implant into hpg mouse, anti-CD4 treatment (low dose) Rat POA implant into hpg mouse, anti-CD4 treatment (high dose)

Testicular weight 30 days (mg)

60 days (mg)

52.98 k 11.7

104.4

+_

9.9

+

92.6

?

13.5

45.9

5.5

et al., 1981). In this strain vasopressinergic innervation of the lateral septum and lateral habenulae cannot be detected immunocytochemically. However, treatment with estradiol elicits an innervation pattern indistinguishable from normal mice (Mayes et al., 1988). It is pertinent to point out that similar sex differences in vasopressinergic innervation have not been observed in the human brain (Fliers et al., 1986). Future perspectives

49.6

?

4.9

12.0

46.8

k

5.1

48.2 k 3.8

?

3.2

Testicular weights at 30 days are comparable in all groups. Note, firstly, that antibody treatment did not impair testicular growth in those animals which received mouse implants and, secondly, that rat implants were able to elicit the same rate of testicular growth as mouse implants. At 60 days testicular weights in groups which received mouse implants were similar. In those mice receiving POA xenografts testicular weights decreased toward hpg levels in the group given low-dose antibody treatment but remained stable at 30 day levels in those mice given high-dose antibody treatment.

sinergic fibre network within the lateral habenulae and lateral septum. Such an innervation is also found in hpg mice possessing physiologically positive POA grafts (Mayes et al., 1988). These studies indicate that the hpg mouse may be of use in investigating other sexually differentiated neurotransmitter systems and provide positive evidence that the vasopressinergic innervation of the lateral septum and lateral habenulae depends upon the presence or absence of normal gonadal steroidogenesis. The work also shows that neonatal exposure of the brain to gonadal steroids is not a prerequisite for the steroid-induced expression of these vasopressinergic inputs in adult mice. There is a second mouse mutant strain in which androgen function is impaired: the androgen receptor-deficient testicular feminised (tfm) mouse (Lyon

Natural mutations, rather than drug- or experimentally-induced lesions provide precise model systems in many fields of study. However, the ability for us specifically to produce our own “mutant” strains in the form of transgenic animal will be a growth area in the next decade. Provided we are dealing with a single copy gene it is possible to replace such a gene by homologous recombination of defined mutant DNA into the site of the original gene (Cappechi, 1989). With this methodology we can not only knock out a gene completely but also ask questions about the importance of 5 ’ and 3 ’ coding sequences by site directed mutagenesis. It is also possible specifically to delete cells expressing a particular gene by introducing toxic genes driven by cell-specific promoters such that when the normal transactivation of the host gene takes place, so does that of the toxic transgene. For example, Behringer et al. (1988) have fused part of the rat growth hormone (GH) promoter with the diphtheria toxin A-chain gene thus eliminating all GH cells from the pituitary gland and producing dwarf mice. Another approach in this area is to introduce genes driven by homologous promotors into transgenic animals which, when activated, kill dividing cells. In this field the thymidine kinase obliteration system has proved of value. For example by driving the herpes virus thymidine kinase gene from the GH promoter, GH cells acquire pharmacological sensitivity to synthetic nucleosides added to the diet such that dividing cells are killed. Borelliet al. (1989) produced dwarf mice by this means by treating pregnant mothers with synthetic nucleosides for most of

330

their pregnancy. Because toxicity depended upon cell division the young were born with drastically reduced numbers of GH cells. There can be no doubt that the techniques of molecular biology are poised to deliver fundamental information concerning brain-pituitary-gonadal interrelationships.

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33 1 LHRH secretion from arcuate nucleus-median eminence fragments in vitro: involvement of an a-adrenergic mechanism. Endocrinology, 127: 243 1 - 2436. Lyon, M.F., Cattenach, B.M. and Charlton, H.M. (1981) Genes affecting sex differentiation in mammals. In: C.R. Austin and R.G. Edwards (Eds.), Mechanisms of Sex Differentiation in Animals and Man, Academic Press, London, pp. 367 - 374. Mason, A.J., Hayflick, J.S., Zoeller, R.T., Scott Young, W., Phillips, H.S., Nikolics, K. and Seeburg, P.H. (1986a)Adeletion truncating the gonadotropin releasing hormone gene is responsible for hypogonadism in the hpg mouse. Science, 234: 1366- 1371. Mason, A. J., Pitts, S.L., Nikolics, K., Szonyi, E., Wilcox, J.N., Seeburg, P.H. and Stewart, T.A. (1986b) The hypogonadal mouse: reproductive functions restored by gene therapy. Science, 234: 1372- 1378. Mayes, C.R., Watts, A.G., McQueen, J.K., Fink, G. and Charlton, H.M. (1988) Gonadal steroids influence neurophysin 11 distribution in the forebrains of normal and mutant mice. Neuroscience, 25: 1013- 1022. McDowell, I.F.W., Morris, J.F., Charlton, H.M. and Fink, G. (1982) Effects of luteinking hormone releasing hormone on the gonadotrophs of hypogonadal (hpg) mice. J. Endocrinol., 95: 311-320. Mellon, P.L., Windle, J.J., Goldsmith, P.C., Padula, C.A., Roberts, J.L. and Weiner, R.I. (1990) Immortalization of hypothalamic GnRH neurons by genetically targeted tumorigenesis. Neuron, 5 : 1 - 10. Moss, R.L. and Dudley, C.A. (1988) LHRH peptidergic signals in the neural integration of female reproductive behaviour. In: J.M. Lakoski, J.R. Perez-Polo and D.K. Rassin (Eds.), Neural Control of Reproductive Function. Proceedings of the 5th Galveston Neuroscience Symposium, pp. 485 - 499. Negro-War, A. (1982) The median eminenceas a model to study pre-synaptic regulation of neural peptide release. Peptides, 3: 305 - 310. Radovick, S., Wray, S., Lee, E., Nicols, D.K., Nakayama, Y., Weintraub, B.D., Westphal, H., Cutlery, G.B., Jr. and Wondisford, F.E. (1991) Migratory arrest of gonadotropin releasing hormone neurons in transgenic mice. Proc. Natl. Acad. Sci. U.S.A., 88: 3402-3406. Schwanzel-Fukuda, M. and Pfaff, D.W. (1989) Origin of luteinising hormone releasing hormone neurons. Nature, 338: 161 - 164. Schwanzel-Fukuda, M., Bick, D. and Pfaff, D.W. (1989) Luteinizing hormone-releasing hormone (LHRH) expressing cells do not migrate normally in an inherited hypogonadal (Kallmann) syndrome. Mol. Brain Res., 6: 31 1 - 326. Scott, IS., Porter Goff, A., Cox, B.S., Charlton, H.M. and Clayton, R.N. (1991) Effect of ovariectomy or estrogen implants upon pituitary function in female hypogonadal mice bearing normal neonatal preoptic area grafts. J. Neuroendocrinol., 3: 303 - 307. Silverman, A. J. and Gibson, M. (1990) Hypothalamic transplan-

tation. Repair of reproductive defects in hypogonadal mice. Trends Endocrinol. Metab., 1: 403 - 407. Silverman, A.J., Zimmerman, E.A., Gibson, M.J., Perlow, M.J., Charlton, H.M., Kokoris, G.J. and Krieger, D.T. (1985) Implantation of normal fetal preoptic area into hypogonadal (hpg) mutant mice. Temporal relationships of the growth of GnRH neurons and the development of the pituitary/testicular axis. Neuroscience, 16: 69 - 84. Silverman, R.C., Gibson, M.J., Charlton, H.M. and Silverman, A.J. (1990) Are neurons of the arcuate nucleus necessary for pathfinding by GnRH fibers arising from third ventricle grafts? Exp. Neurol., 109: 204-210. Sloan, D.J., Wood, M.J. and Charlton, H.M. (1991) The immune response to intracerebral neural grafts. Trends Neurosci., 14: 341 - 346. Ward, B. J. (1981) Some Aspects of the Reproductive Physiology and Behaviour of the Hypogonatal (hpg) Mouse, thesis, Oxford. Ward, B.J. and Charlton, H.M. (1981) Female sexual behaviour in the GnRH deficient hypogonadal (hpg) mouse. Physiol. Behav., 27: 1107 - 1109. Wray, S., Gahwiler, B.H. and Gainer, H. (1988) Slicecultures of LHRH neurons in the presence and absence of brain-stem and pituitary. Peptides, 9: 1151- 1156.

Discussion D.F. Swaab: How far can the hypogonadal (hpg) mouse be considered as a model for Prader-Willi syndrome? Are they also fat, stupid and hypotone? H.M. Charlton: Hpg mice have an isolated GnRH deficiency and, apart from the endocrine and behavioral differences discussed in this chapter they appear to be normal. There is no evidence of obesity for example. D.F. Swaab: Are you sure that the hpg mouse does not produce an active luteinking hormone-releasing hormone (LHRH) during early development? The molecular biology of the deficiency would in theory allow this. H.M. Charlton: Antibodies to both GnRH and the GnRH associated peptide (GAP) fail to detect any immunoreactive cells within the POA of hpg mice and there is no physiological evidence that GnRH is synthesized within the brain; nevertheless there cannot be a dogmatic statement that GnRH can never be made, but this isextremely unlikely, there being no obvious stop codon and polyadenylation site. G.J. Boer: From your grafting results it is clear that the LHRH fibers perfectly find their target sites in the median eminence. Have you got any idea why the grafted cells are able to do so? Have you, e.g., placed them to sites elsewhere than in the third ventricle and what was the outcome? H.M. Charlton: Grafts placed within the lateral ventricle do not extend axons to the median eminence and there is no stimulation of the pituitary and gonads. It may be that some factor associated

332 with the external zone of the median eminence guides axons to this site. Suggestions have been specific glia and/or the pars tuberalis of the pituitary gland. R.Y. Moore: It is fascinating that your animals have become reflex ovulators. In most rodents, the estrous cycle is a function of a set of circadian cycles. Would it seem likely, or possible, that you might restore both cycling and ovarian function if you transplanted suprachiasmatic nucleus and preoptic LHRH neurons? H.M. Charlton: It should certainly be possible to dissect out donor tissue containing LHRH neurons with and without the SCN and to transplant two groups of hpg mice. A comparison of the physiology of these two groups would be interesting. D.W. Pfaff: Following on your success with grafting, we have a neurotropic virus-based DNA vector which allows foreign gene expression in adult neurons (Kaplitt et al., 1991a,b). If the normal mouse GnRH gene were introduced to adult hpg POA cells, would you predict normal reproductive function? H.M. Charlton: First of all, the hpg mouse would allow you to detect all thecells which had been transfected provided your construct was translated and transcribed. You should also be able to follow the projection fields of such neurons. If the hpg LHRH

cells are transfected then I would expect some degree of physiological recovery in hpg mice. In females vaginal opening should occur and, provided cyclic release of LHRH does not depend upon some normal perinatal stimulus, I would also expect regular estrous cycles with spontaneous ovulation, the expression of mating behaviour with subsequent pregnancies. In genetically engineered hpg male mice I would expect the stimulation of spermatogenesis but no mating behaviour. With your technique it might be possible to transfect one side of the brain alone and this might give information on contralateral/ipsilateral projections.

References Kaplitt, M.G., Rabkin, S. and Pfaff, D. (1991a) Defective viral vectors for gene transfer into brain. Current Topics in Neuroendocrinology, Vol. 11, Springer, Heidelberg, in press. Kaplitt, M.G., Pfaus, J.G., Kleopoulos, S., Hanlon, B.A., Rabkin, S. and Pfaff, D. (1991b) HSV-1 based amplicon allows expression of bacterial 0-galactosidase in adult rat brain. Mol. Cell. Neurosci., 2: 320- 330.

D.F.Swaab, M.A. Hofman, M. Mirmiran, R. Ravid and F.W. van Leeuwen (Eds.) Progress m Brain Research, Vol. 93 0 1992 Elsevier Science Publishers B.V. All rights reserved.

333

CHAPTER 24

Genetic, hypothalamic and endocrine features of clinical and experimental obesity George A. Bray Pennington Biomedical Research Center, Baton Rouge, LA 70808, U.S.A.

Introduction A number of models exist for the study of obesity (Brayet al., 1989). Several of the clinical and animal models are summarized in Table I according to the mechanism for their development. Three major mechanistic categories can be identified including neuroendocrine, genetic and dietary. In this paper, the discussion will focus on the endocrine aspects of several of the specific types of obesity listed in Table I. Hypothalamic obesity in animals and man will be discussed along with the endocrine causes which include Cushings disease, the polycystic ovary syndrome (PCO) and castration-induced obesity. Among the genetically transmitted forms of obesity, the focus will be on the Prader-Willi syndrome. The effects of adrenalectomy on the development of obesity in the animals with dominant and recessive inheritance will also be reviewed,

Hypothalamic obesity Anatomy Hypothalamic obesity as a clinical syndrome has been known for nearly 100 years (Bray and Gallagher, 1975). The original descriptions suggested that the problem was of pituitary origin or involved destruction of both the pituitary and hypothalamus (Babinski, 1900; Frohlich, 1901). The classic studies of Smith (1927) clearly distinguished between

hypopituitarism and hypothalamic injury by injecting chromic acid selectively into each area. It was only the injections into the hypothalamus which were associated with obesity. Hetherington and Ranson (1940) showed that obesity could be produced following stereotaxic lesions which injured the ventromedial hypothalamus and produced hyperphagia (Brobeck, 1946). More recently it has been shown that small lesions in either the paraventricular nucleus (Fukushima et al., 1987) or the ventromedial nucleus (Parkinson and Weingarten, 1990) can produce obesity. However, the mechanisms for these obesities are different.

Mechanisms of hypothalamic obesity In the years following the introduction of stereotaxic lesions to produce obesity, the hyperphagic characteristic of these animals lead to the conclusion that the obesity was the result of overeating (Brobeck, 1946). Critical studies showed that with tube-fed animals (Han and Frohman, 1970) or in weanling animals (Frohman et al., 1969) that do not become hyperphagic, obesity still develops. Thus mechanisms other than hyperphagia had to be involved. It is now recognized that there are two distinct syndromes of hypothalamic obesity (Fig. 1; Brayet al., 1982). The first isassociated with injury to the paraventricular nucleus in which hyperphagia is a necessary and sufficient cause for the syndrome (Parkinson and Weingarten, 1990).

334 TABLE I A classification of obesity

Neuroendocrine Genetic

Clinical

Animal

Cushings] Hypothalamic' Polycystic ovary3 Prader-Willi4-

Hypothalamic" Castration''

Cohen7

Recessive (ob;eb;fat; Tub; fa;f~;~p)",'~ Polygene (NZO; KK; P B B ~ 13 ~,

Carpenter'

Dietary

Ahlstrom' Bardet-Biedel" High fat"

Dominant (yellow a ~ ) ~143 ,

High fat" Sucrose solutions" Supermarket"

References: Cushing (1932); Bray (1984); Rebar et al. (1976); Bray et al. (1983); Butler (1990); Cassidy and Ledbetter (1989); Friedman and Sack (1982); Temtamy (1966); Goldstein and Fialkow (1973); lo Stiggelbout (1972); I ' Brayeta1.(1990);" SchemmeletaL(1982); l 3 BrayandYork (1979); l4 Bray et al. (1988).

'

Fig. 1. A model for the syndrome of hypothalamic obesity. Lesions of the paraventricular and ventromedial nucleus are shown on the left. Their effects on food intake, the autonomic nervous system and release of growth hormone are shown along with the consequences of these changes.

When hyperphagia is prevented, neither obesity nor other endocrine nor neurologic defects occur. The second syndrome is associated with injury to the ventromedial nucleus where the necessary and sufficient components are a complex of disturbances of the autonomic nervous system (Bray et al., 1989). Endocrine disturbances Lesions of the ventromedial nucleus are associated with increased activity of the vagal efferent system and reduced activity of the efferent thermogenic component of the sympathetic nervous system (Bray et al., 1990).Acutely following a VMN lesion, the vagal activity increases as reflected in increased insulin secretion which can be abolished by vagotomy (Berthoud and Jeanrenaud, 1979; Tokunaga et al., 1986). Acute hypothalamic lesions also produce an acute reduction in electrical activity of sympathetic nerves to brown fat (Sakaguchi et al., 1988). No corresponding changes occur after lesions of the PVN (Sakaguchi et al., 1988). In addition to an imbalance of the autonomic nervous system, lesions in the ventromedial nucleus are associated with impaired growth hormone secretion (Bernardis and Frohman, 1970), leading to stunted growth in animals and stunting in humans. The VMN is an important site for the production and release of growth hormone-releasing hormone (GHRH). Destruction of the VMN would thus eliminate or reduce this trophic hormone for GH release. Other endocrine disturbances may also occur in hypothalamic obesity. With sufficiently large lesions, the menstrual cycle and ovulatory function in women may be abolished, men may become hypogonadal and estrus cycles are disrupted in animals. Disturbance of the adrenal and thyroid gland is only seen with very large ventromedial hypothalamic lesions. In humans, hypothalamic obesity is often associated with impaired diurnal rhythms, somnolence and abnormalities in temperature control. We have recently demonstrated that the diurnal rhythm for insulin resistance is also lost in individuals with hypothalamic obesity (Lee et al., 1991).

335

Endocrine obesity Cushings disease and hyperadrenalism In 1932, Cushing described the syndrome which bears his name. His patient had a basophilic pituitary adenoma associated with hypertension, modest obesity, stria and central fat location. This complex of symptoms can result from increased pituitary secretion of ACTH, and also from increased steroid secretion by adrenal adenomas, adrenal carcinomas, extra adrenal tumors producing ACTH and from exogenous corticosteroid administration. In patients with Cushings syndrome, the symptom complex depends on the rate of steroid secretion and the particular type of steroids which are secreted. When glucocorticoids predominate, the major symptoms are centrally increased deposition of body fat in the abdominal, supraclavicular and upper thoracic region, thinning of the skin, formation of striae, loss of muscle mass with thinner extremities and hypertension. Removal of the cause for the excess corticosteroids is sufficient to reverse this syndrome. The administration of excess corticosteroids in animals produces an increase in the ratio of fat to lean tissue (Devenport et al., 1989) which is similar to the change in fat and protein that is observed in humans, but steroids do not usually produce gross obesity in animals. As the quantity of administered steroid increases the fat to protein ratio rises in a dose-dependent fashion. However, at higher doses of steroid, the total body protein level is reduced such that body weight is reduced although the percentage of body fat may be increased. Polycystic ovary syndrome The polycystic ovary syndrome (PCO), formerly called the Stein-Leventhal syndrome is a heterogeneous syndrome that has been known for more than 50 years. It is a syndrome of obesity, hirsutism and anovulatory cycles. There are three primary endocrine alterations associated with this syndrome. A first component of the syndrome is increased

adrenal androgen production. This produces increased levels of A-4 androstenedione which can be converted in peripheral tissues to estrone and testosterone. This peripheral conversion and increased adrenal secretion lead to the increased hirsutism and impaired ovarian function associated with this syndrome. The elevated androgens suppress SHBG production by the liver thus leading to adequate estrogenization. The second feature is loss of the usual relationships between FSH and LH. The level of LH is increased and the level of FSH is low or normal resulting in failure of regular ovulation (Rebar et al., 1976). The third endocrine feature is insulin resistance. Independent of the degree of obesity, individuals with the PCO are insulin-resistant (Pasquali et al., 1983). The mechanism for this insulin resistance is presently unclear, although altered steroid secretion would be a likely explanation. In one subgroup of women with the polycystic ovary syndrome, obesity is particularly prominent. They are individuals whose obesity begins in childhood. They show the following characteristics: (1) mild carbohydrate intolerance; (2) high levels of circulating insulin with insulin resistance; (3) acanthosis nigricans is often present as is hyperuricemia; and (4) elevated androgen levels with hirsutism.

Castration-induced obesity Estrogen in rodents plays an important role in modulation of food intake and body fat. During the normal estrus cycle there is an inverse relationship between food intake and estrus cycle. When estrogen levels rise during estrus, there is a decrease in food intake. During diestrus when estrogen levels are low, food intake increases. With castration, the estrogen levels are reduced and food intake rises with an increase in body fat. Replacement injections of estradiol, but not progesterone, will reverse this syndrome. Similarly, adrenalectomy will eliminate the effects on food intake and fat gain of castration. In women there is an increase in food intake and a preference for carbohydrate during the second half of the menstrual cycle (Schemmel et al., 1982).

336

Genetic obesity The Prader- WiNi syndrome Description. A mnemonic for the Prader-Willi syndrome is the H30, meaning hypotonia, hypomentia, hypogonadism and obesity. The characteristic features of this syndrome were originally described by Prader et al. (1956) more than 30 years ago. Since this seminal publication, a large number of cases have been described and this information has been reviewed in a number of sources (Bray et al., 1983; Cassidy and Ledbetter, 1989; Butler, 1990). This syndrome occurs with an incidence of between 1 in 10 000 and 1 in 25 000. It is the result of paternal transmission of a small deletion in the 412 region of the proximal portion of the long arm of chromosome 15 in somewhat more than 50% of the cases. A normal karyotype is the second

most common finding in careful cytogenetic studies of patients with this syndrome. A few patients show chromosomal translocations at chromosome 15 (Bray et al., 1983). When the defect in this region of chromosome 15 occurs by maternal transmission, the Angelman syndrome is produced (Cassidy and Ledbetter, 1989; Butler, 1990). These individuals have more severe mental retardation and do not have obesity. The similar chromosomal location with differing syndrome features when occurring from maternal or paternal transmission has been attributed to imprinting. It is widely believed that major components of this syndrome are the result of disturbances in the hypothalamus (Bray et al., 1982, 1983, 1988). The basic features of the Prader-Willi syndrome appear in utero and are shown in Table 11. The individuals are less mobile and fetal movements are reduced. The frequency of breech delivery is in-

TABLE I1 Manifestations of hypogonadism in Prader-Willi syndrome ~~

Genital hypoplasia

Males

Females

Small phallus Small, then, poorly rugated scrotum Cryptochidism Small clitoris Hypoplastic labia minora

Abnormal puberty

Both sexes

Puberty often delayed Occasional premature pubarche Incomplete puberty Lack of adolescent growth spurt

Adulthood hypogonadism

Both sexes

Sparse pubic, axillary and body hair Short stature for family Lack of sexual interest or activity Infertility Osteoporosis Small penis and testes Poor beard growth Small clitoris and labia minora Amenorrhea or oligomenorrhea

Males Females Laboratory findings

Both sexes Males Females

From Cassidy and Leadbetter (1989).

Low FSH and LH Low bone densitometry Low testosterone Low estradiol

337

creased. At birth, hypotonia is characteristic and the cry is often weak. These infants may remain in the hospital due to difficulties with feeding and may require intragastric intubation. During the first year of life, they tend to be small and thrive less well than normal. Motor development is slowed and cognitive and language problems are common. A number of behavioral features characterize the syndrome. They are often loveable and affectionate children, but frequently have temper tantrums particularly when food is not available. The explanation for these mental changes is unknown (Cassidy and Ledbetter, 1989; Butler, 1990).

Obesity. Some time during the first two years of life, obesity appears. The fat is generally distributed centrally. The obesity occurs for two reasons, first the children tend to be physically inactive and second they are hyperphagic. There is almost no limit to the quantity of food which they will eat when it is available. Moreover, they frequently have temper tantrums when food is not readily available. Because of the relationship of increased activity of the vagus nerve to development of hypothalamic obesity as noted above, vagotomy has been tried in one patient with the Prader-Willi syndrome without success (Fonklesrud and Bray, 1981). Hypogonadism. Hypogonadotropic hypogonadism is a major feature of the Prader-Willi syndrome (Bray et al., 1983). The problem is present in both sexes. Adrenarche may occur early, but pubarche is usually absent. Gonadal response to exogenous gonadotropins has been demonstrated. In addition, the hypothalamic pituitary system can be “turned on” by the estrogen agonist clomiphene which is associated with a rise in LH and in both males and females with this syndrome (Bray et al., 1983). However, when clomiphene is discontinued, the gonadal system reverts to its prepubertal state. ~ y ~ o t h a l a m i c ~ i t ~abnormalities. itary Short stature is characteristic of the Prader-Willi syndrome. However, there is no defect in growth hormone secretion when compared to appropriately matched

obese controls. Random growth hormone levels are generally normal, but nocturnal levels may be low. Treatment of a small number of individuals with growth hormone has resulted in accelerated growth, and it may be that the reduced levels of nocturnal growth hormone plays a role in the short stature which becomes most apparent after age 10 (Lee et al., 1989). The rise in TSH in response to exogenous TRH is at the upper limits of the response for normal controls. The rise in cortisol after insulininduced hypoglycemia is likewise normal.

Animal models of genetic obesity Dominant obesity in the yellow mouse. The obese yellow mouse was originally described more than 100 years ago (Bray and York, 1979). It has long been known that the degree of obesity is associated with the degree of the yellow coat color. Geshwind et al. (1972) showed that injection of a-MSH into the yellow mouse produced darkening of the hair, indicating that the melanin forming system in the hair follicle was responsive to exogenous melanocyte-stimulating hormone. With the discovery of pro-opiomelanocortin (POMC), we suggested that the yellow mouse might be the result of a defect in the processing of POMC (Bray et al., 1989). In pursuing this hypothesis, we have shown that the pituitary of the yellow mouse has an increased concentration of the non-acetylated form of PITUITARY

PERIPHERY

Reduced Acetylation

OBESTIY

t FOOD INTAKE

I

4

YELLOW COAT COLOR

Fig. 2. A model for the genetically obese yellow mice. The defect appears to be a reduction in acetylation of desacetyl MSH during processing of proopiomelanocortin (POMC). This results in a decrease in the level of a-MSH. As a consequence there is increased food intake (d-MSH) and decreased pigmentation with yellow coat color.

338

MSH compared to the acetylated form (Fig. 2). We subsequently showed that the non-acetylated or desacetylated form of MSH (d-MSH) was 30-fold more potent than a-MSH in stimulating food intake and body weight gain. Conversely, blackening of the hair follicles was 30 times more potent with aMSH. Thus, reduced acetylation of d-MSH during processing of POMC could account for both the increased food intake, increased body weight and decreased yellow coat color of the dominantly inherited form of obesity (Bray et al., 1988; Shimizu et al., 1989). Recessively inherited obesity. In both rats and mice, a number of genes have been described which are associated with obesity when present in a recessive form (Bray et al., 1989; Coleman and Eicher, 1990). These include the ob gene on chromosome 6 , the db gene on chromosome 4,the tubby (tub) gene on chromosome 7 and thefat gene on chromosome 8 (Coleman and Eicher, 1990). In addition, there is a fatty rat (chromosome 5) and the corpulent gene cfacP) which are also recessively inherited forms of obesity (Bray et al., 1989). Our laboratory has demonstrated that the development of all of these forms of obesity are dependent upon the adrenal glucocorticoids. Moreover, the genetically obese animals are more sensitive to glucocorticoids than lean animals. The corticotropin-releasing hormone hypothesis (Bray, 1982, 1987) provides one explanation for the effect of adrenalectomy (Fig. 3). This hypothesis suggests

-

I ADRENALECTOMY 1-1 I CORTICOSTEROIDS 1 ”

I

Fig. 3. Corticotrophin hormone-releasing hormone model for the effects of adrenalectomy in obesity. Following adrenalectomy the level of CRH increases with a subsequent decrease in food intake and an increase in activity of the sympathetic nervous system, both of which can be produced by CRH.

that the rise in CRH following adrenalectomy is responsible for reduction of food intake, stimulation of the sympathetic nervous system and inhibition of the parasympathetic nervous system, the three phenomena which occur after adrenalectomy. When CRH is injected into the third ventricle of genetically obese animals, it acutely reduced food intake to an even greater degree than in lean animals (Arase et al., 1989). Chronic infusion of CRH into these animals is associated with a reduction in body weight which is greater in the genetically obese animals than in the lean ones. Recent studies from our laboratory have explored the effects of glucocorticoid replacement in adrenalectomized obese animals. We have observed dose-dependent effects on expression of messenger RNA for two enzymes: malic enzyme in adipose tissue and glycerol-3-phosphate dehydrogenase in liver. These effects in two tissues and on two enzymes led us to propose that the genetic defect in the fatty rat may be associated with loss of a gene product which modulates the action of glucocorticoid receptors as they interact with the glucocorticoid responsive elements of the genome in producing their many effects (Bray, 1989; Bray et al., 1991). Summary and conclusions

Obesity occurs in both clinical and animal forms in a variety of specific models which allow study of its underlining endocrine and mechanistic features. Among the neuroendocrine varieties of obesity, polycystic ovaries are probably the most common. The importance of the gonadal feedback system for regulation of food intake and obesity is indicated by the effects of castration in experimental animals which is a widely used mechanism for producing experimental obesity. Cushing syndrome and hypothalamic obesity are rare clinical syndromes. The current evidence suggests that there are two types of hypothalamic obesity from a mechanistic point of view - one associated with hyperphagia as a necessary and sufficient cause and a disturbance of the autonomic nervous system without hyperphagia as a second mechanism.

339

Although genetic factors underlie most types of human obesity, there are several dymorphic forms of obesity including the Prader-Willy syndrome, Cohen’s syndrome, Carpenter’s syndrome, Ahlstrom’s syndrome and the Bardet-Biedel syndrome. The Prader-Willi syndrome is characterized by obesity hypotonia hypogonadism and mental retardation. In animals, a dominant form of inheritance of obesity is seen in the yellow mouse. Current evidence suggests that this syndrome can be explained by reduced acetylation of MSH in the pituitary and/or hypothalamus. Several recessively inherited forms of obesityexist including the obese mouse, the diabetes mouse, fatty rat, the fat mouse, tubby mouse and the corpulent rat. In addition, there are a number of polygenic types of experimental obesity.

The final mechanistic classification of obesity are those due to dietary manipulation. For both human beings and animals, a highly fat diet appears to be particularly problematic for the development of obesity. In addition, palatability and a variety of foods enhance the likelihood of obesity. Acknowledgements This study was supported in part by NIH grants DK31988 and DK32018. References Arase, K., Shargill, N.S. and Bray, G.A. (1989)Effects of corticotropin releasing factor on genetically obese (fatty) rats. Physiol. Behav., 45: 565 - 570. Babinski, M.J. (1900)Tumeur ducorps pituitairesans acromegalie et avec le developpement des oganes genitaux. Rev. Neurol., 8: 531 - 533. Bernardis, L.L.and Frohman, L.A. (1970)Effect of lesion size in the ventromedial hypothalamus on growth hormone and insulin levels in weanling rats. Neuroendocrinology, 6: 319 - 328. Berthoud, H.R. and Jeanrenaud, B. (1979) Acute hyperinsulinemia and its reversal by vagotomy after lesions of the ventromedial hypothalamus in anesthetized rats. Endocrinology, 105: 146- 151. Bray, G.A. (1982) Regulation of energy balances: studies on genetic, hypothalamic, and dietary obesity. Proc. Nutr. SOC., 41: 95 - 108.

Bray, G.A. (1984)Syndromes of hypothalamic obesity in man. Pediatr. Ann., 13(7): 525 - 536. Bray, G.A. (1987)Obesity - a disease of nutrient or energy balance? Nutr. Rev., 45(2): 33 - 43. Bray, G.A. (1989)1989 McCollum award lecture. Genetic and hypothalamic mechanisms for obesity. Finding the needle in the haystack. A m . J. Clin.Nutr., 50(5): 891 - 902. Bray, G.A. and Gallagher, T.F. (1975) Manifestations of hypothalamic obesity in man: a comprehensive investigation of eight patients and a review of the literature. Medicine, 54: 301 - 330. Bray, G.A. and York, D.A. (1979)Hypothalamic and genetic obesity in experimental animals: an autonomic and endocrine hypothesis. Physiol. Rev., 59(3): 719 - 809. Bray, G.A., Sclafani, A. and Novin, D. (1982)Obesity-inducing hypothalamic knife cuts: effect on lipolysis and blood insulin levels. Am. J. Physiol., 243: R445 - R449. Bray, G.A., Dahms, W.T., Swerdloff, R.A., Fiser, R.H., Atkinson, R.L. and Carrel, R.E. (1983)The Prader-Willi syndrome: a study of 40 patients and a review of the literature. Medicine, 62(2): 59 - 80. Bray, G.A., Shimizu, H., Retzius, T., Shargill, N. and York, D. (1988) Reduced acetylation of melanocyte stirnulatory hormone (MSH): a biochemical explanation for the yellow obese mouse. In: P. Bjorntop (Ed.), Obesity in Europe I . Libbey, London, pp, 259 - 270. Bray, G.A., York, D.A. and Fisler, J.S. (1989)Experimental obesity: a homeostatic failure due to defective stimulation of the sympathetic nervous system. Vitam. Horm., 45: 1 - 125. Bray, G.A., Fisler, J.S. and York, D.A. (1990)Neuroendocrine control of the development of obesity: understanding gained from studies of experimental animal models. Front. Neuroendocrinol., 1 l(2): 128 - 181. Bray, G.A., York, D.A., Lavau, M. and Hainault, I. (1991) Adrenalectomy in the Zucker fatty rat: effect on mRNA for malic enzyme and glyceraldehyde-3-phosphate dehydrogenase. Int. J. Obesity, 15: 703 -709. Brobeck, J.R. (1946)Mechanism of development of obesity in animals with hypothalamic lesions. Physiol. Rev., 26: 541 - 559. Butler, M.G. (1990)Prader-Willi syndrome: current understanding of cause and diagnosis. Am. J. Med. Gen., 35: 319- 332. Cassidy, S.B. and Ledbetter, D.H. (1989) Prader-Willi syndrome. Neurol. Clin.,7(1): 37-56. Coleman, D.L. and Eicher, E.M. (1990) Fat (fat) and Tubby (tub):two autosomal recessive mutations causing obesity syndromes in the mouse. J. Hered., 81: 424-427. Cushing, H.W. (1932)The basophil adenomas of the pituitary body and their clinical manifestations (pituitary basophilism). Bull. Johns Hopkins Hosp., 50: 137 - 195. Devenport, L., Knehans, A., Sundstrom, A. and Thomas, T. (1989)Corticosterone’s dual metabolic actions. Life Sci., 45: 1389- 1396. Fonklesrud, E.W. and Bray, G.A. (1981)Vagotomy for treat-

340 ment of obesity in childhood due to Prader-Willi syndrome. J. Pediatr. Surg., 16: 888 - 889. Friedman, E. and Sack, J. (1982) The Cohen syndrome: report of five new cases and a review of the literature. J. Craniofac. Genet. Dev. Biol., 2: 193 - 200. Frohlich, A. (1901) Ein Fall von Tumor der Hypophysis cerebri ohne Akromegalie. Wien Klin. Rundschr., 15: 883 - 886. Frohman, L.A., Bernardis, L.L., Schnatz, J.D. and Burek, L. (1969) Plasma insulin and triglyceride levels after hypothalamus lesions in weanling rats. A m . J. Physiol., 216: 1496- 1501. Fukushima, M., Tokunaga, K., Lupien, J., Kemnitz, J.W. and Bray, G.A. (1987) Dynamic and static phases of obesity following lesions in PVN and VMH. Am. J. Physiol., 253: R523 - R529. Geshwind, I.I., Huseby, R.A. andNishioka, R. (1972)Theeffect of melanocyte stimulating hormone on coat color in the mouse. Rec. Prog. Horm. Res., 28: 91 - 129. Goldstein, J. L. and Fialkow ,P. J . (1973) The Alstrom syndrome. Report of three cases with further delineation of the clinical, pathophysiological and genetic aspects of the disorder. Medicine, 52: 53 - 71. Han, P.W. andFrohman, L.A. (1970) Hyperinsulinemiain tubefed hypophysectomized rats bearing hypothalamic lesions. A m . J. Physiol., 219: 1632- 1636. Hetherington, A.W. and Ranson, S.W. (1940) Hypothalamic lesions and adiposity in the rat. Anat. Rec., 78: 149- 172. Lee, A.T., Fisler, J.S. and Bray, G.A. (1989) Naloxone (NAL) reduces insulin secretion in essential obesity, but not in hypothalamic obesity. Clin.Res., 37(1): 138. Lee, A.T., Bray, G.A. and Kletzky, 0. (1991) Nocturnal growth hormone secretion does not affect variation in insulin secretion. Metabolism, 4012): 181 - 186. Lee, P.D.K., Wilson, D.M. and Roundtree, L. (1987) Linear growth response to exogenous growth hormone in PraderWilli syndrome. A m . J. Med. Genet., 28: 865-871. Parkinson, W.L. and Weingarten, H.P. (1990) Dissociative analysis of ventromedial hypothalamic obesity syndrome. A m . J. Physiol., 259: R829 - R835. Pasquali, R., Casimurri, F., Venturoli, S., Paradisi, R., Mattuoli, L., Capelli, M., Melchionda, N. and Labo, G. (1983) Insulin resistance in patients with polycystic ovaries: its relationship to body weight and androgen levels. Actu Endocrmol., 104: 110- 116. Prader, A., Labhart, A. and Willi, H. (1956) Ein Syndrom von adipositas, Kleinwuchs, Kryptorchismus, and Oligophrenie nach myotonieartigem Zustand im Neugeborenenalter. Schweiz. Med. Wochenschr., 86: 1260- 1261. Rebar, R., Judd, H.L., Yen, S.C.C., Rakoff, J., Vandenberg, G. and Naftolin, F. (1976) Characterization of the inappropriate gonadotropin secretion in polycystic ovary syndrome. J. Clin. Invest., 57: 1320- 1329. Sakaguchi, T., Bray, G.A. and Eddlestone, G. (1988) Sympathetic activity following paraventricular or ventromedial

hypothalamic lesions in rats. Brain Res. Bull., 20(2): 461 -465. Schemmel, R.A., Teague, R.J. and Bray, G.A. (1982) Obesityin Osborne-Mendel and S 5B/P1 rats: effects of sucrose solutions, castration, and treatment with estradiol and insulin. A m . J. Physiol., 243: R347 - R353. Shimizu, H., Shargill, N., Bray, G.A., Yen, T. and Gesellchen, P. (1989) Effects of MSH on food intake, body weight, and coat color of the yellow obese mouse. Lsfe Sci., 45(6): 543 - 552. Smith, P.E. (1927) The disabilities caused by hypophysectomy and their repair. J. A m . Med. Assoc., 88: 158. Stiggelbout, W. (1972) The Bardet-Biedel syndrome, including Hutchinson-Laurence-Moon syndrome. In: P.J. Vinken and G.W. Bruyn (Eds.), Handbook of Clinical Neurology, Elsevier/North Holland, Amsterdam, pp. 380- 412. Temtamy, S.A. (1966) Carpenter’s syndrome: acrocephalopolysyndactyly. An autosomal recessive syndrome. J. Pediatr., 69: 111 - 120. Tokunaga, K., Fukushima, M., Kemnitz, J.W. and Bray, G.A. (1986) Effect of vagotomy on serum insulin in rats with paraventricular or ventromedial hypothalamic lesions. Endocrinology, 119: 1708- 1711.

Discussion C. Pihoker: What is the effect of corticotropin releasing factor (CRF) infusion on weight gain in animals with paraventricular (PVN) vs. ventromedial nucleus (VMN) lesions? G.A. Bray: CRF infusions in animals with VHM lesions prevent the weight gain, CRF infusions have not been given to animals with PVN lesions. C.B. Saper: I am interested in the dichotomy between ventromedial and paraventricular nucleus lesions in producing obesity. The paraventricular nucleus, of course, innervates both parasympathetic and sympathetic preganglionic neurons, while the ventromedial nucleus does not (Saper et al., 1976). In view of the observations by Rogers and colleagues (Rogers and Hermann, 1986), that paraventricular nucleus oxytocin neurons appear to regulate vagal gastric motor neurons, I wonder what the effect of vagotomywould be on paraventricular obesity. Has this been tested? G.A. Bray: We have compared the effect of PVN lesions and VMN lesions on vagal and sympathetic activity. VMN lesions acutely increase vagally-mediated insulin secretion and decrease sympathetic firing rate of nerves to brown adipose tissue. Conversely acute PVN lesions do not affect sympathetic firing rate of nerves to brown adipose tissue nor do these lesions enhance insulin secretion. PVN lesions do, however, enhance glucagon secretion from the pancreas which can be blocked by vagotomy. D.F. Swaab: Comment: we so far measured (unpublished work of Jan Purba) the cell number in the PVN of twoyrader-Willi syndrome cases. Vasopressin cell number was normal, but ox-

341 ytocin was only 50% of the cell number in the control group. This fits in nicely with Saper’s comments that oxytocin fibers from the PVN might be crucial in eating behavior. Could you please share with us the evidence that genetic imprinting does not hold any longer for Prader-Willi and Angelman’s syndromes (discussion with Dr . Summar). G.A. Bray: At the first coffee break during this meeting, I was discussing the issue of imprinting as explanation for the difference between Prader-Willi syndrome and Angelman’s syndrome. Dr. Summar told me that recent unpublished data did not support this explanation. I do not, however, have more detailed information. A. Querido: I enjoyed your lectureon the diversity of factors that may lead to obesity. The recent identification of the lesion in the ventromedial area, which is linked to depressing sympathetic/thermogenic activity, seems very relevant for the caloric equation. My question concerns your etiological classification of obesity versus the clinical taxonomical one of obesity in man, which I prefer for several reasons. I have in mind the division in two categories: regulatory versus metabolic. Clinical toxonomy aims at established clinical entities and their inevitable multicausality, and uses its own instruments such as skills for skinfold measurement and the age and sex patterns which result (Edwards, 1950, 1951a,b). The advantage for this classification is well illustrated with “neuroendocrine” as etiology for obesity in Cushings disease. In large series of Cushings +_ 30% are overweight. The others have normal skinfolds, loss of muscle tissues, and osteopenia, because of restriction of caloric intake. Besides protein loss they are overhydrated, which probably is at the root of the moonface which disappears in days after a successful intervention. Also the old confusing entity of Frohlich’s syndrome, with late or delayed puberty and lack of exercise is another example.

Within the regulatory group - which covers 80% or more of the patients - factors as psychological and family eating behavior, neuroendocrine and secundary metabolic deviations, intracranial lesions, abnormal distribution patterns such as lipodystrophy, etc., find their place. It is clear that pathophysiological thinking helps, but it leaves little place for the combination of factors. What is your view on the above proposed clinical taxonomy? G.A. Bray: With our advances in understanding the mechanisms involved in obesity, we can provide a better classification than regulatory or metabolic. First, we can give an anatomic classification of normal or hypercellular and gynoid or android fat distribution. Second, we can use an etiologic classification which is the basis for the present discussion. Third, we can provide a functional classification for the presence of various associated problems.

References Edwards, D.A.W. (1950) Observations of the distribution of subcutaneous fat. Clin. Sci., 9. Edwards, D.A. W. (1951a)Observationsof thebehaviourofsubcutaneousfat in lipodystrophy. Clin. Sci., lO(3). Edwards, D.A.W. (1951b) Differences in thedistribution of subcutaneous fat with sex and maturity. Clin. Sci., 10. Rogers, R.C. and Hermann, G.E. (1986) Hypothalamic paraventricular nucleus stimulation-induced gastric acid secretion and bradycardia suppressed by oxytocin antagonist. Peptides, 7 : 695 - 700. Saper, C.B., Loewy, A.D., Swanson, L.W. and Cowan, W .M. (1976) Direct hypothalamo-autonomic connections. Brain Res., 117: 305 - 312.

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0 1992 Elsevier Science Publishers B.V. All rights reserved.

343 CHAPTER 25

Hypothalamic involvement in sexuality and hostility: comparative psychological aspects N.E. Van de Poll and S.H.M. Van Goozen Netherlands Institute for Brain Research, 1105 A 2 Amsterdam, The Netherlands, and Psychonomics, Faculty of Psychology, University of Amsterdam, 1018 WB Amsterdam, The Netherlands

Introduction Considering the functions of the hypothalamus in the study of “brain and behavior” one is confronted with the problem that is fundamental to this area and in fact essential in all functional research of the CNS: the necessity to focus on one particular “subunit” and simultaneously consider the brain as a functional entity. The analysis of behavior and its underlying psychological elements and organization, as one of the main functional outputs of the CNS, constitutes a dilemma which is equally difficult to get hold of and solve. The most challenging questions, -however, arise where behavior and psychological concepts are to be combined with “materialistic” arguments concerning the structure and dynamics of the brain. Indeed, one has to be cautious to consider the underlying intellectual speculation when looking for a concrete substrate within the brain when dealing with psychological concepts such as emotion and motivation or learning and memory. The comparative approach so fundamentally a root of biology and, more recently, evolving with equal momentum within the behavioral sciences, may comfort the scientists in this field for the duration of a successful project or a short series of experiments, but there is the danger of triviality and truism, or even misleading simplification, in many of our extrapolations and “animal models”.

To pursue research on “sexual orientation” as an example: from animal research we have learned a good deal of the execution of copulatory responses and its underlying neural and endocrine principles. It has fascinated many scientists that under certain circumstances copulatory behavior seems to include the execution of patterns of behavior of - or with - the “inappropriate” sex (see for a review, Feder, 1978, 1984; Adkins-Regan, 1988). Gonadal hormones secreted by the fetal or neonatal gonads, as well as those circulating in adulthood, may drastically alter the incidence of these “homo-sexual” behaviors. Somatic, neuroendocrine and anatomical correlates are often presented to question or support and delineate the notion of an altered substrate, implicitly postulating similar mechanisms in human homosexuality (see for a review, Feder, 1978, 1984; Meyer-Bahlburg, 1984; Adkins-Regan, 1988). However, behavior, even such “innate” and fixed action patterns as copulatory responses, is highly susceptible to experiential modulation both in animal and man, and the “organizational” principles often referred to have thus far offered only a very limited understanding of homosexuality, despite an enormous scientific effort during the last three decades (De Jonge and Van de Poll, 1984; Ehrhardt et al., 1985, 1989; Adkins-Regan, 1988; Baum et al., 1990). There is certainly no doubt about the occurrence of “iso-sexual” orientation in man and, under very restricted circumstances, in animals

3 44

too (De Jonge and Van de Poll, 1984; Brand and Slob, 1991a,b). The question, however, is whether concrete and detectable changes within the anatomical substrate and physiological or neuroendocrinological principles underlying sexual behavior, as such, necessarily underlie these phenomena. If, indeed, factors of experience and learning predominate in the etiology of homosexual behavior, the present quest for the biological substrate is not only a waste of time but even worse, because it withdraws our attention and brings the topic within the wrong domain of science. If the problems of research on “brain and behavior” are as perplexing as they now appear to us, how is one to proceed within the field of brain and behavior and, more particulary, the function of the hypothalamus? Despite the diversity of putative hypothalamic functions it is not difficult to document its various involvements in relevant aspects of behavior: feeding and drinking, sleep, body temperature, the expression of sexual and maternal behaviors, as well as various forms of hostile behavior and aggression, emotional behaviors and reactions to stress with its visceral and hormonal concomitants. For the purpose of restriction, the present article selects two broad categories of behavior: sexuality and aggression. On the basis of the diversity of factors that are known to influence these behaviors, the putative hypothalamic contributions will be outlined, thus providing the reader with a picture of the extreme complexity with which the hypothalamus can be involved in the integration of brain mechanisms, and behavior. However, a complete picture cannot be given if behavior is seen only as hypothalamic output, as aspects of behavior have been shown to serve an equally important input in hypothalamic function. Due attention will therefore be paid to behavioral feedback mechanisms in which the hypothalamus seems to play a part. Finally, a general overview of the human female’s PCO syndrome and some preliminary psychological data will be presented as a further illustration of the complexities of hypothalamic function.

Behavior as hypothalamic output

Aggressive behavior As a sequel to several earlier studies started by Hess (1969), an important systematic investigation of hypothalamic involvement in aggressive behavior was started by Kruk et al. (1984). In these studies electrical stimulation was used in order to trace physiological properties of the hypothalamic network underlying aggression. Stimulated rats were confronted with lighter, naive males of a less aggressive strain and threshold current intensities required to induce different behavioral responses were determined for hundreds of electrode placements. Reconstructions and plots provided a generalized picture of responsive areas for eliciting a diversity of behaviors such as bite attack, attack jumps and clinch fights but also grooming and social grooming, digging, exploring and flight motivated locomotion and teeth-chattering. Obviously there are many kinds of aggressive behaviors to be observed in socially interacting animals. Attempts to systematize the study of this complex behavior defined categories of aggression on the basis of response patterns and sensory stimuli activating the various aggression systems. Logically, these models postulate a fundamentally different physiological basis for each form of aggression, e.g., aggression between rival males, predatory attack, irritability, paininduced aggression and maternal aggression in defense of the young (see for a review, Moyer, 1976). The extensive effort of many laboratories to identify neural elements underlying aggression in animals now clearly indicates that various forms of aggressive behavior are differentially represented within the hypothalamus (for a review see, Moyer, 1976; Fonberg, 1979). Stimulation and lesion studies point to a strong lateral hypothalamic involvement in predatory aggression whereas this structure appears to be involved in intermale aggression as well. Fear-induced and irritable aggression seems to be represented in the anterior hypothala-

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mus, as stimulation of this area resulted in aggression only in cornered rats. Ventromedial hypothalamic stimulation may lead to a rage reaction but it remains to be determined whether this behavior is primarily offensive or defensive in nature. Several investigators have reported aggressive behavior along with evidence of considerable sympathetic arousal, upon stimulation of the ventromedial hypothalamus, thus implicating this area in irritable aggression. Hence, subareas can be distinguished which, with some specificity and interspecies consistency, contribute to the various forms of aggression. The neural systems underlying these different forms of aggression seem to converge from various parts of the brain within the hypothalamus (see also Kruk et al., 1984; Lammers et al., 1988).

Sexual behavior The anterior hypothalamus and medial preoptic area have been shown to play a key role in the expression of masculine copulatory behavior in a diversity of species. Consistent results have now shown that stimulation of these areas may drastically activate sexual behavior, whereas lesions reduce or even totally abolish all copulation (Van Dis and Larsson, 1971; Heimer and Larsson, 1976; Van de Poll and Van Dis, 1979; Turkenburg et al., 1988; De Jonge et al., 1989). Cells within these areas have been shown to contain both androgen and estrogen receptors, indicating that they may be sites of gonadal hormonal activation of behavior (Sar and Stumpf, 1975; McEwenet al., 1979, for areview). It isimportant to note that these steroid hormone receptors have been localized in a series of other regions of the brain, including the lateral septum, bed nucleus of the stria terminalis, posterodorsal medial nucleus of the amygdala and ventromedial hypothalamic nucleus, with the medial preoptic area receiving its strongest inputs from all these steroid hormone concentrating areas (Sar and Stumpf, 1975; Akesson et al., 1988). Intracranial implants of testosterone placed unilaterally or bilaterally into the medial preoptic and anterior hypothalamic areas can reinstigate or increase sexual behavior of castrated male rats (see

for a review, Hutchison, 1978). Experiments dealing with the intracranial application of estrogen and lesion studies have indicated that the ventromedial hypothalamus, anterior hypothalamus and preoptic area are involved in mediating the effects of hormones on facilitation of the female’s feminine sexual behavior in rats and other species (Barfield et al., 1983). Similarly, progesterone implants have been shown to affect receptivity (see Etgen, 1984, for a review). If the behavioral relevance of the hypothalamus with respect to sexual behavior is reviewed it has to be stressed that the relatively well studied gonadal hormones are by no means the only factors involved. Luteinizing hormone-releasing hormone (LHRH) produced within the hypothalamus appears to play a more direct and important role with respect to sexual arousal and behavior than has been recognized thus far, and an elaborate network of LHRH-sensitive neurons has only recently been detected which may integrate the hypothalamus within other limbic structures (see for a review, Dornan and Malsbury, 1989). It has been suggested that this peptide - or related fragments - acts as a neurotransmitter or neuromodulator within the brain, influencing behavior that precedes corresponding behavioral patterns induced by gonadal hormones or prepares the animal for these latter behaviors. Geldings that had received LHRH and testosterone exhibited significantly greater flehmen response frequencies and attention durations when confronted with estrous mares, without significant effects upon actual sexual behaviors (McDonnel et al., 1989). It is important to note that these effects, which are most probably mediated by the CNS, occur immediately and within a temporal relation which is totally different from all of the hormonal effects upon sexuality (see section on behavior as hypothalamic input). Finally, direct hypothalamic involvement in sexual behavior is indicated by an interesting series of experiments carried out by Orsini (1981, 1982). Recorded potentials from several forebrain structures showed that neurons localized predominantly within the lateral hypothalamus respond to testos-

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lead to self-stimulation if the animal is given the opportunity to control stimulation. For instance, animals with lateral hypothalamic electrodes inducing fragments of eating, if given the opportunity to stimulate these areas by pressing a lever, may do so with high frequencies. Equally “positive” electrode sites were localized within the rostra1 part of the anterior hypothalamus and preoptic area (Van de Poll and Van Dis, 1971). Interestingly, selfstimulation rates obtained with electrodes placed in these areas were drastically reduced by castration and reinstituted by hormone substitution in male rats (Fig. 1). This did not occur with electrodes placed in the lateral hypothalamus. In some animals there was a clear-cut temporal parallel between the reduction of self-stimulation and sexual behavior, both of which were abolished after castration and reinstigated after a few weeks of testosterone suppletion. Hypothalamic stimulation of the anterior aspect of the lateral hypothalamus in male rhesus monkeys

terone injections with a “short latency” of about 5 min. The author interpreted this response as a “nongenomic effect’, perhaps involving a direct coupling of steroid molecules with membrane receptors, and implicated these neurons in the regulation of sexual motivation. A subsequent study using deoxyglucose (2DG) analysis indicated that male rats exposed to female odors show an increased 2DG uptake in the lateral hypothalamus, possibly related to the behavioral or endocrine activation of these males as a response to the attractant potency of urine odors (Orsini et al., 1985). The hedonic quality of hypothalamic stimulation Ever since the first studies on self-stimulation of the brain, the hypothalamus, with its well localized regions with strong positive or negative effects of electrical stimulation, has been one of the prime areas of interest. In general, stimulation of those parts of the hypothalamus that are associated with behavior of a fundamentally positive quality may

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C M

0

1

2

M

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M 4

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M

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Fig. 1 . Self-stimulationfrequencies k S.E.M. and sexual behavior of amale rat with a bipolar electrode located within the medial preoptic area, before and after castration and after treatment with testosterone propionate (150 pg/animal per day). The electrical stimulus (300 p A , individually selected and kept constant throughout experimentation) was a sine wave train (50 cs) lasting 0.3 sec following each bar press. Abbreviations: C, castration; TP, start testosterone substitution; E, ejaculation; I, intromission; M, mount. (Adapted from Van de Poll and Van Dis, 1971.)

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resulted in vicious directed attack on a dominant male (Robinson et al., 1969). Some of the electrode points producing attack also supported selfstimulation. Kruk et al. (1984), stimulating the hypothalamus in their research on aggression, established colocation of self-stimulation and aggression in some but not in all electrodes. It was shown that there are placements supporting selfstimulation, placements supporting switch-off (negative quality) and placements supporting neither or both of these responses, which were evenly distributed between “aggressive” and “non-aggressive” electrodes. Aggressivity and sexuality: human data With respect to direct hypothalamic influences upon human sexuality and aggressive behavior, there are only scarce and scattered data. The data on aggressiveness and hostile responses are relatively clear-cut and consistent. Tumors in the anterior hypothalamus have been reported to result in a facilitation of hostile impulses (Sano, 1962). Damage to the anterior aspect of the hypothalamus leads to a dramatic change of character and very aggressive and irritable episodes (Alpers, 1937). A female patient with a neoplasm destroying the ventromedial hypothalamus showed symptoms comparable to those of animals with lesions in this area. She developed hyperphagia, became obese and had a very low threshold for aggression (Reeves and Blum, 1969). Sano and Mayanagi (1988) have performed many what they call sedative surgical interventions, involving lesions of the posterior hypothalamus in order to “normalize ergotrophic and tropotrophic balance” (see Sano, 1966; Sano et al., 1970, for a discussion of these hypothalamic circuits). He reports remarkable success with patients showing intractable violent behavior. Electro-cauterization of the posterior hypothalamus has led to patients becoming markedly calm, passive, tractable but with a (temporarily) decreased spontanei ty. Inarecent overviewofdatafrom37cases followed up for 10-25 years, 78% were considered to be “satisfactory” (no violent behavior post-operati-

vely; Sano and Mayanagi, 1988; see also Moyer, 1976, for a discussion). A similar study reports 80% “satisfactory” operations in a long-term evaluation (Schvarcz, 1977). Altered sexual behavior and sexual preference appears to be a highly unusual consequence of focal brain injuries in humans. Miller et al. (1986), describing eight patients manifesting increased sexual activity or altered sexual preference, report that hypothalamic damage was involved in only one case with an infiltrating glioma of the midbrainhypothalamic region. Sexual preference changed in this 50-year-old male patient from heterosexual to an almost single-minded preoccupation with children. There is some evidence to suggest an association of pedophilia and neurological abnormalities. In humans, hypothalamic lesions generally reduce sex drive (Miller et al., 1986). The first stereotaxic operation on the hypothalamus with the aim of eliminating unwanted sexual behavior was carried out by Roeder in West Germany in 1962 (Roeder et al., 1972). Approximately two-thirds of the ventromedial nucleus of a pedophilic homosexual 5 1-year-old man was destroyed. Since than, approximately 70 men (homosexuals and pedophiliacs) have undergone similar surgery on the basis of “sexually abnormal” tendencies. An exclusive and persistent abolishment or weakening of the “distressing pressure of the sexual drive” was reported in several cases. These operations were motivated by the combination of two lines of animal research in which it was shown that: (1) amygdala lesions in various male species resulted in indiscriminate mount attempts, a behavioral tendency that could be counteracted by lesioning parts of the (ventromedial) hypothalamus; and (2) “homosexual tendencies” could be activated in rats by hormonal treatment during development. The legitimacy of extrapolation of these animal data to human psychosexual orientation and subsequent use of operational techniques is discussed in many articles (Rieber and Sigusch, 1979; Adkins-Regan, 1988).

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Psychoneuroendocrinology and psychological functions: behavior as hypothalamic input

Circulating gonadal hormones have been shown to be of crucial significance for male and female sexual behavior in adult animals and humans. According to the concept of the “activating” effects of gonadal hormones, this factor acts to prepare central mediating mechanisms in such a way that relevant stimuli may excite specific brain areas and behavioral responses in the adult (Leshner, 1979). The development of masculine or feminine morphological, endocrine and behavioral characteristics depends to a great extent upon the action of gonadal steroids secreted pre- and early post-natally. They presumably act in concert with developmental peripubertal experiential factors to perpetuate sex-dependent and individual behavioral characteristics. Until now, this research has confined its attention almost exclusively to effects of perinatal hormonal treatment (in rats) upon a rigid selection of masculine and feminine copulatory responses (intromission of the male and lordosis of the female). A few recent studies now have shown that precopulatory behaviors and motivational aspects, such as proceptivity, preference and sexual orientation may also be influenced in this manner, although the underlying endocrine and psychological mechanisms may be much more complex (De Jonge and Van de Poll, 1984; Adkins-Regan, 1988; Baum et al., 1990; Brand and Slob, 1991a,b). The behavioral effects and underlying hormonal mechanisms as described thus far, are based upon remarkably slow-acting and relatively permanent characteristics such as “base-line levels of hormones” and permanent changes within the neural substrate as the result of early hormonal “organizational” activity. A third mode of hormonal effects with behavioral relevance has been proposed by Leshner (1979), who based this principle upon “behavioral feedback” effects observed within both the pituitary-adrenal and pituitary-gonadal axis. In experiments on mice and rats that were made to lose in an agonistic conflict, a clear-cut and immediate activation of the pituitary-adrenal axis was

noted which was significantly higher and longerlasting than was a similar activation in the more aggressive winner. In an elegant series of experiments Leshner could show that these characteristics of momentary hormonal activation are extremely relevant for the outcome of later agonistic interactions. Artificially increasing this hormonal response upon initial losing served to make animals more submissive and increase anxiety in subsequent encounters, whereas its suppression counteracted the emergence of submission in subsequent encounters. Apparently, the animal evaluates the outcome of conflict on the basis of its hormonal response, which as an intrinsic part of experience determines future behavior (see for a review, Leshner et al., 1981). Beach (1956), in one of his most influential articles on sexual motivation, has conceptually dissociated the mechanisms underlying sexual behavior into an arousal and a consummatory component. The arousal mechanism is supposed to determine sexual excitation, prior to the start of sexual behavior, whereas the secondary process comprises different elements of the copulatory act. This model asserts the role of appetizing incentives and stimuli associated with a sexual partner (Singer and Toates, 1987). In the field of psycho-neuroendocrinology this aspect of sexual behavior has been largely neglecfed thus far, although the results of some incidental studies may be taken as an argument to relate acute hormonal changes within the hypothalamo-pituitary gonadal axis to sexual arousal and concomitant behavioral changes.

Sexual arousal and gonadal hormonal activation It has now been well established that the presentation of specific sex-related stimuli may lead to an activation of the males’ gonadal-hormonal axis, as evidenced by an acute and short-term increase of luteinizing hormone (LH) and testosterone (T) (Kame1 and Frankel, 1978). Such effects were ascertained in experiments with a wide variety of animal species and investigations in primates and the human male (Stearns et al., 1973; Lee et al., 1974; Bernstein et al., 1978). It is important to note that not only sexual stimulation as such, but also presumably uncondi-

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tioned sexually relevant stimuli, such as odor from receptive females, may lead to the same endocrine response. In the human male, exposure to sexually explicit film fragments significantly increased plasma LH and T levels (Hellhammer et al., 1985; Rowland et al., 1987). The notion that the activation of the gonadal hormonal axis might be significantly involved in sexual arousal is supported even more by the finding that neutral stimuli associated with presentation of a receptive female may easily obtain control over this process by classical conditioning. Indeed, experimental evidence has shown that repeated exposure to the mating arena or to neutral olfactory stimuli followed by the presentation of a receptive female elicits hormonal changes similar to those of exposure to an estrous female rat after only a few pairings (Kame1 and Frankel, 1978). Some of the inconsistencies in human studies on the influence of psychosexual stimulation upon hormonal responses might therefore result from anticipation of sexual stimulation or related cognitive processes (Lee et al., 1974). Hormonal activation as a response to sexually conditioned stimuli may play a crucial role in mating behavior by facilitating approach tendencies, provoking sexual arousal and preparing the male for sexual activity in adequate situations (Graham and Desjardins, 1980; Van de Poll et al., 1990; Koeman et al., 1991; Fig. 2). A recent series of experiments has provided consistent behavioral evidence that a classically conditioned stimulus associated with unconsummated sexual arousal increases copulation in the rat (Zamble et al., 1985, 1986). Interestingly, a possible hormonal involvement in this behavioral facilitation was suggested, since it was noted that the conditioned stimulus optimally facilitated sexual behavior if it was presented at intervals of 8 - 16 min pre-copulation. As can be derived from the previous paragraphs, a possible involvement of unconditioned and conditioned hormonal activation in sexual arousal presupposes the mediation of behavioral facilitation by an acute increase in the hormone levels of the hypothalamo-pituitary gonadal axis. As the luteini-

zing hormone-releasing hormone (LHRH) surge logically precedes LH- and T-release, this hormone is the first candidate to be studied. Male rats tested in a paradigm supposed to reflect sexual arousal mechanisms have shown increased sexual arousal after intra-cerebro-ventricular injections of LHRH at an interval of 2 h. It has been proposed that this effect results from an interaction of LHRH with the neural substrate of sexual arousal, since plasma levels of LH and T were not affected by the LHRH injections at the time of testing (Dorsa and Smith, 1980). Although the hypothalamus by its putative psychoendocrine integrating role clearly may be involved, other parts of the brain undoubtedly also play an important part in these phenomena. Indeed, LHRH has been localized in limbic structures and may act as an neurotransmitter within complex networks underlying sexuality as such (for a review, see Dornan and Malsbury, 1989).

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In contrast to chronic treatment, there have been very few studies aimed at establishing acute effects of LHRH uponaspects of sexual behavior and arousal of human males with normal pituitary-gonadal function. McAdoo et al. (1978), studying effects of this hormone on the male’s mood and behavior, have noted an increase in self-reported sexual arousal, whereas word stimuli with explicit sexual meaning were sooner perceived when subjects received intravenous LHRH injections. In a similar study, a consistent facilitation of parameters of sexual arousal (shorter latencies, greater degree of tumescence, shorter duration of masturbation) was noted in a small group of males exposed to erotic material within a period of 30 min (Evans and Distiller, 1979).

Activation of the gonadal axis and affect One of the most significant aspects of sexual arousal and subsequent sexual consummatory responses is the subjective positive feeling it is inherently associated with. According to elements of the theoretical model of Singer and Toates (1987), external incentives play a crucial role by “inflaming” motivation and producing affect attached to those stimuli that are of biological relevance. This “innate” positive characteristic of emotion is supposed to be attachable to other stimuli through conditioning. Characteristically, such incentives motivate the tendency to approach. It has been shown now in several experiments that male rats quickly develop a strong and reliable preference for stimuli associated with the presenta-

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tion of a receptive female and subsequent sexual interaction (Mehrara and Baum, 1990). The question whether the administration of hormones of the hypothalamo pituitary-gonadal axis is associated with “reward” has been investigated at our laboratory for some time. The results clearly indicate that male rats develop a significant preference for environmental stimuli which are associated with the state of central activation of the gonadal axis accomplished by LHRH administration (see Fig. 3; De .Beun et al., 1989, 1991). Similar results of testosterone suggest that under natural circumstances this positive effect may derive from LHRH as well as from steroid hormonal activation (De Beun et al., 1992b). It is interesting to note that female rats did not show any significant preference for LHRHassociated stimuli. Estradiol injections could be shown to be of an aversive quality both in males and females, i.e., the animals avoided environmental stimuli associated with administration of this hormone in a placeaversion and taste-aversion paradigm (De Beun et al., 1992~).Furthermore, estradiol treatment was shown to act as an aversive stimulus in both sexes, males and androgenized females being more susceptible to this effect (Gustavson et al., 1989). These data stimulated speculations about the etiology of anorexia nervosa in girls on the basis of three theoretical elements: (1) the estrogen-produced malaise at sexual maturation; (2) disruption of normal neurological organization during fetal development and mild masculinization; and (3) an evolving behavioral starvation inappropiately evoked as a way of coping with this feedback loop (Gustavson et al., 1989). The daily use of androgenic steroid hormones and related compounds has recently been associated with addiction (Brower et al., 1989; Kashkin and Kleber, 1989). There is agrowing interest in psychological effects of androgenic-anabolic steroids such as difficulty in cutting down on use, despite psychological side effects, drug craving and withdrawal symptoms. Withdrawal has been associated with depression syndromes such as anergia, anhedonia, loss of libido and dysphoria with suicidal thoughts

(Pope and Katz, 1988; Brower et al., 1989). Evidently, there seems to be a clear link between these effects of sudden changes in the levels of steroid hormones and such phenomena as post-partum depression and psychological concomitants of menopause in women, which has to be further investigated.

Discriminative properties of gonadal hormones If activation of the gonadal hormonal axis plays an important role in sexual arousal, the question can be raised whether this hormonal component of the psychological state of sexual arousal as such may act as a stimulus for the animal. Consequently, hormonal changes may have internal stimulus characteristics and thus control behavior in certain situations. State-dependent learning and techniques to investigate stimulus properties of centrally acting drugs have been known for many years, and numerous experiments have shown that drug-induced internal alteration may act as a discriminative stimulus in learning. Hardly any experiment, however, has thus far established the discriminative relevance of endogenous, hormonal stimuli, although it has been shown that the steroid hormones testosterone and progesterone may serve as discriminative stimuli in learning paradigms (see for a review Overton, 1985; Heinsbroek et al., 1987; De Beun et al., 1992b,c). By giving daily injections of LHRH or saline in a semi-random order, De Beun et al. (1992a) have trained male rats to discriminate between LHRH and saline in a two-lever drug discrimination paradigm (Fig. 4). Injections with LHRH or saline were given 45 min prior to the discrimination sessions. The females used in this test never learned to discriminate. Self-evidently, these data do not provide any indication of the behavioral significance of such cues and the animals’ readiness to use them. Although the results might suggest that discrimination is based upon an internal state related to sexual arousal, such a conclusion remains to be confirmed. Initially, experiments concentrated upon the involvement of LHRH in aspects of sexual activity as such. However, some evidence now supports a fur-

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Hypothalamus, the polycystic ovarian syndrome and psychological concomitants

ther and more general role of this hormone, as behavioral and biochemical evidence indicates that LHRH may indirectly modulate dopaminergic (DA) activity within the brain (Mora and DiazVelis, 1988). Mesencephalic DA activity has been shown to be involved in many types of behavior associated with reward and reinforcement, such as the reinforcing efficacy of drugs, brain stimulation and food and sexual reinforcement (Robbins et al., 1989; Everitt, 1990).

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PCO a hypothalamic diseuse? The “polycystic-ovary syndrome” (PCO), most often referred to as a clinical and not as a pathophysiological syndrome since the causes encompass different abnormalities with similar and converging symptoms, was first described by Stein and Leventhal (Stein and Leventhal, 1935; Goebels-

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mann, 1979; Vaitukaitis, 1983). The syndrome is associated with obesity, hirsutism and amenorrhea in women with bilaterally enlarged polycystic ovaries. The coincidence of the most common symptoms and concomitant endocrine abnormalities as well as the possible underlying primary defects and causes have been a matter of debate for many years. Even now the question of whether it is an ovarian or a hypothalamic disease can not be answered with any certainty. For many years it has been suggested that the primary defect of PCO has to be localized in endocrine and biochemical abnormalities within the ovaries (Axelrod and Goldzieher, 1961; Short and London, 1961). Hypotheses often stressed the dual abnormalities of pituitary and ovarian dysfunction in PCO, giving rise to a self-perpetuating vicious cycle with abnormal gonadotrophin secretion influencing ovarian function and abnormal sex-steroid synthesisinfluencing pituitary function (Fox, 1990). Evidence for a hypothalamic defect among women with polycystic-ovary syndrome is accumulating. Arguments for this have been derived both from the presumed primary hypothalamic involvement in the etiology of pituitary hormonal abnormalities, such as the inappropiately elevated LH secretion and relatively constant and low FSH release, and from the often observed abnormally high levels of prolactin (for a review, see Adashi, 1988). To a large extent this increase in the levels of LH appears to be due to a hypothalamic setting of the amplitude and frequency of the pulsatile LH release. These findings are in keeping with studies in pubertal patients with presumed PCO in whom desynchronization of the nocturnal LH secretory pattern was demonstrated as a presumptive hypothalamic defect (Zumoff et al., 1983). Despite abnormal LH andFSH levels, patients with PCO appear to retain intact negative and positive feedback mechanisms as has been shown in studies using estrogen or clomifeen to attenuate or provoke gonadotrophin release (Adashi, 1988). Arguments for a functional central depletion of dopamine as part of the syndrome can be found in the literature (Vaitukaitis, 1983; Lobo, 1988). Characteristically for those syndromes in which

the hypothalamus is involved, it is extremely difficult to separate primary and secondary physiological and endocrine abnormalities both in the etiology and course of the disease. Given the many facets of hypothalamic-pituitary, ovarian and adrenal dysfunction, it is not possible as yet to localize causal relations of psychological changes in women with PCO. Stress and its hormonal and psychological concomitants have been mentioned as one of the possible causes of PCO (Lobo, 1988), and of other disturbances in which the hypothalamo-pituitary ovarian axis is involved, such as “functional hypothalamic amenorrhea’’ (Berga and Girton, 1989). Increased androgen levels in adulthood as one characteristic concomitant of PCO have also been reported to occur in women pre-natally exposed to DES, suggesting that pre-natal hormonal disturbances may be involved in the origin of PCO (Wu et al., 1980; Peress et al., 1982).

Studies on the psychological effects of chronically elevated levels of androgens in women Psychologically, PCO has its own “feedback” in the form of primary and secondary psychological defects resulting from amenorrhea, obesity, hirsutism and infertility, which makes it difficult (if not essentially impossible) to separate cause and effect. Women suffering from PCO characteristically show chronically elevated levels of androgenic hormones in adulthood. Psychological measures of such female patients may contribute to our understanding of behavioral effects of androgens in women and much of our research, thus far, has been undertaken from this perspective. The present contribution concentrates on the many possible psychological concomitants of PCO without taking into account problems related to the interpretation of defects as primary, secondary or higher order effects. The overview of different elements of hypothalamic function, as presented in this paper, hopefully broadens our view and approach and stimulates further psychological research in relevant groups of patients and proper controls. Androgenic gonadal hormones are of vital importance for various aspects of the male’s sexual

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behaviors proper, but also for “intermale” aggression (Moyer, 1976). More recent research in rats indicates that the female, if exposed to testosterone in adulthood, may exhibit equal and unexpectedly high levels of aggressive behavior (Van de Poll et al., 1981a,b, 1986). It has been shown, moreover, that in addition to the females’ steroid hormones estradiol and progesterone, testosterone may also affect sexual behavior: this hormone affecting motivational aspects of feminine sexual behavior (De Jonge and Van de Poll, 1986; De Jonge et al., 1986a,b). In female primates testosterone appeared to increase sexual motivation (Keverne, 1976). Not much attention has been paid to possible influences of testosterone upon the adult human female. Psychological studies of women chronically exposed to elevated levels of testosterone, as is the case in PCO patients, are of obvious interest with respect to possible effects of androgens upon women. In a recent pilot study a battery of questionnaires on sexuality (including sexual experience and sexual interest) and aggressive behavior was used to study the psychological concomitants of PCO in a small group of patients (Sitters-Zwolsman et al., 1987). The results of this study showed small but consistent differences between PCO patients and proper controls (total hostility scores were equal but PCO patients scored higher on “assault” and the PCO group scored higher on sexual motivation). PCO patients characterized themselves as more feminine and less masculine than the controls. Although the group of PCO patients had been chronically exposed to high levels of testosterone, they did not stand out as an exceptional group of females. All patients showed a sexual orientation towards men. When biographic data were compared with those of the controls, no gross differences were noted on hobbies, sports or occupational life. Most of these patients were married adults and consulted their physician because of the desire to have children. Gorzynski and Katz (1977) reported increased sexual drive and initiative in a small group of PCO patients(seealsoKo1odnyet al., 1979).Inarecent study on the consequences of wedge resection of the ovaries, nodifferencesinsexlifewere found between

control women and a relatively large group of PCO patients whose plasma testosterone levels were within the normal range after operation (Raboch et al., 1985). According to these patients no pronounced changes occurred after operation but no systematic attempt was made to compare pre- and postoperative sexual life in this latter study. These results, taken together with those of our pilot study, seem to indicate that PCO patients, despite high levels of circulating testosterone, are only marginally more responsive with respect to motivational measures of sexuality. It should be kept in mind, however, that the psychological instruments used to study feminine sexual motivation and hostile behavior are not well validated and that secondary factors associated with chronic exposure to high levels of androgens, such as hirsutism and obesitas, might contribute to results established thus far. A link between higher levels of testosterone and increased sexual motivation in normal females has been suggested by several studies. Women who had undergone both ovariectomy and adrenalectomy exhibited a loss of sexual responsiveness (Waxenberg et al., 1959). It has been reported that the female’s sexual desire, excitement, sexual initiative and responsivity correlate significantly with (midcycle) levels of testosterone (Adams et al., 1978;Persky et al., 1978a,b, 1982; Morris et al., 1987). Exposure of women to exogenous androgens has thus far yielded conflicting results (Carney et al., 1978; Sanders and Bancroft, 1982; Mathews et al., 1983; Sherwin and Gelfand, 1987). Studies relating testosterone levels to moods and personality in women indicate that this hormone might act positively upon both aspects (Persky et al., 1978b, 1982). An association of androgen levels and aggression, though well documented in animal studies on both sexes, has not been clearly ascertained in men (Rose, 1978; Benton, 1981; Mazur, 1983; but see Olweus et al., 1980, 1988). Hardly any such studies have been done in women. Ehlers et al. (1980) investigated female patients of a neurological clinic and established higher levels of testosterone in agroup selected on the basis of the incidence of violent behavior. This group scored high on a questionnaire on “feel-

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ings and acts of violence” but did not differ on “feelings and acts of sex” when compared with a less aggressive group. In another study, testosterone levels were found to correlate with measures of sexual excitement, initiative and responsiveness, whereas no relation was found between the plasma levels of several androgens and testosterone profiles on the one hand, and self-rated anxiety, depression or hostility on the other (Persky et al., 1982). The finding of a positive relation between testosterone levels and scores on the “assault” subscale, in our pilot study on PCO patients, would therefore be worthwhile to investigate more thorougly by a combined effort including experimental measures as well as peer-ratings and direct observation. There is a clear relationship between aspects of aggressive behavior, such as hostility, anger, irritability, and more general aspects of emotion. Whether an individual will behave within these categories of aggression or otherwise depends very much upon the individuals’ disposition, experience and appraisal of the situation. Individual differences can be of a short- (e.g., mood-changes in irritability and quick-temperedness) as well as a long-term character. It should be realized that the items of most questionnaires are aimed at measuring the occurrence of overt aggressive acts without reference to relevant stimuli, situational cues and antecedents. With respect to measures of sexuality, comparable drawbacks can be mentioned. These considerations have now led to an approach in which questionnaires are combined into a battery of tests on different aspects of sexuality (sexual motivation, arousability and attitudes) and aggression (anger-tendency, aggression-tendency, irritability). Due attention is paid in these tests to the various stimuli and possible behavioral responses in situations that are relevant for women. Additionally an anger/aggression arousing paradigm has been developed, consisting of a moderately aversive physical situation in which women have to perform frustrating psychological tests. The reliability and validity of these instruments have been tested in relatively large groups of women such as students and “aggressive” sports women.

This research on hormones and emotion has now been extended not only to include PCO patients but also female-to-male transsexuals, treated with male hormones, influences of the menstrual cycle and studies on different professional groups of females. Especially tests of female-to-male transsexuals, tested prior to treatment and after 3 months of androgen treatment, provide an interesting comparison with PCO patients. On the second test occasion most patients in a group of 11 female-to-male transsexuals reported increased sexual motivation and interest, an increase in sexual fantasies and an increase in masturbation frequency. These results were in accordance with questionnaire responses. A comparison of 15 female-to-male transsexuals before the treatment and 12 PCO patients indicated that the transsexuals scored higher on assault and lower on sexual fantasy. This PCO group did not differ in any respect from a large group of female psychology students. Apparently there are many factors which contribute and complicate the effects of altered hormone levels. Summary and conclusions Evidence presented in this article shows the representation of sexual and aggressive behaviors at the level of the hypothalamus to be more prominent than in all other brain areas involved. Indeed, there are good arguments to attribute a central position to the hypothalamus within larger structural systems encompassing the limbic system, where aspects of the behaviors involved can be influenced. So far, however, the arguments are purely descriptive and factual and do not contribute much to answering questions about hypothalamic function: the grounds for and consequences of this massive representation of apparently almost all emotionally relevant social behavioral complexes, so universally established in a diversity of species, still has to be detected. A second and equally important aspect of hypothalamic function obviously has to be related to its central position within various hormonal systems. The present article concentrated on the acute

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dynamics and behavioral significance of activation of the pituitary-adrenocortical and pituitary-gonadal axes. Evidence indicates that the unconditioned behavioral stimuli or the consequences of behavior, but also stimuli conditioned to emotionally relevant events, may drastically alter hypothalamic hormonal regulation. Most importantly, these hormonal consequences in themselves again seem to determine further behavior and responses in relevant situations. The evidence presented with respect to reward and aversion, associated with alterations of specific hormones of the gonadal axis, may add a new dimension to our understanding of psychoendocrine functions ofthe hypothalamus (see also Gary, 1975; Leshner et al., 1981; Carey, 1987). Psychologically, such data can be taken as an argument for a more thorough study of the relation between memory processes and emotion (Bower et al., 1981). However fragmentary and incomplete this review may be, it will be clear that hypothalamic substrates and directly related areas, as well as affiliated hormonal mechanisms, play a central role in many of the most complex motivational and emotional syndromes and disorders. The prime idea in this is that the psychological concomitants of hypothalamic (dys)function are as much output as input, and as much the consequences as the cause within related syndromes. Such a view places the hypothalamus at the core of psychological theories of emotion and motivation, which from their most early origin have been heavily set towards hormonal and humoral changes and their relationships with psychological experience. The data presented on the psychological significance of hormonal activation, in line with other interpretations of comparable changes within the pituitary-adrenal axis in the experiments of Leshner’s, can therefore be considered as a “posthumous” compliment to the theory of James, if, indeed, this theory is dead (a revival can be noted within psychology). Evidently, the elements of hypothalamic functional involvement as they are presented are by no means pure and isolated aspects of this brain area. On the contrary, there are arguments to localize the rewarding quality of hypothalamic activity or hor-

mones in other substrates (Robbins et al., 1989; Everitt, 1990) as there are arguments to reconstruct neural networks underlying different forms of hostile behavior or sexuality (for a review, see Moyer, 1976; Kruk et al., 1984). Yet, the convergence within hypothalamic substrates and psychoendocrine involvement and its intricate feedback and adaptive mechanisms should give this structure a central position within theoretical conceptualizing mechanisms of the most basic aspects of motivation and emotion. Common and apparently obvious psychological concepts and functions such as emotion, motivation and memory, however well founded in psychological theory, are not necessarily represented as such, and within separated substrates. It is equally well possible that the hypothalamus plays a role in certain forms of learning and memory as well, as indeed hormones derived from or drastically influenced by its nuclei have now been shown to do so (Leshner et al., 1981; Carey, 1987; Spear et al., 1990). Evidence has recently been reported that diencephalic pathology plays a role in human memory disorder (Dusoir et al., 1990). Indeed, the influential theory of James about psychology and emotion, formulated a hundred years ago, is perhaps better balanced in combining physical and psychological elements, brain and behavior, than any other theory developed since then, and it is not surprising that, despite all new developments, psychological thinking is still influenced in many ways by these ideas. A central aspect of Jamesian thinking about emotion is the relevance of direct and unconditioned reactions to psychologically relevant stimuli and the concomitant central and peripheral hormonal processes, emotional behavior and subjective experience being separated by an “evaluation” of these bodily processes. It is striking to note how well many of the data on central hormonal involvement in aversively motivated learning, as well as those of Leshner dealt with in an earlier section of this article, but also those on the acute activation of the gonadal axis as presented in this review, fit within such a theory. At the same time, however, there is a problem in-

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herent in these ideas which seems to be a philosophical flaw but which at the same time may prove to contain one of the biggest challenges of this “brain and behavior” research to articulate and solve in the years to come: “who” is it that is evaluating these processes and base “his” reaction thereupon. In evaluating hypothalamic function we have reached the level of fixed and unconditioned reactions to certain classes of evolutionary relevant stimuli well integrated in a series of hormonal reactions and processes. The individual, the subjective “self”, who is using this source of information or suffering from its imbalances, however, still remains far out of reach.

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SECTION VIII

Hypothalamus and Stress

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0 1992 Elsevier Science Publishers B.V. All rights reserved.

CHAPTER 26

Reexamination of the glucocorticoid hypothesis of stress and aging Bruce S. McEwen Laboratory of Neuroendocrinology, Rockefeller University, New York, N. Y. f0021, U.S.A

Introduction The “glucocorticoid cascade hypothesis” of stress and aging was an attempt to formulate a causal relationship between activity of the hypothalamicpituitary-adrenal axis (HPA axis) and deleterious consequences of the actions of glucocorticoids upon the viability of cells in the brain as well as their structure and function. The hypothesis also noted that there are negative effects related to the actions of excess adrenal steroid secretion on bone, muscle and the immune system (Sapolsky et al., 1986a). A key finding which helped to generate the hypothesis was that adrenal glucocorticoids exert damaging and destructive effects on brain cells, particularly in the hippocampus. Another factor was the discovery that the hippocampus is part of a system which keeps the HPA axis in check, with the effect that progressive destruction of hippocampal neurons results in a progressive disregulation and hyperactivity of the HPA system, leading to progressively accelerated destruction resulting from progressively rising titers of adrenal steroids. The purpose of this chapter is to review the hypothesis in the light of old and new evidence on the aging of the HPA axis and the role of glucocorticoids in the generation of neural damage.

Two lines of evidence for the glucocorticoid cascade hypothesis The glucocorticoid cascade hypothesis relates

glucocorticoid-induced neural damage in the hippocampus to declining hippocampal function, particularly to its role in tonically inhibiting pituitaryadrenal activity (Sapolsky et al., 1986a; Jacobson and Sapolsky, 1991). Moreover, the hypothesis proposes that glucocorticoid-induced damage to hippocampal neurons progressively impairs shut-off of glucocorticoid secretion and leads to progressively higher ambient levels of glucocorticoids, which, in turn, cause more damage, leading to a cascade effect. The hypothesis was based on two lines of evidence: (1) that glucocorticoids cause hippocampal damage; and (2) that hippocampal damage results in pituitary-adrenal hypersecretion. These two lines of evidence will now be summarized. Evidence for glucocorticoid-induced neural damage in hippocampus derives from studies of Aus der Muhlen and Ockenfels (1969) in guinea pigs and studies of Landfield and colleagues (Landfield et al., 1978, 1981); Landfield, 1987) on aging and hippocampal damage in rats. The former study purported to show that repeated injections of ACTH or cortisone over 21 days caused neuronal loss in brain areas, including the hippocampus. The latter investigations established that hippocampal aging is retarded by adrenalectomy of rats in mid-life, suggesting that adrenal secretions may play a role in the age-related loss of pyramidal neurons in the hippocampus. In his Ph.D. thesis work at Rockefeller University, Sapolsky showed that repeated injections of corticosterone to young adult male rats mimicked the effects of aging and caused pyramidal

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neuron loss in the hippocampus (Fig. I ; Sapolsky et al., 1985). Sapolsky’s studies also showed a deficit in adrenal steroid receptors in hippocampus (Sapolsky et al., 1983a)and suggested further that negative feedback by adrenal steroids at the hippocampal level might be impaired. Evidence for an involvement of the hippocampus in negative control of pituitary-adrenal function derives from several lines of evidence. First, the studies by Sapolsky and coworkers showed that aging rats fail to shut off corticosterone secretion after stress as effectively as younger rats (Fig. 2; Sapolsky et al., 1983b, 1984a, 1986a), just as aging rats also fail to shut off adrenaline secretion as effectively as younger rats (Fig. 3; McCarty, 1985). This was true when measuring the time course of recovery of corticosterone (Fig. 2) and adrenaline (Fig. 3) levels after cold stress or restraint stress (Sapolsky et al., 1983b) and when measuring fast and delayed feedback to a histamine challenge (Sapolsky et al., 1986b). Hippocampal lesions made with kainic acid infusion also produced hypersecretion of corticosterone and a retarded shut-off (Sapolsky et al., 1984a). Moreover, hippocampal ablation or fimbria-fornix lesions were shown to lead to increased resistance to dexamethasone suppression of corticosterone secretion (Feldman and Conforti, 1976, 1980; Magarinos et al., 1987), and implants of cor-

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ticosterone in hippocampus suppressed ACTH levels which are elevated after adrenalectomy (Kovacs and Makara, 1988). Furthermore, recent evidence points to a pathway from the hippocampus to the paraventricular nucleus by way of the bed nucleus of the stria terminalis (Herman et al., 1989a,b). Such effects of hippocampal lesions are consistent with studies over several decades by other investigators, although it must be noted that stimulation of sites in the hippocampus produces facilitation as well as inhibition of hypothalamic-pituitaryadrenal (HPA) function (e.g., Dunn and Orr, 1984; reviewed in Sapolsky et al., 1986a; Jacobson and Sapolsky, 1991). Thus there are neural pathways going through the hippocampus which can facilitate as well as inhibit the HPA axis, although the net effect of damaging the hippocampus is to enhance HPA activity. The glucocorticoid cascade hypothesis generated a great deal of interest and research activity, and it is the purpose of the remainder of this article to review that research and reassess the original hypothesis. This will be done by first considering both old and new evidence on HPA function in aging rats and then describing and evaluating old and new evidence regardingglucocorticoid effects on the hippocampus and its adrenal steroid receptors.

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u ) .

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Age-related deficits in HPA function Other investigators have provided evidence which rounds out a picture of how the HPA axis of rats changes with age. In particular, there is a reduction of HPA capacity as well as a decline in negative feedback sensitivity to glucocorticoids. The studies of Riegle and coworkers in the early 1970s showed that 22 - 25-month-old Long Evans rats are deficient in their adrenocortical response to a maximal stimulation by ether or ACTH (Hess and Riegle, 1970; Hess et al., 1970). A similar finding was reported by Tang

and Phillips (1978) for 26-month-old CFY-Sprague Dawley rats, and Brett et al. (1983) reported nonsignificant trends in the same direction. A reduced diurnal peak level of ACTH in 22 - 24-month-old Fisher 344 rats was reported by Sonntag et al. (1987). In addition to the deficiency in HPA response during stress or the diurnal peak, aging Long Evans rats (22 - 32-months-old) were markedly resistant to suppression of corticosterone responses to ether stress by chronic administration of dexamethasone, suggesting that they were deficient in negative feedback control (Riegle and Hess, 1972).A more recent study by Oxenkrug et al. (1984) has provided supporting evidence by showing that even by 6 months of age, male Sprague Dawley rats showed marked resistance to dexamethasone suppression of the HPA axis. There is, however, some disagreement as to whether basal corticosterone levels are altered in aging rats. Neither Hess and Riegle (1970), nor Tang and Phillips (1978), nor Sonntag et al. (1987), nor Brett et al. (1983) found an age-related difference in basal levels of corticosterone. However, other investigators have found age-related elevations of corticosterone levels (e.g., Landfield et al., 1978; DeKosky et al., 1984). Conceivably, basal levels may be elevated in some groups of rats because of ongoing stress, which the aging rats may have greater difficulty in shutting-off. Interestingly, in vitro studies of release of corticotrophin-releasing factor (CRF) from hypothalamic fragments showed that tissue from 25-month-old Sprague Dawley rats releases higher amounts of CRF than tissue from 3month-old rats, both under basal and stimulated conditions (Scaccianoce et al., 1990). Moreover, although maximal ACTH and corticosterone responses to stress may be deficient in aging rats, there are circumstances under which aging rats have been reported to display elevated hormone levels. Lorens et al. (1990) reported an exacerbated corticosterone response to a conditioned emotional response in 22-month-old Fisher 344 rats compared to 7-month-old animals. Moreover, Sapolsky et al. (1983a) reported elevated corticosterone in 27-month-old Fisher 344 rats during

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Fig. 3. The effects of 10 min of 12°C cold water stress on plasma epinephrine response in young v p w s old rats. (Data redrawn from McCarty, 1985.)

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cold stress over several hours (see Fig. 2); the elevations were prolonged compared to younger rats, which appeared to have a more efficient adaptation mechanism. Finally, Van Eekelen et al. (1992) found that 30-month-old brown Norway rats showed elevated and prolonged ACTH secretion following novelty stress and in a conditioned emotional response compared to rats of 3 - 6 months of age; corticosterone levels were less markedly elevated in old vs. young under these conditions. However, plasma levels of transcortin, glucocorticoid-binding globulin (CBG), were markedly reduced in aging rats, indicating that free corticosterone levels were undoubtedly higher in the older animals (Van Eekelen et al., 1992). It should be noted, however, that in another study on Long Evans rats, there were no age-related changes in CBG levels (Issa et al., 1990). With regard to adaptation to repeated stress, Riegle (1973) reported that 4 h of daily restraint stress for 20 days led to greater adaptation of corticosterone responses in 4 - 6-month-old Long Evans rats compared to aging rats of 22 - 28 months. A more recent study on Fisher 344 rats has provided additional information, namely, that at the onset of chronic stress, 12-month-old rats show a greater enhancement of the corticosterone responsiveness to the stressor and then show a greater adaptation of HPA response to prolonged exposure to the chronic stressor compared to the 26-month-old aging rats (Odio and Brodish, 1989). When a novel stressor is applied to chronically stressed rats, there is a priming effect of the previous chronic stress and an enhancement of acute corticosterone stress responses which is greater in younger rats (Brodish and Odio, 1989). At the same time, ACTH responses to the novel stressor were somewhat lower in old animals prior to the chronic stress, and these responses tended to increase and become like those in younger animals as a result of the chronic stress (Brodish and Odio, 1989). Thus adaptation of the HPA axis to repeated stress is deficient in older rats, and this includes both the priming or enhancing effects of the response to novel stressors as well as the habituation to repetition of the same stressor.

Another example of a deficit in adaptation in aging rats is the demonstration that 24 - 26-month-old female Sprague Dawley rats failed to re-entrain corticosterone responses to temporal shifting of availability of water and also failed to shut off corticosterone when water was presented to drink; in contrast, 3 - 5-month-old rats of both sexes as well as aging male rats efficiently entrained as well as shut-off corticosterone responses to water presentation (Brett et al., 1986). A final aspect of aging of the HPA axis in rats concerns individual differences within a rat population. Male Long Evans rats of 23 - 27 months of age were subdivided into two populations which represented extremes within the population: 28% were designated as impaired and 34% as unimpaired, based upon their performance on a spatial learning task in a Morris water maze (Issa et al., 1990). Compared to aged unimpaired rats, the aged impaired rats showed poorer shut-off of the corticosterone response to restraint stress (Fig. 4), as well as elevated basal corticosterone levels and higher plasma ACTH levels in the afternoon (Issa et

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Fig. 4. Mean (-t S.E.M.) plasma corticosterone levels bg/dl) in age-impaired (AI), age-unimpaired (AU), and young control (CTL) rats (n = 10- 12 animals per group) at various times before (Pre), during (0 min) and following (30- 180 min) a 20min period of restraint stress (A1 differs from AU and CTL animals at 30, 60, 90, 120 and 180 min; P < 0.05). (Reprinted from Issa et al., 1990, by permission.)

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al., 1990). In short, the impaired group were more like the aged rats described in some of the studies above. These fascinating observations suggest that one explanation for the variable results of different studies may be the individual characteristics of rats within the population under investigation. This seems to be a more likely explanation than strain differences among rats, because the Issa et al. (1990) study employed Long Evans rats, whereas the studies of Sapolsky utilized Fisher 344 rats and yet found similar types of aging effects (Sapolsky et al., 1986a). One of the most compelling explanations for individual differences among rats is that they are the long-term consequences of differential experiences during development (Meaney et al., 1988, 1991). Handling of newborn rats, a form of gentle and regular stimulation involving separation from the mother during days 1 - 14 of post-natal life, was shown to attenuate the age-related decline in hippocampal neuronal loss and loss of spatial learning ability. An underlying reason for the effects of handling was suggested to be a handling-induced reduction in the magnitude and duration of the HPA response to stressors, resulting in lesser exposure of the hippocampus to the wear and tear of glucocorticoids during adult life. Changes in hippocampal adrenal steroid receptors with aging Evidence obtained in support of the glucocorticoid cascade hypothesis included the observation that adrenal steroid receptors in the hippocampus were decreased in aging rats. The decrease might be related to the cell loss which occurs in the hippocampus with age, or it may be independent of it and related instead to elevated levels of circulating glucocorticoids in the aging animal. We shall summarize this evidence and the follow-up of these observations and then consider their implications for the functioning of the hippocampus in pituitaryadrenal function. The initial studies by Sapolsky et al. (1983a) on 3 vs. 24-month-old Fisher 344rats revealed a deficit in

the older animals in 3H corticosterone uptake and cell nuclear retention in vivo and 3H dexamethasone binding to cytosol in vitro in both hippocampus and amygdala. No changes were found in hypothalamus, cerebral cortex or midbrain. A study by Rigter et al. (1984) on 6 vs. 26-month-old Wistar rats found an age-related deficit in hippocampus in the in vitro binding of 3H corticosterone. Autoradiographic results from in vivo labeling of adrenalectomized young and old rats with 3H corticosterone revealed a deficit in the number of hormone-concentrating pyramidal neurons in the hippocampus (Sapolsky et al., 1984a,b). A subsequent study by Patacchioli et al. (1989) confirmed these autoradiographic findings and showed, furthermore, that 7 months of treatment with acetyl-Lcarnitine retarded the loss of 3H corticosterone uptake sites in the hippocampus. These studies imply that there is a general deficit in hippocampal adrenal steroid receptor binding in the aging rat brain which may be associated, at least in part, with neuronal loss. Shortly after these studies, the brain was shown to contain two types of receptors for adrenal steroids, referred to as type I and type I1 receptors, which differ in affinity and specificity for adrenal steroids (Reul and DeKloet, 1985). Type I receptors have a higher affinity than type I1 receptors for corticosterone and also possess a high affinity for aldosterone, which has led them to be called “mineralocorticoid” receptors, whereas type 11 receptors are called “glucocorticoid” receptors. Both receptor types have been cloned and are products of different genes (Hollenberg et al., 1985; Arriza et al., 1987). Type I receptors are present in high concentrations in neurons of the hippocampus, septum and parts of the amygdala, whereas type I1 receptors are found in high concentrations in neurons and glial cells of many brain areas including the hippocampus (McEwen et al., 1986). Subsequent studies with specific tools for looking at these two receptor types have shed further light on the nature of the agerelated deficit. A study by DeKloet et al. (1987) in 3 vs. 28-month-old Wistar rats revealed deficits in hippocampus of the old animals in both type I and

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type I1 receptors, reflecting decreases in the number of sites of 52% and 28%, respectively. Subsequent autoradiographic studies using in vitro labeling of receptors in brain sections revealed decreases in both type I and type I1 receptors throughout the hippocampal formation as well as in lateral septum (Reul et al., 1988). A later study on 6 vs. 23 - 27month-old Long Evans rats also showed deficits in type I and type I1 receptors in hippocampus in aged rats (Issa et al., 1990), and a recent abstract has reported in 5 vs. 26-month-old Fisher 344 rats an age-related deficit in type I and type I1 receptor mRNA levels as well as receptor binding in the hippocampus (Morano and Akil, 1990). A recent study by Pfeiffer et al. (1991) has followed type I1 receptor binding and mRNA across three ages in Long Evans rats. Regionally, only the hippocampus showed decreased type I1 receptor binding and mRNA at 24 months of age compared to 6 and 12 months of age; other brain areas like amygdala, hypothalamus, frontal cortex and pituitary gland did not show age-related decreases. One puzzling finding was that type I1 receptor mRNA levels actually increased in a number of brain areas at 12 months of age relative to the other two ages. This occurred in pituitary, amygdala, frontal cortex and hippocampus, whereas hypothalamus showed an increased type I1 receptor mRNA level at both 12 and 24 months of age relative to 6-month-old animals (Pfeiffer et al., 1991). However, not all studies have produced such consistent results. Lorenset al. (1990)studied7 vs. 17.5month-old Fisher 344 rats and found deficits in hippocampal type I receptors, but not any age-related decrease in type I1 receptor levels. Conceivably, the lesser age of the rats in this study could have been a factor. Van Eekelen et al. (1992) studied 3 - 6 vs. 30 - 33-month-old Brown Norway rats and reported that mRNA levels for type I1 receptors were decreased in the hippocampal formation, with the exception of CAI, whereas type I receptor mRNA was not decreased in the hippocampus. Rachmanin et al. (1989) reported no decrease of either type I or type I1 receptor binding in hippocampus in Fisher 344 or Long Evans rats of 24 - 26-month-old com-

pared to 3 - 5-month-old animals. Finally, the studies of Eldridge et al. (1989a) on 3 vs. 24-monthold Fisher 344 rats raised an issue not reported in other studies, namely, that there was a progressive increase in the age-related deficit in type I and type I1 receptor binding capacities with time after adrenalectomy. In fact, these studies did not find an age-related deficit in type I1 receptor binding at the shortest time intervals after ADX and concluded that aging rats may be deficient in the ability to upregulate adrenal steroid receptor levels after ADX. In contrast, Morano and Akil (1990) reported that older rats in their study showed an up-regulation of type I and type I1 receptor mRNAs after adrenalectomy, whereas younger rats did not show an upregulation of either receptor mRNA level in the hippocampus. It should also be noted that Landfield and Eldridge (1989) reported that aging rats had Kd’s (a measure of receptor affinity) for type I1 receptors which were significantly lower than the younger rats. In contrast, there was no sign of an age-related difference in K d in the study of DeKloet et al., (1987). The reasons for these discrepancies are unclear.

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CA1 CA3 Fig. 5 . Pyramidal neuron density in hippocampus of young control (CTL) and aging-cognitively impaired (AI) and agingcognitively unimpaired (AU) rats. Asterisks indicate significant reductions: * from CTL and ** between A1 and AU at P < 0.01. (Reprinted from Issa et al., 1990, by permission.)

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Another aspect of age-related deficits in adrenal steroid receptors was demonstrated in one study on 23 - 27-month-old Long Evans rats, namely, that rats which showed such deficits are ones which also showed deficits in spatial memory and a failure to shut off pituitary adrenal function efficiently after stress; these same aged, impaired rats gave evidence of neuronal loss in the hippocampus (Fig. 5 ; Issa et al., 1990). Thus the functional deficits and the neuroanatomical and neuroendocrine deficits are co-present in the same subgroup of aging rats, but how are they related to each other? One of the tenets of the glucocorticoid cascade hypothesis is that the long-term elevation in glucocorticoid levels accelerate the destructive processes which culminate in neuronal loss within the hippocampus. In the original version of this hypothesis, glucocorticoid exposure in stress was seen to result in down-regulation of glucocorticoid receptors in the hippocampus, which brain structure is more susceptible to such down-regulation than other brain regions. According to this version of the hypothesis, such down-regulation reduced the impact of circulating glucocorticoids on the shut-off of the stress response and contributed to the progressive rise in the glucocorticoid levels which, in turn, accelerated the destructive processes occurring in the hippocampus. However, there are several problems with this version of the hypothesis. First, the down-regulation by stress-induced glucocorticoid secretion of hippocampal type I and type I1 receptors does not seem to occur in healthy rats except under the most extreme conditions involving prolonged daily stress over many weeks (Fig. 6 ; Eldridge et al., 1989b) or debilitation of the animal’s health, leading to the conclusion that when stress-induced down-regulation occurs in the hippocampus it may be more of a pathological than a normal process (McEwen et al., 1990). Second, down-regulation would appear to reduce the susceptibility of hippocampal neurons to the destructive effects of glucocorticoids, which would be an adaptive and protective response. This point will be dealt with in the next section. Third, the actual evidence is that as rats age the ability of pro-

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0 MID AGED YOUNG Fig. 6. Type I1 adrenal steroid receptor level in hippocampal cytosol 1 day and 3 weeks after termination of 6 months of daily training in footshock-escape stress. Each bar represents the mean & S.E.M. of data from 8 rats. Ages at beginning of 6 months of stress: young, 6 months; mid, 12 months; aged, 18 months. Rats killed at 1 day or 3 weeks following final training session. Main effects: age, F(2.71) = 9.963, P < 0.001; stress, F(2.71) = 3.700, P < 0.05. (Reprinted from Eldridge et al., 1989b, by permission.)

longed stress to cause down-regulation of the type I1 receptor population in hippocampus declines (Fig. 6; Eldridge et al., 1989b), as also does the upregulation of type I1 receptors which occurs after adrenalectomy (Eldridge et al., 1989a). Thus, the adrenal steroid receptor system appears to become less plastic with increasing age, in parallel with the loss of adaptability of the pituitary-adrenal axis to repeated stress with increasing age (see above). As is summarized in Fig. 7, such a loss in the ability to down-regulate receptors in response to stress and elevated glucocorticoids might actually increase the vulnerability of hippocampal neurons to the damaging effects of glucocorticoids.

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Fig. 7. Pyramidal cell density in hippocampus expressed as number of nucleoli (mean f S.E.M.) per 100 pm of stratum pyramidale length, for young, mid-aged and aged (non-stressed vs. stressed) rats. See legend to Fig. 6 for explanation of stress. Main effects of age were observed, and chronic stress resulted in an increase in cell loss only for the aged group. (Reprinted from Kerr et al., 1991, by permission.)

Neuronal degenerative changes during aging and the possible role of glucocorticoids The hippocampus is prominent among brain regions in showing degenerative changes with aging (Coleman and Flood, 1987). The progressive accumulation of lipofuchsin deposits, reduced density of pyramidal neurons, and increased astrocyte hypertrophy are among the most consistent and prominent features (Landfield, 1987). Aging is also associated with a decreased capacity of the nervous system to respond with synaptic sprouting after damage (e.g., DeKosky et al., 1984) and with the formation of new capillaries after environmental manipulations which promote neuronal and glial plasticity (Black et al., 1989). As in other aspects of aging reviewed above, the reports of neuroanatomical changes with age also show variability and some inconsistency from laboratory to laboratory (for review, see Landfield, 1987). There are at least two possible reasons for these differences. First, there are strain differences in rats in the degeneration of the septohippocampal

cholinergic system which may be related to the degree of stress responsiveness (Gilad et al., 1987); conceivably such differences may extend to other aspects of brain aging, Second, within strains of rats, there are individual variations in the degree of cognitive, neuroendocrine and neuroanatomical impairment which are found as rats age (Figs. 4 and 5 ; Issa et al., 1990). Since these impairments appear to cluster together and may even be causally-related to each other, it is important to screen animals for behavioral deficits prior to making neuroanatomical assessments; no such screening was done in any of the studies reporting absence of neuroanatomical changes with age. There are a number of studies which did assess behavioral impairments and which also found degenerative changes in the hippocampus. One of the first studies compared 4 month and 26-month-old Fisher 344 rats and found in the rats impaired for spatial maze learning a loss of perforated axospinous synapses in the dentate gyrus (Geinesmanet al., 1986). Inanotherofthese studies, a silver degeneration stain revealed degenerating fibers in the hippocampus of 28 - 32-month-old Fisher 344 rats compared to 6 - 8-month-old rats (Greene and Naranjo, 1987). In a third study, behaviorally-impaired 23 - 27-month-old Long Evans rats showed loss of hippocampal CAI and CA3 neuron density compared to 6 - 7-month-old rats, whereas behaviorally-unimpaired rats showed a lesser loss of neuron density (Fig. 5 ; Issa et al., 1990). Finally, Wistar rats of 3, 12, 17 and 24 months of age showed progressive impairment of working memory in an 8 arm radial maze and progressive accumulation in lipofuchsin deposits in the hippocampus; CA3 neurons were reduced in both number and size with increasing age (Kadar et al., 1990). The loss of neuron size and density in the CA3 field of the hippocampus was a prominent feature of the findings of Sapolsky et al. (1985). Moreover, there was a loss in the uptake and retention of 3H corticosterone by the remaining CA3 neurons in 28month-old Fisher 344 rats (Sapolsky et al., 1985). Corticosterone administration at a dose of 5 mg

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every day for 12 weeks to young adult rats caused a decrease in the size of pyramidal neurons in the CA3 subfield as well as a loss of the number of the largest neurons (Sapolsky et al., 1985). These results are complemented by a more recent study showing that local administration of cortisol pellets into the hippocampal formation of vervet monkeys resulted in hippocampal degeneration which was especially prominent in CA3 (Sapolsky et al., 1990). We shall consider below the possible reasons for the vulnerability of the CA3 region. Before moving ahead, it should also be noted that there is other evidence that the rest of the hippocampal formation besides the CA3 region, can show considerable loss of 3H corticosterone uptake with age. In the recent study of Patacchioli et al. (1989), there was loss of 3H corticosterone uptake by

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Fig. 8. Schematic summary of relationship between corticosterone elevation by repeated stress and down-regulation of type I1 glucocorticoid receptors (GR) which requires severe and prolonged stress. This scheme recognizes the fact that aging is accompanied by a resistance of type I1 receptors to undergo down-regulation as a result of repeated daily stress and by an enhancement of stress-induced loss of hippocampal pyramidal neurons. This scheme suggests that the down-regulation of hippocampal type 11 receptors, which are implicated in the death of pyramidal neurons (Sapolsky, 1990), may be a protective mechanism against cell death. The difference from the original version of the glucocorticoid cascade hypothesis (Sapolsky et al., 1986a) is that receptor down-regulation now appears to be the product of severe, prolonged stress, rather than stress over a shorter time period. Moreover, the capacity of the hippocampus to show such down-regulation declines with age (Eldridge et al., 1989b), while at the same time aging permits severe, prolonged stress to accelerate hippocampal neuronal loss (Kerr et al., 1991).

autoradiography in CA1, CA2 and CA3 as well as in the dentate gyrus. If glucocorticoid administration works to accelerate neuronal destruction, then what about stress? Prolonged, severe and ultimately fatal social stress in vervet monkeys led in one opportunistic study to some degeneration within the CA3 field of the hippocampus (Uno et al., 1989). Moreover, 6 months of a chronic, daily stress in a shuttle box resulted in significantly greater pyramidal neuron loss in the hippocampus of Fisher 344 rats which had been stressed between 19 and 25 months of age than in rats which had been stressed for 6 months at two younger ages (Fig. 8; Kerr et al., 1991). This result is consistent with a role of stress-induced glucocorticoid secretion in aging changes but it also indicates that there is something about the aging rat which makes it more susceptible than younger ones to the effects of stress and/or glucocorticoids. The lesser plasticity of the brain in adapting to repeated stress as well as the lesser ability of the aging brain to show a down-regulation of hippocampal glucocorticoid receptors after repeated stress, which were both described above, may be important factors in this age-related increase in susceptibility to stress and the damaging actions of glucocorticoids. Does the glucocorticoid cascade hypothesis apply to the human hippocampus? There is far less information available regarding the interactions between glucocorticoids and the human hippocampus; nevertheless, what is available for both infra-human primates and humans is consistent with the applicability of the glucocorticoid cascade during stress and aging. First, it is known that the rhesus monkey hippocampus has adrenal steroid receptors with a distribution like that in the rat (Gerlach et a1.,1976). Second, lesions of the rhesus monkey hippocampus result in adrenal steroid hypersecretion and failure to shut off the pituitary-adrenal stress response (reviewed in Jacobson and Sapolsky, 1991). Third, opportunistic studies of vervet monkeys exposed to severe social stress has produced preliminary evidence for

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Fig. 9. Average plasma cortisol levels in patients ( + S.E.M.) 120 min after glucose infusion as a function of the severity of the hippocampal lesion. (Reprinted from DeLeon et al., 1988, by permission.)

stress-related damage to CA3 pyramidal neurons (Uno et al., 1989). Thus, the infra-human primate hippocampus has many of the features of the rat hippocampus as far as involvement in pituitary-adrenal regulation and sensitivity to adrenal steroids and stress. How do such interactions play out in the human aging process? Unfortunately, there is very little systematic information on normal human aging, although a few case reports exist of hypercortisolism in elderly human subjects (see Jacobson and Sapolsky, 1991). However, evidence from the study of Alzheimer’s disease has suggested a possible relationship between hippocampal damage and cortisol hypersecretion under the conditions of a glucose tolerance test. As shown in Fig. 9, the larger the hippocampal lesion determined by a CAT scan the higher was the cortisol response in the aftermath of a bolus of glucose (DeLeon et al., 1988). DeLeon and colleagues have taken this analysis further by validating the predictive value of the CAT or MRI information about hippocampal atrophy in terms of actual neuroanatomical damage at post-mortem autopsy; and the CAT or MRI data on hippocampal atrophy provide an early warning of incipient Alzheimer’s disease with a high predictive value (DeLeon et al., 1989, 1992). It has been reported that Alzheimer’s disease is associated with cortisol hypersecretion (Davis et al.,

1986). However, one cannot be sure that hippocampal damage is the only or even the main responsible factor for this hypersecretion; rather, non-specific factors such as the stress of the disease process on the individual and the interaction with hidher family, may play some role. Nor is it possible to conclude at this stage in our knowledge that cortisol contributes to the progressive pathology of the hippocampus in Alzheimer’s disease (e.g., DeLeon et al., 1989). This, however, remains aviable possibility in light of the evidence from experiments on rats which were reviewed above. Possible mechanisms of degenerative effects of glucocorticoids on the hippocampus How do glucocorticoids bring about degenerative changes in the hippocampus and why is the CA3 region especially vulnerable? The studies of Sapolsky and coworkers have shown that excitatory amino acids figure prominently in the destructive actions of glucocorticoids and that the hormonal influence increases the vulnerability of neurons to other degeneration-inducing agents (Sapolsky, 1990). The synergy between glucocorticoids and excitatory amino acid neurotoxicity has been demonstrated for the application of kainic acid as well as transient ischemia, and it has been demonstrated in hippocampal neurons in culture as well as in vivo (Sapolsky, 1990). The effects of the neurotoxin MDMA on serotonergic neurons are also enhanced by circulating glucocorticoids (Johnsonet al., 1989), although the effects of another neurotoxin, trimethyltin, on hippocampal degeneration is not enhanced by glucocorticoids (J. O’Callaghan, personal communication). One of the key elements in glucocorticoidinduced vulnerability to excitotoxins is their ability to inhibit glucose transport (Horner et al., 1990); and, indeed, glucose and other metabolizable sugars overcome the toxic effects of excitotoxins both in vitro and in vivo (Sapolsky, 1990). Another aspect of excitotoxin actions involves calcium ions, because free calcium is able to activate proteolytic enzymes which enhance the generation

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of free radicals that, in turn, damage membranes and destroy cells (Siesjo and Bengtsson, 1989; McEwen and Gould, 1990). Hippocampal neurons of aging rats show an increase in a calcium-mediated after-hyperpolarization which is dependent on circulating glucocorticoids; the magnitude of the effect of adrenalectomy on this electrical response increases with aging which suggests that there is an age-related increase in the effect of glucocorticoids on some calcium-mediated brain process (Kerr et al., 1989). Of course, the progressively elevated levels of circulating glucocorticoids which are seen in many studies of aging rats (see above) may also be a factor in these results. Whatever the mechanism, 6 months of chronic stress in young, middle age and aging rats produced evidence of accelerating the aging process. Young and middle age rats undergoing stress through a daily shock-escape/avoidance training session over 6 months duration demonstrated not only stressinduced loss of pyramidal neurons (Fig. 8) but also

depression of thresholds for eliciting EPSPs and population spikes; however, the stress effect was in the same direction as the changes with aging, and the oldest rats appeared to have reached a floor and did not show a further suppression of thresholds to the chronic stress (Kerr et al., 1991). At the same time, chronic stress had the ability to suppress frequency potentiation of the EPSP, and this ability was evident only in the younger rats undergoing stress and was lost in the middle age and most aged rats undergoing stress indicating another type of agerelated loss of plasticity (Kerr et al., 1991). In many of the neuroanatomical studies of aging changes, the CA3 subfield of the hippocampus shows the greatest evidence of degenerative changes. This may well be related to the fact that the CA3 pyramidal neurons receive the mossy fiber innervation from the dentate gyrus, since both kainic acid damage and perforant pathway-induced damage are most evident in the CA3 region and are blocked by lesioning of the dentate gyrus (Nadler and Cuth-

Fig. 10. Schematic summary of the interactions between adrenal steroids and the dentate gyrus and CA3 and CA1 pyramidal neurons. Neuronal activation of the dentate gyrus via the perforant pathway causes mossy fiber activity, which activates CA3 pyramidal neurons and then CAI pyramidal neurons via the Schaeffer collaterals. Adrenal steroids interact with dentate gyrus neurons to promote their viability and conceivably also their activity. Adrenal steroids also act synergistically with released excitatory amino acids to promote atrophy of pyramidal neurons and eventually to accelerate their destruction. The inherent activity of this neural system during normal as well as stress-induced behavior, together with the presence of adrenals steroids, creates a situation where normal “wear and tear” may be affecting pyramidal neuron structure; the “wear and tear” is accelerated under conditions of high neural activation coupled with high levels of adrenal steroids in the blood, as in chronic stress.

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bertson, 1980; Sloviter, 1983). In addition, we have recently found that corticosterone treatment which leads ultimately to destruction of pyramidal neurons in the CA3 subfield produces within just 2 weeks evidence for atrophy of the apical dendrites of the CA3 pyramidal neurons; this could well be the result of intense excitatory amino acid release from the mossy fiber system which heavily innervates the apical dendrites (Fig. 10; Woolley et al., 1990). Not only is the mossy fiber innervation a source of excitatory amino acids, but it also appears to be the recipient of a different kind of glucocorticoid influence, which is opposite to that seen on the pyramidal neurons. Adrenal steroids appear to maintain and perhaps even to stimulate the morphological features of dentate gyrus neurons (Gould et al., 1990), and the absence of adrenal steroids leads within days to a massive increase in death of dentate gyrus neurons which can be prevented by low doses of adrenals steroids in the drinking water or by other routes of administration (Sloviter et al., 1989; Gould et al., 1990). It was the low dose of corticosterone in the drinking water in the aging studies of Landfield et al. (1981) which precluded any loss of dentate gyrus neurons. It should be noted as yet another sign of the loss of plasticity of the aging brain that the loss of dentate gyrus neurons after adrenalectomy declines with age when rats approach 1year (E. Gould and C. Woolley, unpublished observations).

Can age-relatedneuronal degenerationbe retarded? The apparently central role of glucocorticoids in manifestations of the effects of excitatory amino acids and excitotoxins raises the important question as to whether the age-related degeneration represents a wear and tear on the hippocampus related to the normal operation of the pituitary-adrenal axis as well as the endogenous exicatory amino acids and the calcium homeostasis mechanism of the hippocampus (McEwen, 1991; Swaab, 1991). Most of the evidence cited above is at least consistent with this notion. An important issue raised by the notion of wear

and tear and the putative role of glucocorticoids is whether age-related degeneration can be retarded or prevented. There have been three types of interventions described thus far, namely, the use of certain drugs, dietary restriction, and developmental manipulations, and each treatment has had some limited success on various measures of hippocampal aging. Moreover, each of the treatments provides further information pertinent to the mechanism. The initial evidence for adrenal involvement in the aging process was provided by studies involving bilateral adrenalectomy of Fisher 344 rats in midlife and maintenance of these animals on low to moderate levels of corticosterone replacement in the drinking water (Landfield et al., 1981). Although the primary focus has been upon the glucocorticoids secreted in response to stress for their deleterious effects on hippocampal neurons, there are other hormonal factors which may be involved, namely, the elevation of pituitary hormones like ACTH and Pendorphin in adrenalectomized animals. ASa result, Landfield et al. (1981), Rigter et al. (1984) and Landfield (1987) described experiments in which some sparing of brain aging was obtained by treating aging Fisher or Wistar rats with Org 2766, a long-acting peptide analog of ACTH(4 - 9). The Landfield studies showed improvement in maze learning and hippocampal neuroanatomical markers after 9 months of treatment with Org 2766, whereas Rigter et al. (1984) used only 2 weeks of Org 2766 treatment and did not alter behavioral responses but did increase binding of 3H corticosterone to receptors in hippocampus above the reduced levels normally seen in the 26-month-old animal. In another study, a 2-week treatment with ginsenoside, a plant extract used in the Orient, caused type I (mineralocorticoid) receptor levels to increase in the aging rat hippocampus without affecting type I1 receptor levels; no studies were carried out on morphological or behavioral measures of aging (DeKloet et al., 1987). Another approach to retarding the aging process has been to use the naturally occurring relative of acetylcholine, acetyl-L-carnitine (ALCAR). In one study already cited above, ALCAR treatment for 7

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months retarded the loss of 3H corticosterone uptake in the brains of 28-month-old Wistar rats (Patacchioli et al., 1989). In another study, a 6month treatment of Sprague Dawley rats with ALCAR retarded the decrease area of the hippocampal mossy fiber field in the CA3 region of Ammons horn, measured at 22 months of age, and increased the density of the Timm’s stain for zinc in the mossy fibers relative to 22-month-old control rats not given ALCAR (Ricci et al., 1989). Besides ACTH analogs, ginsenoside and acetyl-Lcarnitine, more holistic, natural manipulations have been carried out in an attempt to alter aging by influencing the pituitary-adrenal axis. Dietary restriction has been proposed as a means of slowing the aging process, and food restriction does appear to increase longevity in rats and other species (Barrows and Kokkonen, 1978). Two studies have shown that food restriction counteracts neuroendocrine and neuroanatomical signs of aging. In a neuroendocrine study, life-long food restriction to 60% of ad libitum food intake in Fisher 344 rats resulted at 24 months of age in a reduction of corticosterone levels over 24 h; these results appear paradoxical because food restriction did increase glucocorticoid output in anticipation of food, but the long-term effect of the treatment over many months actually decreased total 24 h corticosterone production even though the AM corticosterone levels continued to be elevated by the food restriction (Stewart et al., 1988). These results are consistent with the glucocorticoid cascade hypothesis by showing that a procedure which slows aging also lowers the average daily exposure to glucocorticoids later in life. In a Golgi study on neurons in parietal cortex, male Wistar rats showed a loss of dendritic spines at 24 and 30 months of age compared to 6-month-old animals; food restriction by every-other-day feeding prevented the loss of spines at 24 months when food restriction had started at 19 months and at 30 months when food restriction had begun at weaning (Moroi-Fetters et al., 1989).There was no indication in this study of what happens to neurons in the hippocampus under these circumstances. Moreover, one can only speculate, in light of the neuroen-

docrine results of Stewart et al. (1989), that food restriction-induced reduction in the loss of spines during aging might be the indirect consequence of reduced overall exposure to glucocorticoids. Another means of bringing about a reduced overall secretion of glucocorticoids is the procedure of “handling” of newborn rats, which is a gentle procedure of stimulating and mildly stressing them at regular intervals over the first several weeks after birth (e.g., Levine et al., 1967). This procedure results in animals which show lower levels of corticosterone in response to stress and more efficient shut-off of the secretion following acute stress at 6, 12and24monthsofage(Meaneyet al., 1988,1991). Not only is neuroendocrine aging retarded by neonatal handling, but also the decline of cognitive ability and the loss of hippocampal neurons are retarded in both male and female Long Evans rats by neonatal handling (Meaney et al., 1988, 1991). These findings are perhaps the most comprehensive and convincing data in support of the glucocorticoid cascade hypothesis. Moreover, the fact that the Meaney et al. studies were conducted on males and females revealed that both sexes undergo agerelated loss of hippocampal neurons, HPA regulation and spatial learning ability and that both sexes benefit from the effects of neonatal handling. Conclusions The glucocorticoid cascade hypothesis of stress and aging has stimulated much research on the role of the HPA axis and involvement of the hippocampus in the aging process. Some of the basic features of the hypothesis have been supported, while others need to be revised on the basis of new information. There are five features of the results which stand out. First, as a rat ages, there is a general decline in the plasticity of the brain to respond to environmental challenges. Not only is the capacity to produce hormones in the HPA axis reduced with aging, but so is also the ability to shut off their secretion in the aftermath of at least certain types of stressors. The enhancement as well as the habituation of the HPA

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axis to stress, depending on novelty and repetition, respectively, appears to be somewhat impaired in aging rats. Likewise, aging rats show impaired neural adaptive responses to environmental challenge, e.g., less angiogenesis in response to stimuli which cause neural growth, less down-regulation of glucocorticoid receptors in hippocampus to repeated stress, and less modulation of LTP in hippocampus by repeated stress. Second, with increasing age, there is increased vulnerability to stress-induced loss of hippocampal neurons. This may be due to such factors as the higher glucocorticoid receptor levels as a result of lesser down-regulation by the repeated stress or the greater efficacy in aging rats of glucocorticoids to affect calcium mobilization. Third, the original glucocorticoid cascade hypothesis proposed that hippocampal adrenal steroid receptor down-regulation by stress is part of the process which gives rise to glucocorticoid hypersecretion. Moreover, the original hypothesis held that down-regulation was a rather normal part of repeated stress. Based upon more recent information, such down-regulation appears to apply only in cases of very severe and prolonged stress. Thus, under normal circumstances, glucocorticoid receptors need to be present to mediate adaptive processes and shut-off of glucocorticoid secretion. However, excess glucocorticoids eventually can cause neural damage, but only under extreme conditions. Since extreme conditions of repeated stress can evidently lead to receptor down-regulation, this downregulation could be regarded - as in the original version of the glucocorticoid cascade hypothesis as a line of defense which reduces the impact of circulating glucocorticoids to damage the brain. As noted above, the potential to show hippocampal receptor down-regulation as a result of severe stress appears to decline as the rat ages; thus, as it ages, the brain is becoming less adaptable to handling extreme stress; indeed, as noted above, the aging brain appears to be more susceptible to neuronal loss from prolonged repeated stress, according to the work of Landfield and coworkers.

Fourth, there are important individual differences within a rat population, with a clustering of characteristics that indicate declining hippocampal function, impaired HPA shut-off, and damage to hippocampal neural structure. Studies of age-related changes in choline uptake, choline acetyltransferase activity and monoamines such as dopamine and noradrenaline have also shown that the behaviorally-impaired rats show the neurochemical changes (Gallagher and Pelleymounter, 1988; Gallagher et al., 1990). Such individual differences may help to explain some inconsistencies across studies to the degree to which there are reported age-related impairments in hippocampal structure, HPA activity, neurochemistry and spatial ability. These individual differences may have their origin in developmental events, as revealed by the neonatal handling studies of Meaney and coworkers. Fifth, procedures which have been reported to alter the rate of brain aging include dietary restriction and the application of the fragments of ACTH molecule and acetyl-L-carnitine, as well as neonataI handling of rat pups. There are indications that dietary restriction and neonatal handling may both reduce the overall exposure of the hippocampus of glucocorticoids and thus reduce the “wear and tear” which these steroids exert on this important brain structure during the lifetime of the individual animal. In conclusion, judging from the last 5 years, the glucocorticoid cascade hypothesis of stress and aging has served well as a stimulus and guide for research on endocrine factors in the aging of the brain, but much more needs to be learned to uncover basic cellular mechanisms and to account for the sometimes disturbing variability among studies and among individual animals in their susceptibility to negative effects of stress and in degenerative changes in the brain. Acknowledgements Work in the author’s laboratory referred to in this review is supported by NIMH Grant MH 41256.

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381 Pfeiffer, A., Barden, N. and Meaney, M. (1991) Age-related changes in glucocorticoid receptor binding and mRNA levels in the rat brain and pituitary. Neurobiol. Aging, 12: 475 - 419. Rachmanin, G., Luttge, W., Hunter, B. and Walker, D. (1989) Neither chronic exposure to ethanol nor aging affects type I or type I1 corticosteroid receptors in rat hippocampus. Exp. Neurol., 106: 164- 171. Reul, J.M. and DeKloet, E.R. (1985) Two receptor systems for corticosterone in rat brain: microdistribution and differential occupation. Endocrinology, 117: 2505 - 25 11. Reul, J., Tonnaer, J . and DeKloet, E. (1988) Neurotrophic ACTH analogue promotes plasticity of type I corticosteroid receptor in brain of senescent male rats. Neurobiol. Aging, 9: 253 - 260. Ricci, A., Ramacci, M.T., Ghirardi, 0. and Amenta, F. (1989) Age-related changes of the mossy fiber system in the rat hippocampus: effect of long term acetyl-L-carnitine treatment. Arch. Gerontol. Geriatr., 8: 63 - 71. Riegle, G . (1973) Chronic stress effects on adrenocortical responsiveness in young and aged rats. Neuroendocrinology, 11: 1-10. Riegle, G . and Hess, G. (1972) Chronic and acute dexamethasone suppression of stress activation of the adrenal cortex in young and aged rats. Neuroendocrinology, 9: 175 - 187. Rigter, H., Veldhuis, H. and DeKloet, R. (1984) Spatial learning and the hippocampal corticosterone receptor system of old rats: effect of the ACTH(4-9) analogue ORG 2766. Brain Res., 309: 393 - 398. Sapolsky, R. (1990) Glucocorticoid hippocampal damage and the glutamatergic synapse. In: P.D. Coleman, G.A. Higgins and C.H. Phelps (Eds.), Molecular and Cellular Mechanisms of Neuronal Plasticity in Normal Aging and Alzheimer’s Disease - Progress in Brain Research, Vol. 86, Elsevier, Amsterdam, pp. 13 -23. Sapolsky, R., Krey, L. and McEwen, B.S. (1983a) Corticosterone receptors decline in a site-specific manner in the aged rat brain. Brain Res., 289: 235 - 240. Sapolsky, R., Krey, L. and McEwen, B.S (1983b) The adrenocortical stress-response in the aged male rat: impairment of recovery from stress. Exp. Gerontol., 18: 55 - 64. Sapolsky, R., Krey, L. and McEwen, B.S. (1984a) Glucocorticoid-sensitive hippocampal neurons are involved in terminating the adrenocortical stress response. Proc. Natl. Acad. Sci. U.S.A., 81: 6174-6177. Sapolsky, R., Krey, L. McEwen, B.S. and Rainbow, T. (1984b) Do vasopressin-related peptides induce hippocampal corticosterone receptors? Implications for aging. J . Neurosci., 4: 1479- 1485. Sapolsky, R., Krey, L. and McEwen, B.S. (1985) Prolonged glucocorticoid exposure reduces hippocampal neuron

number: implications for aging. J. Neurosci., 5 : 1222- 1227. Sapolsky, R., Krey, L. and McEwen, B.S. (1986a) The neuroendocrinology of stress and aging: the glucocorticoid cascade hypothesis. Endocr. Rev., 7: 284 - 301. Sapolsky, R., Krey, L. and McEwen, B.S. (1986b) The adrenocortical axis in the aged rat: impaired sensitivity to both fast and delayed feedback inhibition. Neurobiol. Aging, 7: 331-335. Sapolsky, R., Uno, H., Rebert, C. and Finch, C. (1990) Hippocampal damage associated with prolonged glucocorticoid exposure in primates. J. Neurosci., 10: 2897- 2902. Scaccianoce, S., Sciullo, A. and Angelucci, L. (1990) Agerelated changes in hypothalamo-pituitary-adrenocorticalaxis activity in the rat. Neuroendocrinology, 52: 150- 155. Siesjo, B. and Bengtsson, F. (1989) Calcium fluxes, calcium antagonists and calcium-related pathology in brain ischemia, hypoglycemia and spreading depression: a unifying hypothesis. J. Cereb. Blood Flow Metab., 9: 127- 140. Sloviter, R. (1983) “Epileptic” brain damage in rats induced by sustained electrical stimulation of the perforant path. I. Acute electrophysiological and light microscopic studies. Brain Res. Bull., 10: 675 - 697. Sloviter, R., Valiquette, G . , Abrams, G., Ronk, E., Sollas, A., Paul, L. and Neubort, S. (1989) Selective loss of hippocampal granule cells in the mature rat brain after adrenalectomy. Science, 243: 535 - 538. Sonntag, W., Goliszek, A , , Brodish, A. and Eldridge, J.C. (1987) Diminished diurnal secretion of adrenocorticotropin (ACTH), but not corticosterone, in old male rats: possible relation to increased adrenal sensitivity to ACTH in vivo. Endocrinology, 120: 2308-2315. Stewart, J., Meaney, M., Aitken, D., Jensen, L. and Kalant, N. (1988) The effects of acute and life-long food restriction on basal and stress-induced serum corticosterone levels in young and aged rats. Endocrinology, 123: 1934- 1941. Swaab, D.F. (1991) Brain aging and Alzheimer’s disease “wear and tear” versus “use it or lose it”. Neurobiol. Aging, 12: 317 - 324. Tang, F. and Phillips, J. (1978) Some age-related changes in pituitary-adrenal function in the male laboratory rat. J. Gerontol., 33: 377 - 382. Uno, H., Ross, T., Else, J., Suleman, M. and Sapolsky, R. (1989) Hippocampal damage associated with prolonged and fatal stress in primates. J. Neurosci., 9: 1705 - 1711. Van Eekelen, A,, Rots, N.Y., Sutanto, W. and DeKloet, E.R. (1992) The effect of aging on stress-responsiveness and central corticosteroid receptors in the Brown Norway rat. Neurobiol. Aging, 13: 159- 170. Woolley, C., Could, E. and McEwen, B.S. (1990) Exposure to excess glucocorticoids alters dendritic morphology of adult hippocampal pyramidal neurons. Brain Res., 53 1: 225 - 231.

Discussion E. Goudsmit: I was a little puzzled by the Landfield experiment which showed a loss of corticosteroid receptor down-regulation in aged rats. The control groups in this experiment showed an increase in receptor number with aging. Does this refute the results by, e.g., Reul et al. (1988) on a loss of corticosteroid receptors with aging? Might this increase in receptor number be related to the fact that these aged animals respond to stress with cell loss, in contrast to the young rats? B.S. McEwen: You have sharp eyes. Landfield’s group (Eldridge et al., 1989) used intact rats, not ADX rats, because you can measure type I1 receptors without adrenalectomy and avoid the complications associated with adrenalectomy. They probably ran binding assays on their different age groups at different times, so that, in looking at their results, comparisons across ages may not be as meaningful as the effects of stress at each age. Moreover, unlike the Meaney group, they did not evaluate the cognitive or neuroendocrine state of their rats in relation to binding results, i.e., rats that show lowest receptor levels are also the most impaired cognitively and in terms of shut-off of corticosterone secretion after stress. So I would not abandon the general finding in many laboratories that type I1 and also type I receptors decline with age and cognitive and neuroendocrine impairment in the hippocampus of rats. And, yes, I would underscore your point about failure to downregulate with stress in age, as Eldridge et al. (1989) show, as increasing the vulnerability of the hippocampus to corticosteroidinduced cell loss. R. Ravid: (1) What is the evolutionary advantage of stress in aging? (2) Is there any evidence for different prevalence of Alzheimer’s disease in patients suffering from pathological adrenal condition? B.S. McEwen: (1) That is an impossible question to answer there is certainly no advantage as far as reproduction, because that has presumably occurred earlier in the life span. (2) No study has yet related adrenal size or corticosterone levels in a longitudinal way to the time course or prevalence of Alzheimer’s disease. D.F. Swaab: A comment to the start of your lecture. The salmon has often been used to illustrate the importance of the adrenal in premature aging. I understood, however, that the salmon is dying from an explosive increase in parasites that they carry with them and consequently are liable to die because of the activation of the adrenal. This means it would be death by disease and it does not have anything to do with premature aging. B.S. McEwen: Migrating salmon die after first spawning from causes related to toxic levels of adrenal corticosteroids, and this is prevented by castration before maturity (reviewed by Finch, 1976). Failure to resist parasites as well as involution of the gastrointestinal tract, thus preventing nutrition, may be among the actual pathophysiological causes of death. Males of the

marsupial mouse, Antechinus, also die after mating due to toxic levels of adrenal corticosteroids brought about by high androgen levels as a consequence of a mating frenzy (Cockburn and Lee, 1988). Gastrointestinal ulcers and immunosuppression are among the primary causes of death. M. Mirmiran: (1) There is a profound diurnal circadian variation in plasma levels of corticosterone and levels of CRF in CSF; do you think that there will be concomitant changes in density or affinity of glucocorticoid receptors as a function of time of day? (2) You have indicated individual differences during aging in stress-response deficiency. There is also evidence for individual differences in cognitive functions in elderly people. Do you think that the two phenomena are causally related? B.S. McEwen: ( I ) Type I and type I1 receptors are occupied to varying degrees as a function of the diurnal variation of circulating glucocorticoids, and this will produce effects on their distribution within the cell (i.e., occupied receptors tend to be in the cell nucleus). Diurnal variations in type I and type I1 receptor mRNAs have been reported in regions of the hippocampus and other brain areas (Herman et al., 1991). (2) I am sure there are individual differences in humans in aging and cognitive function, but I do not know whether they are causally related to changes in adrenal function as they appear to be in the rat. N. Kopp: Comment: There is a paper showing in magnetic resonance imaging anomalies of the hippocampus in schizophrenia and depression (Swayze et al., 1990). E.R. DeKloet: In your concluding statement you mentioned that the glucocorticoid cascade hypothesis has been useful to examine how glucocorticoids, stress and the hippocampus are linked in the aging process. Based on the new data you presented do you think it is still tenable that the action of glucocorticoids in promoting brain aging can be generalized? B.S. McEwen: Generalized to what? To humans, maybe (see answer to question by Dr. Mirmiran). But what I think you are referring to is whether the glucocorticoid influence can be generalized across individuals in a rat or human population? The enormous individual variations in aging documented especially well for the rat by the group of Michael Meaney point to the role of experience from early life onwards in determining the rate of aging. In my response to Dr. Swaab’s comment, I noted the role of adrenal steroids in terminating the life span of the salmon and the marsupial mouse - if these animals are castrated or (in the case of the salmon) prevented from migrating, then the environmental causes of the adrenocortical hyperfunction are removed, and the animals will survive longer. From this I think we have to conclude that adrenal steroid influences on aging are not inevitable, although some wear and tear may be an inevitable part of life (McEwen, 1991; Swaab, 1991). In fact, we need our adrenal cortex to survive. But individual experiences, particularly stressful ones, may have an accelerating function. J.J.F. Taljaard: Could you please comment on prolonged in-

383 crease of cortisol in many major depressive episode patients and how that could differ from Sapolsky’s experiments with intermittent corticosterone increase. B.S. McEwen: Prolonged hypercorticism in depression may well alter brain structure but there have been as yet no studies with MRI on CI which have specifically looked at the vulnerable areas of the brain such as the hippocampus. These are important studies to do.

References Cockburn, A. and Lee, A.K. (1988) Marsupial femmes fatales: males of a small Australian mammal species pay a high price for fatherhood. Natural History, 97: 41 -46. Eldridge, J.C., Fleenor, D., Kerr, D.S. and Landfield, P . (1989) Impaired up-regulation of type I1 corticosteroid receptors in hippocampus of aged rats. Brain Res., 478: 248 - 256. Finch, C.E. (1976) The regulation of physiological changes during mammalian aging. Q.Rev. Biol., 51: 49- 83.

Herman, J.P., McEwen, B.S., Chao, H., Coirini, H. and Watson, S.J. (1991) Diurnal rhythms of glucocorticoid and mineralocorticoid mRNA expression in the hippocampal formation: regional specificity and steroid dependence, SOC. Neurosci. Abstr., 17: 620.2. McEwen, B.S. (1991) When is stimulation too much of a good thing? Neurobiol. Aging, 12: 346- 348. Red, J.M.H.M., Tonnaer, J.A.D.M. and DeKloet, E.R. (1988) Neurotrophic ACTH analogue promotes plasticity of type I corticosteroid receptor in brain of senescent male rats. Neurobiol. Aging, 9: 253 - 260. Swaab, D. (1991) Brain aging and Alzheimer’s disease: “wear and tear” versus “use it or lose it”. Neurobiol. Aging, 12: 317 - 324. Swayze, V.W., Andreasen, N.C., Alliger, R.J., Ehrhardt, J.C. and Yuh, W.T.C. (1990) Structural brain abnormalities in bipolar affective disorders; ventricular enlargement and focal signal hyperintensities. Arch. Gen. Psychiatry, 47: 1054- 1059.

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D.F. Swaab, M . A . Hofman, M. Mirmiran, R. Ravid and F.W. van Leeuwen (Eds.) Progress in Bruin Research, Vol. 93

0 1992 Elsevier Science Publishers B . V . All rights reserved.

385 CHAPTER 21

The role of corticotropin-releasing hormone in the pathogenesis of Cushing's disease, anorexia nervosa, alcoholism, affective disorders and dementia Florian Holsboer, Dietmar Spengler and Isabella Heuser Max Planck Institute of Psychiatry, Clinical Institute, 0-8000Munich 40, Germany

Introduction

More than 40 years ago Harris (1948) proposed that pituitary adrenocorticotrophic hormone (ACTH) secretion might be regulated by humoral factors released from the hypothalamus. The first support for this historic suggestion, which defined the hypothalamus as a key link between the nervous system and the endocrine systems in reaction to the environment, was provided by Saffran et al. (1955) and Guillemin and Rosenberg (1955). These investigators used crude extracts of the stalk median eminence to demonstrate the presence of an ACTHreleasing component, which was initially named corticotropin-releasing factor (CRF) and is now usually called corticotropin-releasing hormone (CRH). Ten years ago the group led by Vale succeeded in determining the structure of ovine CRH and in synthesizingthe peptide according to the postulated structure (Vale et al., 1981). Soon after, the amino acid sequence of human CRH was deduced after sequencing of the CRH gene located on chromosome 8 (Furutani et al., 1983; Shibahara et al., 1983; Arbiser et al., 1988). These studies demonstrated that the 41-amino-acid straight-chain peptide sequence is highly conserved across a number of species. Human CRH has a 100% homology with rat CRH and an 83% homology with ovine CRH, differing by seven amino acid residues. The past 10 years have

been marked by a surge of activity in basic and clinical research aimed at elucidating the role of CRH in stress physiology and in the pathophysiology of disorders of the hypothalamic-pituitaryadrenocortical (HPA) system. The purpose of this chapter is to review the accumulated data and to demonstrate that a broad concept of the role of CRH as a mediator of adaptive response to all forms of stress has now emerged. Functional neuroanatomy and regulation of the CRH neuron

Central nervous system distribution of CRH The identified and synthetically available CRH41 molecule is present in the median eminence, can be measured in hypothalamic-pituitary venous blood and stimulates specifically ACTH and other proopiomelanocortin (P0MC)-derived compounds from the corticotrophic anterior pituitary cells (Rivier and Plotsky, 1986). Thus, it fulfills many of the criteria for a CRH. However, systematic studies have shown it to be less potent in releasing ACTH than the crude hypothalamic extracts or less specific stressors such as hypoglycemia. Consequently, the full corticotropin-releasing potency includes secretagogues other than CRH. As is discussed later in more detail, the major co-releaser of ACTH, potentiating or synergizing the effect of CRH, is vaso-

386

pressin, and, under certain conditions, also oxytocin. The distribution of these neuropeptides and other neurotransmitters involved is important to an understanding of the full scope of CRH effects in the central nervous system (CNS). In keeping with Harris’s concept, HPA activity is regulated by numerous afferent pathways that relay information about the internal and external environment and that converge in the basal hypothalamus. There the input is transformed into a multifactorial humoral signal by neurons of the parvicellular division of the nucleus paraventricularis (PVN). These neurosecretory cells, projecting to the median eminence, from where the neuropeptides are released into the portal system, are the primary source of CRH, arginine vasopressin (AVP) and other possible regulators of pituitary-adrenocortical activity. Furthermore, other cell divisions (magnocellular and descending neurons) of the PVN contain CRH and AVP. However, they appear not to be involved in neurohumoral adaptation to stress. This conclusion is derived from the observation that corticosterone, the major negative feedback signal in the rat, suppresses messenger ribonucleic acid (mRNA) of CRH and AVP only in the parvicellular neurons, whereas the same steroids may increase the CRH gene transcription in descending projections, in certain magnocellular neurosecretory neurons, and in a part of the central nucleus of the amygdala, or may leave it unaffected as in the rostra1lateral hypothalamic area. Thus, in different types of CRH neurons, corticosterone may increase, decrease or have no influence on CRH gene transcription (Swanson and Simmons, 1989). Other regions of the rat brain containing relatively high concentrations of CRH were found in the prefrontal cortex, nucleus amygdala, olfactory bulb, certain thalamic nuclei, locus coeruleus (LC) and certain areas of the cerebellum (Swanson et al., 1983). These localizations of CRH immunoreactivity point to a rather diverse role of CRH and provide an anatomical basis for understanding the endocrine and non-endocrine effects involved in the mediation of a concerted adaptation program to internal and external challenges. Of particular func-

tional significance is the occurrence of two types of paraventricular CRH neurosecretory cells: AVPcontaining and AVP-deficient ones (Whitnall et al., 1985, 1987). There is evidence that differential activation of these subpopulations of CRH axons provides a mechanism by which the levels of CRH and AVP in portal plasma can be fine-tuned independently (Whitnall, 1989). Differential activation from centers above the hypothalamus determines the portal CRH: AVP ratio and AVP secretory neurons specifically mediate the ACTH response to short-term stress. The relative amount of CRH and AVP determines the effect on corticotrophic cells because theeffect of CRH is strongly potentiated by AVP (Gillies et al., 1982).

Release of CRH and its negative feedback control At the corticotrophic level the stimulatory effect of CRH on ACTH release is initiated by high-affinity (KD = 0.1 - 0.5 nM) receptor binding and subsequent activation of adenylate cyclase via a stimulatory G-protein. The concept that intracellular cyclo-adenosine-monophosphate (CAMP) is the major signal for ACTH release is supported by studies in which forskolin and choleratoxin, both activators of CAMP, were found to release ACTH from cultured pituitary cells (Aguilera et al., 1983). Intracellular signaling of membranous AVP-receptor binding is mediated by phospholipase C activation. This effect can be mimicked by phorbol esters, which directly activate protein kinase C. At which level the synergizing effect between AVP and CRH occurs is still unclear, and it has been suggested that there is a separate mechanism or cell type for AVPstimulated ACTH secretion distinct from that responsible for CRH-stimulated ACTH release. The synergy of both secretagogues possibly involves a paracrine action (Schwartz and Vale, 1988). The regulation of CRH- and AVP-mediated effects on the synthesis and release of POMC-related peptides is extremely complex and involves neural and neurohormonal factors, which are summarized in Fig. 1 . Of central importance in the regulation of ACTH is the negative feedback by adrenal glucocorticoids, which primarily act at the level of the hip-

387

Fig. 1. External cognitive (e.g., psychosocial stressor) or non-cognitive (e.g., infective) stimuli and emotional disturbances arising from brain dysfunctions that underly affective syndromes or panic anxiety all activate various brain areas, which finally use the hypothalamus as a relay station to translate the brain activity pattern into changes of autonomic system function and hormonal secretion. While CRH is the permissive key hormone activating the HPA system many other transmitters and peptides serve to modulate dynamically the secretory activity of pituitary corticotrophic cells. At each level immunopeptides including interleukins derived from glia tissue (not shown in this figure) are involved in the regulatory circuits. Abbreviations: MR, mineralocorticosteroid receptor; GR, glucocorticosteroid receptor; CRH, corticotropin-releasing hormone; CCK, cholecystokinin; VIP, vasointestinal peptide; ANP, atrial natriureticpeptide; BBB, blood-brain barrier; ACTH, corticotropin; IFN, y,interferongamma; IL1,2,6, interleukin 1,2,6; TNF, tumor necrosis factor.

388

pocampus, hypothalamus and pituitary over multiple time domains. The existence of feedback effects is not apparent after adrenalectomy, which is followed by enhanced CRH and AVP in the parvocellular division of the PVN along with induction of the immediate early c-fosgene in these cells (Dallman et al., 1985; Jacobsen et al., 1990). In the portal plasma CRH and AVP are increased and corticotrophic POMC mRNA and peripheral plasma levels of ACTH and other POMC-derived products are enhanced. These changes are reversed by glucocorticoid replacement. Under physiological conditions the adrenal corticosteroids and their central receptors play a critical role in determining the set-point of initiation and termination of the pituitary-adrenocortical response to stress (De Kloet, 1991). Different kinds of stress, e.g., foot-shock, audiovisual stress or insulin-induced hypoglycemia, lead to different relative quantities of AVP and CRH in the portal circulation and differential suppression by glucocorticoids (Canny et al., 1989). The negative feedback mechanism of corticosteroids on CRH and other central elements of the HPA system is fundamental for our understanding of clinical findings in endocrine disorders and in mental illness and is therefore discussed here in some detail. The combined genomic and non-genomic effects of adrenal steroids result in three distinguishable modes of negative feedback action: (1) fast feedback (up to 10 min) including the control of CRH, AVP and other ACTH secretagogues from the median eminence; (2) intermediate feedback (up to 1 - 2 h) involving gene-mediated steroid effects on stimulus-secretion coupling, excitability and intracellular signal transduction pathways; and (3) slow feedback (several hours) including suppression of stress-induced CRH and AVP gene expression in the parvocellular PVN and POMC gene expression in corticotrophic cells. These mechanisms are primarily exerted via intracellular glucocorticoid receptors which after hormone-ligand binding transform into transcription factors and act directly or indirectly at the CRH and AVP gene. Of course, they may also act on other genes that are involved in

related transcriptional and post-transcriptional processes. Pharmacological studies indicate the presence of two distinct receptor systems, the mineralocorticosteroid receptor (MR) and the glucocorticoid receptor (GR). Whereas GRs are distributed throughout the brain, MRs predominate in the hippocampus, where the receptors are colocalized in the same neurons. Both receptors bind corticosterone, but MRs with a 6 - 10-fold higher affinity than GRs. Whereas the MR occupancy varies between 70 and 90%, that of GR varies between 10 and 90% in parallel with changing levels of corticosterone. Thus, steroid binding at MRs varies over a much lower range than that at GRs. Occupation of hippocampal MRs maintains excitability and limbic inhibitory control over the HPA system. GR activation induced by higher levels of corticosterone during stress or circadian surges counteracts this MR-driven effect. These antagonistic effects of MRs and GRs on neuronal excitability make the degree of hippocampal inhibition of the HPA drive dependent on the level of circulating corticosteroids: higher hormone levels correspond with an increased number of occupied GRs relative to MRs and a decreased inhibitory influence on parvocellular PVN neurons. Under physiological conditions this limbic disinhibition is counterregulated by GR-mediated effects on CRH and AVP synthesis and release from the hypothalamus and POMC gene expression in the anterior pituitary. The antagonistic effect of hippocampal MRs may block the inhibiting effect under baseline conditions, thus enhancing the HPA responsiveness to stimulation. On the other hand, GR antagonism may prolong HPA overactivity because the system becomes unable to initiate negative feedback at the level of the hypothalamus and pituitary. Ratka et al. (1989) developed this hypothesis after applying the specific antimineralocorticoid RU 283 18 and the antiglucocorticoid RU 38486. If their hypothesis is correct and if we assume that the MR/GR concept formulated by De Kloet (1991) holds true in humans, an increased number of MRs in the hippo-

389

campus relative to GRs would reduce the reactivity to stressors. If so, this would provide a possible lead for development of stress-dampening drugs designed to stimulate limbic MRs. Such drugs could have a clinical potential for anxiolysis without the simultaneous adverse effects currently limiting the application of anxiolytics such as the benzodiazepines.

Neurotransmitter control of CRH neurons Major afferents to the PVN originate from (1) ascending mono- and polysynaptic pathways, from brain-stem nuclei and (2) descending pathways from the cortex, hippocampus and amygdala through the bed nucleus of the stria terminalis. The neurotransmitter effects on CRH were reviewed by Antoni et al. (1986) and are briefly summarized in Table I. We discuss here only those neurotransmitter effects directly related to the clinical chapters of this volume. In the PVN, norepinephrine (NE) terminals projecting from the LC are present on CRH-producing neurons. Conversely, increased density of CRH neurons was detected at the LC, pointing to a bidirectional interaction. In fact, CRH elevates norepinephrine and this, in turn, has an excitatory effect on CRH neurons that occurs at dosages of 0.1 -0.5 nM via al-adrenoceptors. At very high

dosages (10 - 50nM) the effect of NE on hypothalamic CRH is reversed, and this inhibition is mediated via a2- and @-receptors(Plotsky, 1987). Corticosteroids themselves have a variety of effects on adrenoceptors, thus influencing the CRH neuron activity by this route also. For example, adrenalectomy decreases and corticosterone administration increases a2-adrenoceptor density, but the al-adrenoceptors remain unaffected (Jhanwar-Uniyal and Leibowitz, 1986). Another effect is that glucocorticoids enhance @-adrenoceptorsynthesis, and still another is that glucocorticoids enhance @-adrenoceptor capacity following dorsal bundle lesions (Roberts and Bloom, 1981). The raphe nuclei, where GRs are abundantly present, innervate frontal brain regions including the hippocampal target neurons containing MRs and GRs. Most evidence supports the hypothesis that serotonin, produced in these nuclei, is an excitatory neurotransmitter on the CRH neuron acting through 5HTlA- and 5HT2-receptors. Glucocorticoids themselves have an effect on 5HT-receptors because adrenalectomy increases 5HT1-receptor density in the dorsal raphe nucleus and hippocampus (CAI region), and corticosterone replacement compensates for this effect (De Kloet et al., 1986). Acetylcholine is also a stimulatory neurotransmitter acting through both the nicotinergic and

TABLE I Neurotransmitter regulation of hypothalamic CRH release Transmitter Acetylcholine Angiotensin IJ Dynorphin 1 - 13 P-Endorphin Epinephrine GABA Norepinephrine Norepinephrine Serotonin

Dose (nmol)

CRH

Receptor specificity

0.1 - 10.0

t t

Muscarinic/nicotinic Block with saralasin x/P

0.1 - 1.0

0.1 - 10.0 0.1 - 10.0 0.1 - 30.0 0.1- 1.0 0.1- 5.0 10.0- 50.0 0.1 - 10.0

+ +

1

Ir.

t

I

a,-/P-Adrenergic Block with bicuculline a -Adrenergic a2-/P-Adrenergic

T

~HTIA

1

I

390

muscarinergic receptor systems. In turn, glucocorticoids seem to stimulate acetylcholine release in the hippocampus (Gilad et al., 1987). Regulation of the human CRH (hCRH) gene promoter by CAMP and glucocorticoids

Structure and organization of the hCRH gene A comparison of the hCRH gene (Shibahara et al., 1983) with the cloned rat (Thompson et al., 1987) and sheep gene (Roche et al., 1988) reveals a similar structural arrangement and extensive sequence homology. The human, rat and sheep CRH genes each contain one intron of 800, 700 and 744 nucleotides (nt) respectively. The protein coding and the 3 ’ untranslated region of the human preproCRH mRNA is uninterrupted. The 3 ’ untranslated region of the human prepro-CRH mRNA contains two copies of the sequence AUAAA (Shibahara et al., 1983), which serve as targets for poly(A) addition to enhance mRNA stability. The human CRH intron starts with a GT and ends with an AG dinucleotide, following the rule of exonintron boundaries. The nucleotide sequences at the boundaries are consistent with the splicejunction sequences observed for other genes and are conserved in the human and rat gene. The human intron provides additional putative donor and acceptor sequences (for example residues - 712 to - 704 and - 250 to - 242 as donors; residues - 350 to - 355 and -205 to - 190 as acceptors) for alternative mRNA splicing (Shibahara et al., 1983). However, in vitro studies of different brain tissues revealed so far only a single mRNA species of about 1400 bp in the rat (Thompson et al., 1987). Interestingly, CRH transcripts in the adrenal rat and testis are longer by about 200 and 500 bp, respectively, and provide evidence for additional transcription initiation sites in peripheral tissues. Tissue-specific differences have been reported for a number of other genes including the POMC (Drouin et al., 1989) and the rat prodynorphin gene (Civelli et al., 1985). Human prepro-CRH consists of 196 amino acid residues. The sequence of the amino-terminal 24 amino acid residues displays characteristics of the

signal peptide of secretory proteins. A possible site for cleavage of the signal peptide of human preproCRH is located after the alanine residue at position 24 (Shibahara et al., 1983). The functional importance of additional putative cleavage products during the release of the 41 amino acid residues presenting CRH is unknown. The expression of eukaryotic genes is controlled through cis-acting DNA elements flanking a gene coding region. For genes transcribed by RNA polymerase I1 these elements include the promoter, upstream regulatory elements and enhancers, which may be located in a position- and orientation-independent manner in the transcription unit. Interestingly the promotor region of the three cloned CRH genes is more conserved than their protein coding region. The 5 ’ flanking region of the human, rat and sheep gene share 90% nucleotide homology from the transcriptional start site to 350 nt upstream. In contrast, the overall protein regions reveal 80% homology. The homology of the promoter region is evenly distributed, with a maximum of 98% for the nucleotide sequence - 303 to - 155 upstream of the start site. In contrast, numerous other genes subjected to transient and/or tissue-specific expression lack such highly conserved promoter regions. The POMC gene, for instance, is expressed strictly in a tissue-specific manner, but there is only 75% and 70% homology between the promoter region (350 nt) of the human and bovine and human and mouse genes, respectively. The high conservation of CRH promoter sequences between different species suggests a strong selected pressure on these regions and possibly regulatory elements. In addition, the extensive homology between the 5 ’ flanking regions of the ovine, rat and human genes points to common cisacting DNA-elements, which interact with multiple general and/or tissue-specific transcription factors. However, the significance of this close homology and the presumed conservation of cis-acting DNA elements remains to be established. Promoters are composed of adenine (A)- and thymidine (T)-rich sequences (boxes), which are considered to establish the start site of transcription.

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The hCRH gene contains two separate regions of TATAA and CAAT box sequences. The two proximal CAAT boxes and the TATAA box are located at positions - 93, - 58 and - 23, respectively, to the capping site. In addition a distal CAAT and TATAA box are observed in positions -309 and - 203 in the human CRH promoter. Thus initiation of transcription could occur at multiple start sites as indicated by the longer mRNA transcript sizes in peripheral tissues of the rat. These promoter elements are conserved within the human, rat and sheep genes and suggest a functional significance. However, primer extension analysis of ovine hypothalamic RNA indicates that the major transcription initiation site is 30 nt downstream to the proximal CAAT box (Roche et al., 1988). Despite the highly tissue-specific expression of the CRH gene, in vitro studies based on transient transfection of a 1400bp rat or a 760 bp human CRH promoter fragment into different neuroendocrine cell lines (Seasholtz et al., 1988; Phi Van et al., 1990) achieved expression of the transfected promoter under basal and stimulated conditions. In agreement with these results Adler et al. (1987) obtained expression of a stably transfected human CRH gene INHIBITION

OF THE CRH GENE PROMOTOR BY GC TRANSFORMED GRs

s

x 100 2 c.

c

3Ac Cm ‘Ac Cm

b

z

c

e a -

50 Cm

E! CAMP IMX DEX

+ +

-

+ + 50nM

+

+ lOOnM

Fig. 2. CRH-gene promoter activity is increased by a cyclic-AMP analog and a phosphodiesterase inhibitor (IMX). The promoter is linked to a gene, coding for an enzyme that acetylates chloramphenicol (chloramphenicolacetyltransferase,CAT). The activity of the promoter is measured by the amount of chloramphenicol acetylation under various experimental conditions. As illustrated, the activity of the CRH gene-promoter can be reduced by dexamethasone in a dose-dependent mode. (From Phi Van et al., 1990.)

including 6 kb of 5 ’ flanking region in a mouse anterior pituitary (AtT20) cell line. In contrast, the expression of the transfected intact human or rat POMC promoter depends on introduction into the AtT20 cell line, which constitutively expresses the endogenous POMC gene and presumed tissue-specific transcription factors (Drouin et al., 1989). Apparently, transcription factors involved in tissuespecific expression of the CRH gene in the brain play an unknown role for the expression of the CRH promoter fragments investigated in vitro by transfection studies. Alternatively tissue-specific elements for CRH expression could reside in further upstream positions of the promoter and will require further refined studies. Regulation of the hCRH gene promoter by CAMP (Fig. 2) In the nervous system a broad variety of neuropeptides and neurotransmitters regulate cellular activity through receptors coupled to adenylate cyclase. Early studies on the regulation of neuropeptide gene expression focused primarily on the second messenger CAMP. In 1986, Montminy et al. demonstrated that somatostatin mRNA levels increased in primary diencephalic cultures after administration of forskolin, a post-receptor activator of adenylate cyclase. The DNA element responsible for CAMPregulated expression was isolated by deletion analysis of a somatostatin fusion gene (Montminy et al., 1986). The identified palindromic motif ( 5 ‘ TGAC GTCA-3 ’ ) was designated CAMP-responsive element (CRE). Subsequently, several other cellular and viral genes were reported to contain functional CRE elements (for review: Goodman, 1990; Montminy et al., 1990). In addition, two recent studies investigated the regulation of expression of the CRH gene by CAMP: Seasholtz et al. (1988) analyzed the rat CRH gene by transient transfection of a pheochromocytomaderived cell line (PC12) with a chimeric gene containing a 1.4 kb promoter fragment fused to the bacterial receptor gene chloramphenicolacetyltransferase (CAT). Cyclic AMP analogs and ac-. tivators of adenylate cyclase positively regulated the

392

expression of this chimeric gene. Similar results were obtained by Phi Van et al. (1990) on transfection of a mouse anterior pituitary cell line (AtT20) with a chimericgene composed of a 760 bp promoter fragment of the human CRH gene. By sequence comparison of the promoter region a consensus motif for a CAMP-responsiveelement was suggested at position - 221 bp relative to the capping site in the human and rat genes. The functional importance of this CRE element is indicated by the finding that upon fusion to an otherwise unresponsive heterologous promoter cAMP inducibility is achieved (Seasholtz et al., 1988; Phi Van et al., 1990). The presence of a functional cAMP element in the human and rat CRH promoter provides the molecular basis for modulation of CRH transcription in vivo by various neurotransmitter systems coupled to the adenylate cyclase via stimulatory or inhibitory G-proteins (Gs and Gi). Agonist-induced activation of the Gs-adenylate cyclase complex results in the release of CAMP, which binds to protein kinase A. The activated protein kinase A phosphorylates in turn the prebound cAMP response element binding protein (CREB) and leads to an increased transcription rate of the target gene (for review: Goodman, 1990; Montminy et al., 1990). Modulation of CRH gene transcription by neurotransmitter pathways in vivo is supported by the observation that unilateral surgical transection of ascending norepinephrinecontaining neurons leads to an ipsilateral decrease in paraventricular CRH content. This finding indicates that catecholamines tonically stimulate CRH synthesis (Sawchenko, 1988). Recently, Herman et al. (1989) reported that dissection of neuronal pathways from the hippocampus enhanced the expression of CRH mRNA in the PVN. These hippocampal afferents could contribute to mediate the effects of circulating steroids on CRH gene transcription via hippocampal mineralo- and glucocortico-steroid receptors. However, the precise role of this inhibitive neurotransmitter pathway and its influence on repression of hypothalamic CRH transcription remains to be established.

Repression of the hCRH gene promoter by glucocorticoids The synthesis of CRH is apotential site of the central feedback regulation by glucocorticoids. Adrenalectomy (ADX) leads to three-fold increases of CRH mRNAin the PVN, which can be prevented by administration of corticosteroids to the animal (Beyer et al., 1988). In principle, a direct humoral and/or an indirect neuronal feedback by glucocorticoids on parvicellular neurons could account for this observation: repression of CRH mRNA synthesis could result from activation of steroid receptors expressed on the CRH neuron in the PVN or indirectly via suppression of tonic neuronal stimulatory afferents from the brain-stem. In support of this view, catecholaminergic neurons in the brainstem projecting to the PVN were reported to contain a high number of glucocorticoid receptors, which modulate neuronal activity. In addition, repression of hypothalamic CRH synthesis might result from stimulation of inhibitive hippocampal afferents via hippocampal glucocorticoid and mineralocorticoid receptors (Herman et al., 1989). However, recent studies by Kovacs and Mezey (1987) suggest that ADX-induced increases in CRH mRNA in the paraventricular nucleus can be prevented by local implants of dexamethasone pellets. This observation suggests a direct impact of hypothalamic glucocorticoid receptors on CRH gene regulation. This hypothesis is strengthened by the observation that the inhibitory effect of glucocorticoids on CRH expression in the PVN is preserved in rats with brainstem hemisections that deprive PVN neurons of catecholaminergic inputs (Sawchenko, 1988). Therefore we suggest that in vivo repression of CRH gene transcription in the intact animal results from a complex interaction of brain-stem and hippocampus-derived neuronal afferents and hypothalamic corticosteroid receptors. Molecular events involved in repression of target genes by glucocorticoids are poorly understood in mammalian systems. In order to establish an in vitro model for CRH gene repression by glucocorticoids Adler et al. (1987) stably transfected AtT20 cells

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with an 8 kb human CRH clone containing the entire human CRH gene as well as 6 kb of the 5 ’ sequence. Dexamethasone (1 pM) treatment for 24 - 96 h caused a specific decrease in CRH mRNA and peptide levels of 40 - 50%. This observation indicates that repression of CRH gene transcription can occur independently of tissue-specific transcription factors and involves an interaction of the transformed glucocorticoid receptor with the transcription machinery. In agreement with this observation Phi Van et al. (1990) reported a two-fold repression of CAMP-induced CRH promoter activity of a chimeric CRH-CAT fusion gene transiently transfected in AtT20 cells. However, the hCRH promoter fragment used in this study does not exhibit any motives related to positive or negative glucocorticoid response elements (GRE) (for review: Beato, 1989), raising the intriguing question whether the observed glucocorticoid receptor-mediated effects are due to a direct interaction of the ligand-activated glucocorticoid receptor with the hCRH promoter or more indirectly via protein-protein interactions. This type of transcriptional regulatory effects have been recently described for interactions between the glucocorticoid receptor and the Fos complex (Jonat et al., 1990; Lucibello et al., 1990; Schiile et al., 1990; Yang-Yen et al., 1990). In addition, repression of target genes by glucocorticoids has been attributed to competition for common DNAbinding sites by positively acting transcription factors and the glucocorticoid receptor (Akerblom et al., 1988; for review, Levine and Manley, 1989). In conclusion, the in vitro results from stable and transient transfection experiments of the CRH promoter (Adler et al., 1987; Phi Van et al., 1990) support the hypothesis that the CRH gene in the PVN is repressed by glucocorticoid receptors expressed in the CRH neuron. However, the molecular mechanisms involved in CRH repression are still unresolved and need further investigation.

CRH receptors Using CRH and CRH analogs, De Souza and colleagues (De Souza et d., 1986; De Souza, 1987)

demonstrated the existence of a saturable, reversible ligand binding to CRH receptors with a dissociation constant (KD)in the nanomolar range (0.1 - 0.2 nM). CRH binding stimulates activation of adenylate cyclase and increases intracellular CAMP,which acts as a second messenger of CRH-mediated effects in brain and pituitary (Labrie et al., 1982; Wynn et al., 1984). Reisine et al. (1985) showed that cAMP increases are essential for CRH-induced ACTH release from pituitary adenoma cells because blockade of cAMP formation by a protein kinase inhibitor prevented the effect of CRH on POMC gene expression and release of ACTH. After adrenalectomy ACTH levels are increased because of the absence of negative glucocorticoid feedback (Wynn et al., 1985), which results in an increase release of CRH and a decrease in the number of CRH receptors at the pituitary (Hauger et al., 1988). Likewise, continuous administration of CRH decreases the pituitary CRH receptor concentration in a dosedependent mode. These changes are accompanied by a decrease in CRH-stimulated adenylate cyclase activity (Wynn et al., 1988). Adrenalectomy-induced decreases in CRH receptors are much larger than those induced by chronic CRH infusions. It is unlikely that different amounts of CRH released from the hypothalamus are responsible for these differential effects. Rather, co-release of other hypothalamic peptides after adrenalectomy may play a role. Wynn et al. (1988) suggested that vasopressin, which is elevated by adrenalectomy but not after CRH infusion, may be involved because adrenalectomy also increases hypothalamic vasopressin release, whereas medial basal hypothalamic deafferentation abolishes the effect of adrenalectomy on pituitary CRH receptors. Importantly, CRH receptor down-regulation does not result in corticotroph hyporesponsiveness, probably owing to the coordinate synergy between vasopressin and CRH. In contrast to adrenalectomy-induced sustained hyperactivation of the hypothalamo-pituitary system, prolonged immobilization stress is characterized by an initial rise of several hours’ duration, followed by a decrease to almost baseline

394

levels (Chappell et al., 1986). This finding points to a role of adrenal glucocorticoids and their receptors in the adaptive response to chronic stress. Glucocorticoids may suppress the corticotrophic response to CRH not only by their effects on synthesis and release of POMC peptides but also by decreasing the number of CRH receptors at pituitary corticotrophs. The latter effect is absent in patients with pituitary corticotrophic adenomas (Grino et al., 1988). The regulation of brain CRH receptors appears to be different from that at the pituitary. After chronic stress CRH receptors in several areas of the brain remained unchanged (Hauger et al., 1988) and, in contrast to the pituitary, glucocorticoids failed to affect CRH receptors in a number of brain areas studied by Hauger et al. (1987). Recently a CRH-binding protein that binds hum a d r a t CRH with an affinity similar to that of the CRH receptor has been isolated and characterized (Potter et al., 1991). This CRH-binding protein binds ovine CRH with a much lower affinity (250 nM), suggesting that one or more of the seven amino acids that differ between the humanhat and ovine forms of CRH are involved in the binding to CRHbinding protein and the pituitary CRH receptor. At these receptors ovine or humadrat CRH have equally good binding properties (KD= 0.1 - 0.5 nM), suggesting that there is a major structural difference between CRH-binding protein and CRH receptors, both of which play an important role in coordinating the neuroendocrine response to stress. Because of the suggested role of CRH as a mediator of anxiety and depression, the group led by De Souza investigated whether psychotropic drugs produce their effects by suppressing CRH secretion, resulting in up-regulation of CRH receptors. A marked increase in CRH receptors in the brain-stem has been observed in response to long-term imipramine treatment (Grigoriadis et al., 1989). In view of the central role of the LC for the cerebral noradrenergic system and its activation after intracerebroventricular administration of CRH, this finding supports an interaction of antidepressants and CRH neuron activity.

Preclinical studies A large number of preclinical studies support the view that CRH is involved not only in the coordinate neuroendocrine reflex after actual or perceived stressors but also in a variety of autonomic and behavioral responses. The majority of these studies tested the hypothesis that CRH is a mediator of anxiety and other fear-related aspects of stress (review: Dunn and Berridge, 1990) and possibly also of depression (Nemeroff et al., 1988). Although no attempt is made to be exhaustive, a few characteristic studies are reviewed that support a possible role of CRH also in non-endocrine behavior related to stress and affective disorder. Exposure to a novel environment induces several exploratory behaviors that are reduced by i.c.v. CRH in the same way as they are by white noise stress during testing in rats, mice (Berridge and Dunn, 1986)and monkeys (Kalin et al., 1983,1989). Moreover, in conflict tests and other tests of anxiety CRH increased the anxiogenic effect of the test condition, and these effects could be reversed either by CRH antagonists or by anxiolytic drugs such as benzodiazepines (Britton et al., 1982, 1985, 1986). Because the LC has been implicated in the pathogenesis of anxiety, the group led by Nemeroff microinfused CRH to this brain-stem nucleus and assessed the anxiogenic activity in rats placed in an open field containing a small darkened area. In a dosedependent mode CRH increased the time spent in the non-threatening dark area. This effect is sitespecific as the same dosages infused into the cerebral aqueduct produced no such effects (Butler et al., 1990). The anxiogenic effect of CRH was compensated for by the triazolo-benzodiazepines alprazolam and adinazolam. Other paradigms employed to test the anxiogenic role of CRH also supported this hypothesis (Dunn and Berridge, 1990). Another interesting effect of CRH is that on sexual behavior. Normally the human reproductive function is inhibited in emotional or physical stress (Schweiger et al., 1988) and also in depression (Steiger et al., 1991). In this context it is of interest

395

that Almeida et al. (1988) and Nikolarakis et al. (1988) found that CRH induces luteinizinghormone suppression via an opioid mechanism, whereas Sirinathsinghji (1986, 1987) showed that exogenous CRH suppresses several reproductive behaviors in male and female rats. Loss of appetite is another typical symptom seen during stress and in major affectivedisorders. Therefore, it is of interest that central administration of CRH produces a decrease in food intake in rats (Levine et al., 1983) and monkeys (Glowa and Gold, 1991). This effect of CRH is specific for an action at the hypothalamic paraventricular nucleus (Krahn et al., 1988). These and other behavioral effects induced by

CRH show some similarities with signs and symptoms of major depression according to the diagnostic scheme published by the American Psychiatric Association. A comparison of these symptoms, given in Table 11, can be regarded as a wellfounded justification to further test the HPA-hyperdrive hypothesis of anxiety and depression. Clinical studies Normal subjects The pituitary adrenocortical system is characterized by a prominent circadian rhythmicity as well as a pulsatile mode of hormone release. The diurnal

TABLE I1 Similarities between signs and symptoms of major depression (DSM 111-R criteria) and the behavioral effect of centrally administered CRH in laboratory animals DSM 111-Rmajor depression

Effects of centrally administered CRH

Depressed mood (irritable mood in children and adolescents) most of the day, nearly every day, as indicated either by subjective account or observations by others

Mimics the behavioral despair syndrome observed after maternal separation in rhesus monkey infants (Kalin et al., 1983, 1989)

Markedly diminished interest or pleasure in all or almost all activities most of the day, nearly every day

Diminishes sexual behavior in male and female rats (Sirinathsinghji, 1986, 1987)

Significant weight loss or weight gain when not dieting or decrease or increase in appetite nearly every day

Decreases food consumption in rats (Levine et al., 1983; Glowa and Gold, 1991)

Insomnia of hypersomnia nearly every day

Disrupts normal sleep patterns with concomitant EEG changes (Ehlers et al., 1986; Holsboer et al., 1988; i.v. administered in humans)

Psychomotor agitation or retardation nearly every day

Increases locomotor activity in a familiar environment and produces “stress-like” alterations in locomotion in a novel environment (Koob and Bloom, 1985)

Fatigue or loss of energy nearly every day

No data

Feelings of worthlessness or excessive or inappropriate guilt nearly every day

No data

Diminished ability to think or concentrate or indecisiveness nearly every day

No data

Recurrent thoughts or death, recurrent suicidal ideation or a suicide attempt

No data

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variation in the amplitude of ACTH secretory bursts, but not in the frequency, determines the 24-h rhythm in plasma ACTH concentrations. Intensive (every 10 min) blood sampling over a 24-h period revealed 40 + 1.5 ACTH secretory burstsl24 h, with a mean interburst interval of 39 f 2.3 rnin (Veldhuis et al., 1990). Endogenous ACTH has an estimated half-life of 15 k 1.2 rnin and its daily production rate is 0.96 f 0.16 ng/ml (0.21 f 0.035 nmol/l). These episodic ACTH stimuli give rise to cortisol peaks that occur with a time lag of about 10 - 20 rnin after pituitary release, resulting in a daily cortisol production rate of 9.9 f 2.7 mg/ml (27.3 k 7.5 pmol/l) (Estaban et al., 1991). The plasma levels of CRH in normal subjects were found to vary between 2 and 28 pg/ml (0.4 - 6.0 pmol/l). Although increases in plasma CRH levels after insulin-induced hypoglycemia or metyrapone administration and suppression of CRH during glucocorticoid treatment have been observed, the diurnal variations have not been consistently reported (Suda et al., 1985; Stalla et al., 1986b; Sasaki et al., 1987). The reasons may be: (1) uncertainties about the assay techniques used by different investigators; (2) extra-pituitary sources of CRH; (3) different circulating levels of CRH-binding protein, with possible reduction in the amount of bioactive CRH, particularly during pregnancy (Stalla et al., 1989), when both CRH and CRH-binding protein levels are high (Potter et al., 1991); and (4) the co-release of peptides other than CRH, which modulate the amount of ACTH release by CRH at different times of day. In the cerebrospinal fluid (CSF) immunoassayable CRH has been detected ranging from 20 to 60 pg/ml. The source of this CSF peptide has not yet been established. However, since in primates an inverse relationship between CRH in the CSF and circulating plasma cortisol has been documented, it can be assumed that the CSF content reflects at least partly neuroendocrinologically active CRH (Garrick et al., 1987). Intravenous administration of ovine or human CRH prompts an increase in plasma ACTH within minutes, followed by a more gradual rise in plasma cortisol that peaks at 30 - 60 rnin (Grossman et al.,

1982; Stalla et al., 1984). Human CRH elicits a hormonal response that is similar in magnitude but shorter in duration than that seen after an equimolar dose of ovine CRH (Schuermeyer et al., 1985; Stalla et al., 1986a). The difference in the duration of action is probably related to different pharmacokinetics. Whereas ovine CRH has an initial half-life of 6 - 12 rnin (representing distributioninto the plasma volume) followed by a second half-life of 45 - 70 min (representing metabolic clearance), the corresponding values for human CRH are 5.3 k 0.2 rnin ACTH AND CORTISOL LEVELS AFTER PRETREATMEN1 WITH INCREASING DOSES OF DEXAMETHASONE ACTH pglml

4 -

3 2 1 -

0.25mg

0.50mg

1.0 mg

1.5mg

DEXAMETHASONE

CORTISOL ng/ml

251

0.25mg

0.50mg

1.0 rng

1.5mg

DEXAMETHASONE

Fig. 3. Increasing dosages (0.25mg, 0.5 mg, 1 .O mg, 1.5 mg) of dexamethasone administered at 23:OO h to 7 healthy controls in randomized order resulted in decreased amounts of released ACTH and cortisol after hCRH injection at 15:OO h the following day. The corresponding areas under time course curves that were registered following hCRH were: 0.25 mg dexamethasone: cortisol, 18.4 f 4.1 mg x min x 1000/ml (AUC), ACTH 3.9 & 1.6 pg X rnin x 1000/ml; 0.5 mg dexamethasone: cortisol, 11.4 k 2.6 AUC, ACTH, 4.1 k 2.0 AUC; 1.0mg dexamethasone: cortisol, 6.4 f 3.3 AUC, ACTH, 2.2 f 0.5 AUC; 1.5 mg dexamethasone: cortisol, 2.3 k 1.4 AUC, ACTH, 1.5 ? 1.0 AUC. (From U. von Bardeleben, I . Heuser and F. Holsboer, unpublished data.)

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and 25.3 f 1.O min respectively. The second halflife time for hCRH reflects the 3 -4-fold greater metabolic clearance rate, which correlates better and more appropriately with the naturally occurring episodes of secretion of ACTH and cortisol. The ACTH response is unaffected by gender. There are age effects but theyareonlysubtle. Theyarediscussed later in the section on the dementias. The hormonal response pattern is dependent on the time of day, with a stronger increase in ACTH and cortisol .in the evening, when baseline levels are low, suggesting that pituitary-adrenocortical hormones modulate the ability of corticotrophs to respond to CRH. This is supported by: (1) the inverse relationship between baseline cortisol and ACTH response toCRH(Hermusetal., 1986; Holsboeretal., 1986); (2) the augmented ACTH response to CRH after pre-treatment with metyrapone (Von Bardeleben et al., 1988b; Lisanski et al., 1989); (3) thedose-dependent suppressibility of the ACTH response to CRH by dexamethasone (Fig. 3; Von Bardeleben et al., 1992); (4) the enhanced ACTH response after pretreatment with naloxone; and ( 5 ) suppression of the ACTH response by morphine sulfate and the metenkephalin analog FK 33-824 (Allolio et al., 1986). Clinically the most important factor modulating the effect of CRH at the pituitary level is AVP, which considerably potentiates the pituitary-adrenocortical response to CRH. This has been shown by hypertonic saline stimulation of endogenous AVP (Rittmaster et al., 1987)and co-administration of CRH and AVP (Liu et al., 1983; Lamberts et al., 1984). In addition, the dexamethasone suppression of ACTH and cortisol can be overridden by simultaneous administration of AVP and CRH (Von Bardeleben et al., 1985). Recently Kellner et al. (1992) suggested that atrial natriuretic factor may suppress the ACTH-releasing effect of CRH. The intravenous administration of CRH induces only moderate and transient side effects such as flushing, metallic taste, tachycardia and tachypnea. These side effects appear to be more pronounced when higher dosages of the ovine analog of CRH are used, Since the greatest hormonal r/esponseoccurs at a dosage of 100 pg, most of the investigators chose

dosages of 1 - 2 pg/kg for neuroendocrine HPA testing. Continuous infusion of ovine CRH at a dosage of 1 pg/kg per hour for 24 h in normal men resulted in ACTH and cortisol levels that were elevated but that maintained their diurnal rhythm. This suggests that corticotrophic cells may have a variable sensitivity to CRH because of the variable presence of factors that modulate the CRH effect at the pituitary level (Schulte et al., 1985). Moreover, a variable sensitivity of glucocorticoid negative feedback mediated by the binary corticosteroid receptor system in the brain may play a critical role (De Kloet, 1991). The endocrine effects of CRH have been less thoroughly studied in clinical than in preclinical investigations. Observations in primates, where high dosages of CRH induced a marked drop in both peripheral vascular resistance and mean systemic blood pressure, led to the suggestion that CRH may have use as a peripheral hernodynamic regulator, e.g., as a selective splanchnic vasodilator (Udelsman et al., 1984). Recently the effect of CRH on sleep electroencephalography in men has been explored. Episodic administration of human CRH reduced the amount of slow wave sleep (SWS) in the second half of the night (Holsboer et al., 1988). This phenomenon is not secondary to CRH-induced elevation of cortisol because corresponding studies with cortisol showed the opposite, i.e., SWS-increasing effect (Born et al., 1987; Von Bardeleben et al., 1988a). Furthermore, the possible effects of ACTH were indirectly ruled out by administration of the behaviorally active but endocrinologically inert ACTH(4 - 9) fragment, which did not affect SWS (Steiger et al., 1991a). Finally, administration of RU 486, an antiglucocorticosteroid, that forcefully enhances CRH production by suppressing corticosteroid-mediated negative feedback, led to deterioration of the physiological sleep structure including abolishment of SWS (Fig. 4; Wiedemann et al., 1992). The provisional conclusion from this study is that CRH is actively involved in modulation of sleep and suppresses SWS, which is in keeping with its presumed

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EFFECT OF GR ANTAGONIST RU486 UPON HUMAN SLEEP-EEG

nm

01I

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MIFEPRISTON ( RU 486)

T

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Fig. 4.Antagonism of glucocorticosteroid receptors deteriorates structure of the EEG sleep in a human male control. Associated phenomena are a hyperdriven HPA system and a blunted nocturnal GH surge. (From Wiedemann et al., 1992.)

role as coordinator of the stress response and with corresponding experiments in the rat (Ehlers et al., 1986). Cushing ’s syndrome Patients with Cushing’s syndrome can be divided into two classes: (1) those with ACTH-dependent, i.e., pituitary-related overproduction of ACTH (Cushing’s disease), or ectopic sources of ACTH or CRH; and (2) those with ACTH-independent processes such as adrenal adenoma or carcinoma. The differential diagnosis of Cushing’s syndrome has profited considerably from the availability of CRH(MUlleretal., 1982;Orthet al., 1982). Patients with Cushing’s disease tend to show an exaggerated ACTH and cortisol response to CRH, whereas those with ectopic ACTH syndrome fail to respond (Gil-

lies and Grossman, 1985). Depending on the criterion values for increased response and the number of tests performed, 80 - 90Yo of patients with histologically proven pituitary-dependent Cushing’s disease respond to CRH (Grossman et al., 1988). In Cushing’s disease responsivity of ACTH to CRH and the lack of suppressibility by dexamethasone are related phenomena (Hermus et al., 1986), and the complementary testing with dexamethasone is still indispensable as an additional differential diagnostic test in patients with Cushing’s syndrome (Biemond et al., 1990). In the light of the concurrent elevation of ACTH and cortisol levels the possible role of CRH in the pathogenesis of Cushing’s disease has also been investigated. As demonstrated by Tomori et at. (1983) andKlinget al. (1991), theCRHlevelsintheCSFare lowered in patients with Cushing’s disease. Moreover, as mentioned earlier, continuous infusions of CRH at high dosages in normal men fail to stimulate the ACTH-cortisol pattern seen among patients with Cushing’s disease. In these patients the pattern is characterized by much higher cortisol levels and a flattened or abolished diurnal rhythm. Furthermore, the brisk ACTH response to CRH in untreated patients with Cushing’s disease speaks against a continuous CRH overproduction because in that case the CRH receptors should be desensitized. However, these endocrine findings do not rule out the possibility that the disease is initially triggered by corticotrophic overexposure to CRH. In support of this idea is a study by Sonino et al. (1988) in which stressful life events, neuroendocrinologically characterized by an excessive CRH drive, were frequently found to precede the development of Cushing’s disease. This provocative finding can be viewed as another example of how endocrine disease and psychopathology can be placed on a phenomenological continuum. In patients who have had successful removal of an ACTH-secreting microadenoma a prolonged HPA hypofunction occurs that is secondary to the prolonged suppression of the CRH neuron, that developed while the patients were excessively hypercortisolemic (Schrell et al., 1987).

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Anorexia nervosa Anorexia nervosa and bulimia nervosa are syndromes that occur predominantly in adolescent girls and young women. The patients achieve low body weight by restricting their food intake and increasing their physical activity. Bulimia nervosa is characterized by episodic binge eating and may exist separately or in combination with anorexia. Underweight patients with anorexia nervosa are hypercortisolemic but not necessarily cushingoid. The pathophysiology underlying this endocrine state is poorly understood. From extensive studies the following HPA abnormalities in these patients have been identified: (1) when the patients were chronically underweight, their plasma cortisol levels were not adequately suppressed by dexamethasone; (2) ACTH and cortisol responses to ovine CRH were blunted; (3) 6 - 24 months after normal weight was achieved the hormonal response to CRH had normalized; and (4) CRH levels in the CSF were elevated in the active phase of the disease and returned to the normal range after clinical recovery and appropriate weight gain (Gold et al., 1986a; Hotta et al., 1986). Several animal studies have provided strong evidence that CRH has an anorectic effect per se (Levine et al., 1983; Krahn et al., 1988; Glowa and Gold, 1991). Therefore, the blunted ACTH response to CRH may be caused by excessive CRH release from the hypothalamus, leading to CRH receptor desensitization and making the corticotrophic cells refractory to CRH stimulation. However, since active weight loss may also activate the HPA system, caution is needed when patients are studied who have not stabilized their current body weight (Fichter et al., 1986).

Alcoholism Acute and chronic drinking of beverages containing ethanol activates the HPA system through secretion of CRH into the hypophyseal portal blood vessels (Rivier et al., 1984). In some actively drinking alcoholics the neuroendocrine abnormality can reach dimensions that are clinically relevant and that may account for some of the Fushingoid physical stigmata frequently observed in patients with

alcoholism. Furthermore, withdrawal from ethanol elevates ACTH and cortisol, and both return to normal levels only after prolonged abstinence. The possible role of excessive CRH secretion as a mediator of enhanced release of ACTH and cortisol was explored in a number of studies performed during acute withdrawal, after medium-term abstention and after long-term abstention from alcohol. The major findings of these studies were (Fig. 5 ) : (1) the hypercortisolemia in patients after acute cessation of drinking is associated with a blunted ACTH response to CRH (Heuser et al., 1988); (2) in patients who have abstained from drinking ethanol for a period of between 14 and 42 days the plasma cortisol secretion, estimated by the mean plasma concentration between 2:OO and 5:OO p.m., was normalized, while the ACTH responses to CRH were still decreased (Von Bardeleben et al., 1989); and (3) patients who were carefully observed not to drink ethanol for at least 6 months had baseline cortisol h-CRH STIMULATION RESPONSE AFTER TERMINATION OF ALCOHOL ABUSE ACUTE WITHDRAWAL ACTH Ipg/rnll

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Fig. 5. hCRH stimulation test response after termination of alcohol abuse. ACTH responses were found to be blunted among patients with alcoholism (acute withdrawal (1 - 2 days): 1.5 k 0.9 pg/ml per minute x lo3;medium-term (3 - 4 weeks) abstention: 1.8 k 0.5; long-term (over 6 months) abstention: 3.5 k 1.9). This effect is not necessarily explained by concurrent hypercortisolism since baseline (2 - 5 p.m.) plasma cortisol secretion was only significantly elevated after acute withdrawal (acute withdrawal: 102.8 k 42 ng/ml; medium-term abstention: 64 k 27.3 ng/ml; long-term abstention: 55.8 k 22.5 ng/ml). 1.5 pg/ml Normal control data (shaded areas): ACTH: 5.8 per minute x lo3; cortisol (2-5 p.m.): 49.3 k 15.9 ng/ml. (From Heuser et al., 1988; Von Bardeleben et al., 1989; and Holsboer, 1989.)

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levels that were indistinguishable from those of normal controls and their ACTH responses were stronger than those in patients after medium-term abstinence or acute withdrawal but still weaker than those in matched controls (Holsboer, 1989). Because the medium-term abstinence group had normal plasma cortisol baseline levels a pituitary or suprapituitary mechanism appears to be involved. The possibility of hippocampal down-regulation of MR and GR was rejected by Spencer and McEwen (1990), who reported that in chronic ethanolstressed rats tolerance develops. These animals show a normal shut-off of the corticosterone response to acute stress. Corticosterone release after ethanol is mediated by hypothalamic CRH and the observed tolerance is probably due to reduced hypothalamic CRH release and/or reduced pituitary response to CRH. However, this finding does not rule out the possibility that after steroid binding corticosteroid receptors aggravate the neurotoxicity of ethanol. The most likely explanation for our findings in acute alcohol withdrawal and medium-term and long-term sobriety is that a reduced response of the pituitary to CRH occurs under these conditions. This view is supported by a study that showed reduced pituitary CRH binding in rats after exposure to ethanol over prolonged time periods (Dave et al., 1986). These changes were accompanied by decreased adenylate cyclase activity and subsequently by reduced production of POMC mRNA. To what degree the above-mentioned changes in the HPA system may be related to an increased risk of relapse in alcoholism is currently under study.

Affective disorders Depression. In studies on the diurnal pulsatile secretion pattern of ACTH and cortisol in depressed subjects the amplitude of cortisol secretory episodes was increased in both the men and the women studied, whereas the ACTH pulse frequency was increased only in the women (Mortola et al., 1987), but not in the men (Linkowski et al., 1985). The fact that the men studied were hospitalized and hence more severely ill means that they probably had a heightened adrenal responsiveness to ACTH and

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Fig. 6 . Patients with elevated plasma cortisol levels at baseline (left) show significantly blunted ACTH release (right) following an intravenous test dose of 100 pg CRH at 19:OO h. Note that despite decreased ACTH secretion depressives have plasma cortisol surges that are indistinguishable from normal controls. This finding suggests that among depressives with hypercortisolism several adaptive changes at each level of the brain-pituitary adrenal system have occurred that result in an altered pituitary corticotroph response pattern to CRH, changes in feedback regulation and increased adrenocorticol sensitivity. (From Holsboer et al., 1986.)

that this is the most likely explanation for the different findings. However, since both studies used only small sample sizes no firm statements regarding the pulse frequencies and amplitudes of ACTH and cortisol and the dependency on severity and duration of illness are justified. The first series of reports in which hCRH was used in depressed patients revealed a blunted ACTH response after i.v. administration of a test dose of hCRH (Holsboer et al., 1984a, 1986, 1987a). The baseline cortisol secretion prior to hCRH stimulation was significantly higher in the depressed patients than in normal control subjects and inversely related to the amount of ACTH produced by stimulation (Fig. 6). Moreover, the cortisol responses in the patient group were indistinguishable from those in the control group despite significantly lower ACTH release. This points toward a hypersensitive adrenal cortex resulting from long-term overexposure to ACTH, and confirms conclusions drawn from ACTH and cortisol profiles at baseline and after ACTH challenges. Furthermore, pre-treatment of depressed patients with metyrapone, which

401

suppresses cortisol biosynthesis, resulted in normalized ACTH release after hCRH stimulation (Von Bardeleben et al., 1988b). These data suggest that elevated circulating cortisol is the main abnormality preventing adequate ACTH response via negative feedback. Exaggerated secretory activity of adrenocortical cells may reflect a suprapituitary abnormality. This interpretation is consistent with earlier studies with the ovine heterologue CRH (Holsboer, 1983; Gold et al., 1984, 1986b; Holsboer et al., 1984b, 1985; Amsterdam et al., 1987). In addition to baseline hypercorticolism, other mechanisms that might account for blunted ACTH response to CRH must be considered. Among these are altered processing and storage of ACTH precursors, desensitized CRH receptors at pituitary corticotrophs and alternative processing of POMC, the precursor of ACTH and ,&endorphin. For example, Rupprecht et al. (1989) recently reported a dissociation of ACTH and &endorphin responses after CRH stimulation in depression. Young et al. (1990) applied a low-dosage ovine CRH challenge to depressed patients and control subjects and measured 0-endorphin, b-lipotropin and cortisol to obtain information on the acute feedback regulation and current CRH receptor sensitivity. They found a decreased total 0-endorphin and b-lipotropin response and a normal adrenal cortisol response in the depressed group, which is in line with our original findings on ACTH and cortisol after hCRH. The @endorphin response pattern was found to be biphasic, with an initial release of 0-endorphin preceding the cortisol surge and a second period of 0-endorphin increase while cortisol was elevated but stable. This pattern is consistent with the concept of two separate feedback phases (Keller Wood and Dallman, 1985): (1) an early termination of corticotroph secretion during the period of rapidly increasing corticosteroid levels; and (2) a silent period in corticosteroid feedback while high adrenocortical hormone levels are still increasing but at a much slower rate than during the period immediately after stimulation. The postulated increase in CRH secretion in depression is also supported by studies in which im-

munoreactive CRH was measured in the CSF of depressed patients. The authors consistently found normal or elevated levels of CZH (Nemeroff et al., 1984; Banki et al., 1987; Roy et al., 1987; Kling et al., 1991). Within the limitations warranted when interpreting CSF studies, one can conclude that CRH is overproduced in these brain regions that contribute to CSF peptide content. Among these are hypothalamic and presumably other limbic CRH neurons, which are obviously refractory to the negative feedback effect of elevated circulating glucocorticoids. In Cushing’s disease such a refractoriness is clearly absent because elevated glucocorticoid levels occur together with decreased CRH levels in the CSF (Tomori et al., 1983). The hypothesis of enhanced central production of CRH in depression is also supported by further studies by Nemeroff and associates: (1) CRH receptors were found to be down-regulated in the frontal cortex of suicide victims (Bmax in suicides: 521 f 43 fmoles/mg protein; B,, in controls: 680 + 51 fmoles/mg protein; P
402

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Fig. 7. Pre-medication of normal controls with 1.5 mg dexamethasone prevents the activation of HPA hormones following hCRH. Depressed patients show a paradoxical rise of cortisol and ACTH in the combined dexamethasone-CRH challenge indicating changes at the glucocorticosteroid receptor level. (From Von Bardeleben and Holsboer, 1989, 1991, and unpublished data.)

creased levels of corticosteroids these receptors are assumed to be involved in enhancing the risk for neurodegeneration of hippocampal pyramidal cells after insults such as hypoxia (Sapolsky and Pulsinelli, 1985). However, these steroid hormones and their receptors are also essential for maintaining the structural integrity of the hippocampus because adrenalectomy results in granule cell loss (Sloviter et al., 1989). These corticosteroid receptors also mediate the attenuation of evoked norepinephrine release (Joels and De Kloet, 1989). In contrast to the GRs, which control the termination of the stress response, the MKs, located almost exclusively in the hippocampus, mediate basal and HPA activity by determining the level of stress responsiveness (Ratka et al., 1989). In other words, lowered MR availability increases the onset and degree of release of CRH, AVP and other neuropeptides that are transcriptionally controlled by corticosteroids. In contrast, lowered levels of GR cause a protracted return of CRH and AVP to baseline. Prolonged periods of stress or major depression result in long-term activation of the HPA system, which in turn has effects on the receptor molecule itself. As described earlier, upon hormone activation the GR is rapidly translocated into the nucleus, where it either binds to glu-

cocorticoid response elements to promote transcription or, in the case of negative regulation, interacts competitively with other transcription factors in the promoter region of target genes. Expression of target genes reaches a maximum within a short period; after prolonged hormone stimulation the effect is gradually reduced (Yamamoto, 1985). This receptor desensitization or down-regulation suggests a negative feedback of the activated GR on its own expression. The consequence for genes such as those coding for CRH or AVP is that this down-regulation results in disinhibited transcription. Because chronic stress activates CRH- and AVP-containing neurons (Whitnall, 1989) and HPA hyperactivity produces down-regulation of GR in the rat (Sapolsky et al., 1986) and in primates (Uno et al., 1989), similar adaptive changes in corticosteroid receptors probably also occur in humans with depression. As a result of such a relatively decreased number of hippocampal and hypothalamic GRs, the capacity of these GRs to shut off HPA hyperactivity would be reduced. This holds true for the multisynaptic hippocampal control of the hypothalamus, which after deafferentation overproduces CRH (Herman et al., 1989), and for hypothalamic synthesis of POMC-stimulating peptides such as CRH and AVP. We recently postulated that the finding of an exaggerated ACTH and cortisol response to a DEXCRH challenge in depression is primarily caused by inadequate suppression of AVP secondary to downregulation of GRs, limiting the inhibitory effect of dexamethasone (Von Bardeleben and Holsboer, 1989, 1991). This notion is supported by animal studies which have shown that AVP concentrations in hypophyseal portal blood react most sensitively to minor changes in circulating corticosteroids (Fink et al., 1988). The finding of Von Bardeleben et al. (1985) that combined administration of CRH and AVP is needed to override the dexamethasone suppression of the HPA system lends further credence to the hypothesis of a concerted action of both peptides, CRH and AVP, to produce exaggerated HPA activity and negative feedback resistance in depression. A further aspect in the difference between high

403

circulating levels of cortisol, which decrease corticotrophic responsivity to CRH, and dexamethasone, which acts differently from the naturally occurring corticosteroids, is explained by the differential binding properties of these steroids at pituitary and brain receptors. In contrast to cortisol and other adrenocortical steroids dexamethasone does not bind to corticosteroid-binding globulin, which is present in the pituitary but not in the brain. This is one reason why after systemic administration dexamethasone acts primarily at the anterior pituitary to block the HPA system (De Kloet et al., 1974). Recently, Ferrara et al. (1991) proposed an additional mechanism possibly involved in decreased sensitivity to dexamethasone feedback. These investigators found evidence that infusion of antisomatostatin y-globulin into the hippocampal dentate gyrus and CA3 region blocks the HPA-suppressive effect of dexamethasone. Inasmuch as somatostatin was found to inhibit CRH and is decreased in the CSF of depressed patients (Rubinow, 1986), it would be important to know whether there is a causal link between impaired somatostatin secretion and dexamethasone resistance in depression. Effect of antidepressants. It has been shown that the HPA dysregulation gradually becomes normal in the course of antidepressant treatment (Holsboer et al., 1982, 1987a; Greden et al., 1983;Amsterdam et al., 1988; Holsboer-Trachsler et al., 1991). From these studies data have also emerged which suggest that the failure to normalize on various HPA function tests indicates a poor prognosis, supporting the idea of a link between actions of antidepressants upon neuroendocrine systems and their therapeutic effect. As assessed by in situ hybridization, treatment of rats with imipramine for 2 months leads to decreased (37%) CRH mRNA in the PVN and to decreased (40%)tyrosine hydroxylase (TH) mRNA in the LC. Antidepressants decrease the intracellular pool of CAMP, which, after binding to a specific protein, activates the CRH gene transcription via specific response elements at the gene promoter. Therefore, decreased intracellular cAMP results in decreased gene transcription. After CRH binding to

its specific corticotrophic receptors, cAMP is activated as a second messenger, and antidepressants possibly impede this process, too, thus attenuating the CRH-induced POMC gene transcription. The noradrenergic axons projecting from the LC to the PVN were found to activate CRH production (Swanson et al., 1983). If TH, the rate-limiting enzyme for biosynthesis of noradrenaline, is decreased because antidepressants mute the expression of the TH gene, one would also expect an attenuation of CRH release. Finally, some recent results suggest that corticosteroid receptor biosynthesis is enhanced by antidepressants. Pepin et al. (1989), for example, found GR mRNA to be increased in neuroblastoma cells exposed to amitriptyline and Brady et al. (1991) found MR mRNA to be increased in the hippocampus of rats that were treated for 8 weeks with imipramine. The relevance of these findings for human depression has not yet been established. However, the coincidence of clinical remission and normalization of HPA hyperdrive during antidepressant treatment suggests a causal link. Anxiety disorders. States of anxiety or panic attacks are associated with increased noradrenaline secretion and a hyperactive HPA system. The anxiolytic benzodiazepines reduce circulating levels of ACTH and cortisol, whereas inverse agonists stimulate them (Insel et al., 1984). Recently, Calogero et al. (1988) reported that the benzodiazepine alprazolam attenuates stimulated CRH release from isolated hypothalami. Furthermore, Owens et al. (1989) found that benzodiazepines prevent the release of CRH from the median eminence. Interestingly, the CRH content of the LC decreased after treatment with benzodiazepines and this effect could be blocked by benzodiazepine antagonists. CRH activates the LC and, at least in animal studies, this mechanism is involved in behavioral anxiety, suggesting that the anxiolytic effects of benzodiazepines may occur through dampening of CRH neurons. These data suggest that panic and anxiety disorders are precipitated by a rapidly progressing dysregulation between CRH neurons and noradrenergic neurons at the LC. This would result

404

in a positive feedback circuit, leading temporarily to explosive LC-CRH disinhibition and mutually triggering panic anxiety and a variety of LC- and HPArelated autonomic (cardiovascular) system and neuroendocrine changes. This rather speculative hypothesis is supported by the observed suppression of CRH activity by benzodiazepines, and by electrophysiological studies in rats and studies with inverse benzodiazepine agonists in primates, but needs strengthening by neuroendocrine measurements during spontaneous panic attacks in humans. Although such data are not presently available, two recent studies with CRH in patients with panic disorders support the idea of episodic CRH bursts. Roy-Byrne et al. (1986) and workers in our laboratory (Holsboer et al., 1987b) found that the ACTH responses following ovine and human CRH probes, respectively, were blunted. In the study by RoyByrne et al. baseline cortisol levels were elevated, probably due to the preceding insertion of the intravenous catheter and resulting anticipatory anxiety. In our study we inserted the catheter 5 h prior to the CRH challenge and used a “through-the-wall” technique, so that all manipulations were out of the patients’ view. Our finding of a diminished ACTH response after hCRH in the absence of hypercortisolism suggests desensitized CRH receptors at anterior pituitary corticotrophs. The receptor changes are probably due to excessive overexposure to hypothalamic CRH as part of the complex symptom pattern during recurrent panic attacks. It must be noted that the reported absence of elevated HPA measures during lactate-induced panic attacks is not inconsistent with this hypothesis. Lactate infusions induce a significant osmotic and volume load, enhancing atrial natriuretic peptide secretion, and the peptide than acts as a functional AVP antagonist (Dayanithi and Antoni, 1990). Because AVP is the most important dynamic factor potentiating the effect of CRH at the pituitary level, blockade of its action during the lactate paradigm would reduce the capacity of CRH to release ACTH. Thus, even if lactate-induced panic attacks were driven by central CRH, the counterregulatory mechanisms at the pituitary would compensate for its peripheral endocrine effect.

Aging, dementia and Alzheimer’s disease The neuroendocrine system plays a major role in controlling certain aging processes and may be involved in the development of pathology, which becomes more likely with increasing age. The HPA system has been implicated in the development of neurodegenerative mechanisms associated with aging because with increasing age the ACTH and corticosteroid response to stressors increase and elevated adrenal steroids may impede neuronal capacity to survive coincident insults such as hypoxia or hypoglycemia (Sapolsky et al., 1986). Studies in aged rats showed increased circulating levels of glucocorticoids, impaired adrenocortical recovery from stress and a substantial neuronal loss in the hippocampal formation (Sapolsky et al., 1983). Similar morphological changes, most pronounced in the hippocampal CA3 region, were also reported in hypercortisolemic primates (Uno et al., 1989). Several mechanisms are involved in these changes. Recently, Scaccianoce et al. (1990) attempted to determine which component of the HPA system contributes to age-related HPA hyperdrive and found exaggerated in vitro hypothalamic activity as evidenced by stimulated release of bioactive CRH. In contrast, they saw no functional changes at the pituitary and adrenal cortex, although other investigators have also proposed age-related increases in adrenal sensitivity to ACTH (Sonntag et al., 1987; Brodish and Odio, 1989). Reaven and coworkers (1988) reported that adrenal cortical cells from oIder rats do not produce corticosterone as efficiently as cells from younger animals. To compensate for this, structural changes in the adrenals might occur with aging, which allow young and old animals to secret similar concentrations of corticosterone after stimulation with secretagogues. The activity of hypothalamic CRH neurons is under coordinate control of hippocampal MRs and GRs, which are colocalized in hippocampal neurons. At the cellular level ligand-activated MRs maintain the response to excitatory input, whereas activated GRs oppose these effects when excitability is exaggerated. On a systemic level the effects

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mediated by MRs control basal HPA activity and the responsivity to stress, whereas GRs in the hippocampus suppress these effects in case of an overshoot. At the hypothalamus and pituitary GRs mediate the negative effect of glucocorticoids on biosynthesis and release of CRH and POMC-derived peptides. The progressive reduction of hippocampal MR capacity may be responsible for elevated CRH release. Down-regulation of GR in the hippocampus, hypothalamus and pituitary in concert with the hippocampal down-regulation of MRs are essential to determine the set-point of homeostatic control of stress (Reul et al., 1988). The mechanism underlying these receptor changes responsible for CRH overactivity are not yet clear. Armanini et al. (1990) recently suggested that glucocorticoids endanger hippocampal neurons at the dentate gyms by inhibiting glucose uptake and by exacerbating the effects of excitatory amino acids, which after binding to N-methy1-D-aspartate (NMDA) receptors cause elevation of free cytosolic calcium ions. The latter effect is deleterious for neuronal survival. The observed changes in receptor density can also be regarded as protective against corticosteroidtriggered hippocampal damage (Eldridge et al., 1989). According to this hypothesis, aging increases the affinity of GR for corticosterone and also causes impaired adrenalectomy-induced up-regulation of GR binding sites and resistance to down-regulation in response to chronic stress (Eldridge et al., 1989). Experimental evidence strongly supports the notion that with aging homeostatic adaption of HPAaxis activity to chronic stress conditions gets progressively more impaired (Odio and Brodish; 1989) and the hypothesis was advanced that environmental factors play a crucial role in modulating these age-related HPA-axis alterations. Interestingly, a recent study in female rats found that the degree of age-related hippocampal GR loss and consequently the amount of stress-induced corticosterone secretion was determined by early environmental manipulations: if rats had been handled during infancy they showed less hippocampal neuronal loss, lower HPA-axis activity and - even more importantly - preservation of spatial mem-

ory capacity during aging in comparison to nonhandled rats (Meaney et al., 1991). Within this context, it could be demonstrated that chronic stress has differential effects upon hippocampal physiology: chronically stressed young rats developed advanced age-like electrophysiological changes but no hippocampal neuronal loss, whereas old animals exhibited marked additional neuronal degeneration with chronic stress (Kerr et al., 1991). Human studies did not reveal that aging is automatically associated with basal HPA hyperactivity (Ohashi et al., 1986; Pavlov et al., 1986; Dodt et ACTH AND CORTISOL RESPONSE TO A COMBINED DEXICRH-TEST IN MALE RUNNERS, DEPRESSIVES AND CONTROLS

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Fig. 8. While basal plasma cortisol concentrations among depressed patients were higher than among age-matched runners (43 +_ 15 nMol/l versus 25 +_ 8) their net cortisol output did not differ much (AUC: depressives 4.9 f 1.3; runners: 5.7 + 1.7; controls: 1.4 f 0.5) (top). In contrast to adrenocortical responses ACTH secretion after DEX/hCRH stimulation was higher in depressed patients (AUC; 1.9 k 0.6) than in runners (AUC: 1.2 k 0.3) and controls (AUC: 0.5 f 0.3). (From Heuser et al., 1991.)

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al., 1991). Two recent studies showed that cumulative exposure to severe stress may accelerate the development of age-related HPA hyperdrive. Heuser et al. (1991) studied elderly endurance athletes with a combined dexamethasone/hCRH probe and found that they responded with elevated ACTH and cortisol secretion (Fig. 8). Interestingly, similar observations were made in depressed patients (Von Bardeleben and Holsboer, 1989, 1991). In these patients the degree of HPA disturbance correlated with the patients’ age. From these studies it was concluded that a negative feedback disturbance exists, resulting in the presence of factors that suppress the effect of dexamethasone and synergize the stimulating effect of exogenous hCRH. It was postulated that this factor is vasopressin because concomitant administration of both peptides overcomes dexamethasone suppression in normal controls (Von Bardeleben et al., 1985). Moreover, several studies showed that vasopressinergic neurons become activated during aging. In aged rats increased activity of neurosecretory vasopressin cells (Fliers and Swaab, 1983), probably secondary to decreased sensitivity of renal vasopressin receptors (Ravid et al., 1987), has been reported. Also studies in humans suggest increased activity of vasopressin cells in the supraoptic nucleus and paraventricular nucleus (Goudsmit et al., 1990). For example, morphological changes of vasopressin cells within these nuclei of senescent humans (Fliers et al., 1985) point to their activation along with increased plasma vasopressin levels (Frolkis et al., 1982; Kirkland et al., 1984; 0 s et al., 1987) and a probably diminished responsiveness of the aging kidney to vasopressin. A reduced perception of thirst in senescence may also play a role (Phillips et al., 1984). Thus far, the complexity between AVP and glucocorticoid regulation is ill understood. In vitro experiments in cultured diencephalic neurons have shown an increase of AVP mRNA in the magnocellular cells of the PVN by glucocorticoid antagonization. Conversely, in vivo studies found adrenalectomy to result in an augmentation of AVP expression in the parvocellular neurons of the PVN only (Davis et al., 1986; Schilling et al., 1991). It remains

unclear, how these results can be related to clinical studies in hypercortisolemic patients; that is: how elevated cortisol levels modulate endogenous AVP synthesis and/or release. In Alzheimer’s disease a reduction in CRH immunoreactivity in the frontal and temporal cortices (50%), caudate nucleus (30%)(Bissette et al., 1985) and occipital cortex (80%) has been reported (Whitehouse et al., 1987; De Souza et al., 1990). These observations have been complemented by receptor studies showing that a decrease in CRH concentration corresponds with increased density of post-synaptic CRH receptors (De Souza et al., 1990). In the cerebrospinal fluid CRH levels were either normal (Nemeroff et al., 1984; Pomaraet al., 1989) or decreased (15 - 30%) (Mouradian et al., 1986; May et al., 1987) and the degree of CRH decrease seemed to depend on the severity of neuropsychological impairment. Heuser et al. (1989) compared the ACTH and cortisol response to CRH in patients with Alzheimer’s disease and in controls. Baseline values of cortisol were significantly higher in Alzheimer patients. However, CRH-stimulated secretion of ACTH and cortisol did not differ between patients and controls. They concluded that possibly it is not the pituitary secretory capacity that is disturbed but rather the time until stress-elevated cortisol levels return to baseline, which would suggest altered negative feedback regulation in these patients. Dodt et al. (1991) reported that old controls respond more rigorously to a combined vasopressin/CRH stimulus than young controls or patients with Alzheimer’s disease. This finding and the finding by Heuser et al. (1989) indicate that there are significant changes in either corticosteroid or vasopressin receptors or both. The regulatory changes in the HPA system in old age and dementia are probably related to the neurotransmitter changes observed in this population. In a number of dementing illnesses cholinacetyltransferase activity (CAT), a marker of cholinergic neuronal activity in the cortex and hippocampus, is decreased and the reductions in cortical CRH concentrations ar positively correlated with reductions in CAT activity. Consistent with this, there is

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a negative association with CAT activity and cortical CRH receptor binding. Although the degeneration of cholinergic neurons corresponds with the severity of dementia, both phenomena also correspond with the decrease in CRH neuronal activity, although a high rate of ACTH and cortisol nonsuppression after dexamethasone was reported (Krishnan et al., 1988). This discrepancy points to an involvement of other neurotransmitters and/or neuropeptides in the regulation of the HPA system. For example, the decrease in somatostatin, which is the most consistent neuropeptide change in Alzheimer’s disease and which is also implicated in HPA neuroregulation, may play a key role in HPA system abnormalities in dementia (Davies et al., 1980; Serby et al., 1984; Sunderland et al., 1987; but: Whitford et al., 1988). A recent study in rats supports this view by evidencing that reduction of hippocampal somatostatin content reduced the corticosterone-suppressing effect of dexamethasone (Ferrara et a!., 1991). An earlier investigation by Wolkowitz and coworkers (1987) explored the relationship between somatostatin and glucocorticoids from a different angle: they administered prednisolone (80 mg daily for 5 days) to healthy volunteers and found significant reductions in CSF-somatostatin-like immunoreactivity. These studies demonstrate convincingly the mutual interdependencies between somatostatin and glucocorticoid regulation, the causal relationship of which still remains obscure. However, these findings provide an explanation for the high incidence of DST non-suppression found in Alzheimer’s disease. Hypothalamic somatostatin inhibits growth hormone (GH) release from the anterior pituitary and counterbalances the stimulating effects of growth hormone-releasing hormone (GHRH) upon GH secretion (Reichlin, 1987). Provided that the somatostatinergic system is compromised in Alzheimer’s disease one would expect dysfunctional GH regulation in patients with this dementing disorder. Indeed, several studies confirmed an either overshooting or delayed GH response to GHRH challenge in Alzheimer’s disease patients in comparison to age-matched control subjects (Cacabelos

et al., 1988; Nemeroff et al., 1989). A more exhaustive exploration of the functional integrity of GH secretion in Alzheimer’s disease revealed similar 24-h secretory profiles of spontaneous GH secretion in patients and age-matched controls and indistinguishable GH responses to a clonidine and GHRH challenge between the two groups; that is, no prominent changes in GH regulation were seen. However, patients with an early onset of the dementing disorder had a larger GH response to GHRH when compared to those with late onset dementia (Heuser et al., 1992). From this and the above-mentioned studies it can be concluded that Alzheimer’s disease patients of more advanced stages of the disorder have subtle disturbances of G H regulation, most likely due to an abnormality in somatostatinergic neurons of the hypothalamus. How the abnormalities of the HPA axis with regard to the CRH-ACTHcortisol loop and the somatostatin-GH system are entwined in dementia is subject of current research. With regard to normal human aging, a continuous decrease of the amount of GH released per secretory burst (most prominent during the night) has been reported (for review, see Meites, 1990). It has been hypothesized that the age-related decline of hypothalamic catecholamines, which in turn modulate GHRH and somatostatin release, is ultimately responsible for an increase of somatostatinergic tone and a decrease of GHRH activity in aged individuals (Sonntag et al., 1986; Hornykiewicz, 1987). However, replenishment of brain catecholamine stores was unsuccessful in restoring “normal” GH responses to GHRH in old rats (Lima et al., 1989). Preclinical data suggest that with aging, for reasons unknown, high-affinity GHRH-binding sites at the rat somatotrophs markedly decreased in capacity paralleled by diminution of GH secretion, whereas low-affinity GHRH-binding sites increased in capacity (Abribat et al., 1991). These differential effects of aging upon low- and high-affinity binding sites may explain the fact that repetitive injections of GHRH in older humans were capable of restoring “normal” GH responses (Iovino et al., 1989). Re: cently, the efficacy of medium-term (6 months) of

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subcutaneous GH administration to elderly men as a preventive measure against some age-related metabolic and endocrine changes has been explored with promising perspectives (Rudman et al., 1990). However, untoward and possibly grave side effects of long-term administration of GH need to be carefully evaluated and balanced against the still not sufficiently proven beneficial outcome of such a treatment. Further, central effects of varying brain levels of GHRH, GH and/or somatostatin are largely unexplored. There is limited evidence that GHRH might facilitate learning in rats in a dose-dependent manner (Cacabelos et al., 1987). An acute trial with rather high doses of a long-acting somatostatin analog in Alzheimer’s patients was unsuccessful in improving cognitive function in these patients although CSF levels of somatostatin markedly increased through peripheral infusion of this neuropeptide hormone (Mouradian et al., 1991). Taken together, in aging and dementia neuroendocrine changes occur, which in turn might propel forward further pathophysiological changes frequently observable in aging, e.g., by modulating neurotransmitter systems or the capacity of peripheral glands (pancreas, thyroid, gonads).

Conclusions The progress and availability of molecular and cell biology, recent developments in animal behavioral research and increasingly sophisticated clinical research have greatly advanced our understanding of brain function. This is true regarding the brain as regulator and target of hormones under physiological conditions such as stress, and pathological conditions such as Cushing’s disease. It is also true for research on behavior, the most complex form of biological organization. The disorders discussed in this chapter can be viewed as experiments by nature, which despite the diversity of their clinical phenotypes share one common feature: a hyperdriven HPA system, allowing for a concerted effort in basic and clinical research. From the evidence presented here it seems fair to propose that: (1) CRH integrates not only the hormonal, but also the physiological

and behavioral pattern in response to environmental and endogenous challenges perceived as stress; and (2) continuous alterations in the fine-tuned neuroendocrine pathways result in overt psychopathology, perpetuate clinical symptoms and may lower the threshold for development of full-blown clinical syndromes in individuals carrying a genetic risk for psychiatric disorders. While it seems plausible that inappropriate regulation of CRH neurons plays a major role in the pathogenesis of the diseases discussed here, we do not know which factors cause this disturbance. The situation is particularly blurred by what we like to call the “bumerang” effects of the consistent attempts of the HPA system to maintain homeostasis or functional integrity. For example, autonomous CRH hypersecretion, adaptational activation of the HPA system to severe stressors or - for that matter - recurrent episodes of major psychiatric illnesses ultimately result in increased plasma cortisol concentrations, which might trigger a host of harmful upstream (brain) or downstream (peripheral tissues, e.g., immune cells) derangements. The studies reviewed in this chapter lead to the conclusion that the neuroendocrine and behavioral “systems” are inextricably intertwined. Given this bidirectional loop, distinctions between “cause” and “effect” are clearly only useful within a limited experimental context. This implies that basic neuroscientists need to maintain a continuous dialogue with clinical investigators and vice versa. Keeping this in mind, the ambitious goal of understanding how neural and endocrine systems regulate behavior and how psychopathology develops may yet be achieved.

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CHAPTER 28

Endogenous pyrogens in the CNS: role in the febrile response Clifford B. Saper and Christopher D. Breder Departments of Pharmacological and Physiological Sciences and Neurology, the Committee on Neurobiology, and the Brain Research Institute, University of Chicago, Chicago, IL 60637, U.S.A.

Introduction

The febrile reaction, or the cerebral component of the acute phase reaction, is an integrated autonomic, endocrine and behavioral response that is coordinated by the hypothalamus. It includes an increase in body temperature, due in large part to redistribution of blood flow to deep vascular beds; alterations in the secretion of a number of hormones, including vasopressin and corticosteroids; and malaise, sleepiness, shivering and anorexia (Dinarello, 1984). All of these responses are thought to be due to the actions on the brain of circulating pyrogenic cytokines, including interleukin-1 (IL-1), tumor necrosis factor (TNF), interleukin-6 and a2-interferon (Krueger et al., 1987; Shibataet al., 1989; Blatteis et al., 1990). Because the cytokines are relatively large proteins (15000 - 25000 kDa molecular weight), they are excluded from the blood-brain barrier. In fact, studies tracing the fate of labeled cytokines indicate that they do not penetrate the brain, except at the circumventricular organs (Hashimoto et al., 1991). The circumventricular organs are small, specialized neural regions, including the organum vasculosum of the lamina terminalis (OVLT) and subfornical organ (at the rostra1 end of the third ventricle) and the area postrema (at the caudal end of the fourth ventricle) adjacent to the ventricular system in mammalian brains, that have fenestrated capillaries, and hence no blood-brain-barrier (Broadwell

and Brightman, 1976; Gross et al., 1986). Neurons in these regions respond to a variety of circulating hormones, and project into the brain, influencing a range of autonomic, endocrine and behavioral responses. For example, angiotensin I1 excites neurons in the subfornical organ, which then project into the hypothalamus and basal forebrain, to control blood pressure, vasopressin secretion and drinking behavior (Lind and Johnson, 1982; Lind et al., 1984). It is remarkable that some of these angiotensin-responsive neurons also are immunoreactive for angiotensin 11, and are thought to use the hormone as a neuromodulator in the CNS control of blood pressure and volume. Similarly, other systemic hormones have recently been found to act as neuromodulators in the brain, performing functions that are coordinated with their roles as hormones. For example, atrial natriuretic peptide as a CNS neuropeptide opposes the actions of angiotensin I1 on blood pressure, vasopressin secretion and drinking behavior (Antunes-Rodrigues et al., 1985; Samson, 1985; Standaert et al., 1987; Levin et al., 1989). Cholecystokinin is found in central pathways involved in gastrointestinal regulation, and has been implicated in the central control of feeding (Fulwiler and Saper, 1985). Local application of cytokines within the CNS produce responses similar to those that are seen during systemic cytokine injections. Specifically, IL-10 causes corticotropin-releasing hormone (CRH) cells to secrete CRH (Sapolsky et al., 1987; Tsagarakis et

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al., 1989; Ohgo et al., 1991). Local application of IL-lp causes cold-responsive neurons in the medial preoptic area to increase firing and warm-responsive neurons to decrease firing (Hori et al., 1988; Nakashima et al., 1989; Shibata et al., 1989). Similarly, iontophoresis of IL-10 or TNFa into the lateral hypothalamus suppresses the firing of glucose-inhibited neurons (Plata-Salaman et al., 1988), whereas iontophoresis of a2-interferon or IL-la stimulates the firing of glucose-excited neurons in the ventromedial nucleus of the hypothalamus (Kuriyama et al., 1990). Thus, although circulating cytokines may never enter the CNS, there are central responses to cytokines that are coordinated with their systemic roles. These observations suggest the existence of a central cytokine system that may be involved in regulation of the febrile response. Role of prostaglandins as mediators in the febrile reaction

There is considerable evidence that prostaglandins (PGs), particularly of the E2 series, play a critical role in mediating the febrile response. Administration of inhibitors of PG synthesis, such as aspirin or indomethacin, can block most of the components of the febrile reaction (including the increased body temperature and corticosteroid secretion, as well as the behavioral effects; see Sobrado et al., 1983; Morimoto et al., 1988, 1989; Tsagarakis et al., 1989). Similarly, the responses of thermosensitive, glucose-responsive or CRH-secreting neurons to local application of cytokines can be blocked by administration of indomethacin (Hori et al., 1988; Plata-Salaman et al., 1988; Kuriyama et al., 1990; Navarra et al., 1991). Furthermore, central administration of PGs, particularly of the E2 series, can reproduce the febrile response (Milton and Weindlandt, 1971; Morimoto et al., 1988). Local application of PGs within the brain indicate that the most sensitive areas are within the hypothalamus (Stitt, 1985, 1986, 1991). While the entire febrile reaction can be elicited by PGE2 injection into the medial preoptic region, and the highest density of PGE2 receptors in the brain is in the median

preoptic nucleus (Matsumura et al., 1990), individual components of the febrile response may be elicited by local application of PGE2 at some distance from the preoptic region. For example, the highest body temperatures are reached with administration of PGE2 into the tuberal hypothalamus (Morimoto et al., 1988). Perhaps the most persuasive evidence for the key role of the preoptic region is that the febrile response to circulating cytokines can be blocked with injection of indomethacin directly into the medial preoptic region (Stitt, 1986). The time course of the febrile response also indicates the role of an intermediary messenger, such as PGs, as an obligate step. Following the injection of cytokines into the circulation, there is an interval of about 5 - 10 min before the febrile response begins (Dinarello, 1984). Similarly, following the local administration of cytokines into the brain, there is typically a 5 - 10 min lag time before neuronal responses are seen (Hori et al., 1988; Plata-Salaman et al., 1988; but see Saphier and Ovadia, 1990, who found responses of putative CRH cells within 1 - 2 min). These delays are consistent with the generation of an intermediate, such as PGE2, from arachidonic acid, but are probably not long enough for the synthesis of new enzyme (Raz et al., 1988). Hence, any cells that synthesize PGs in the initiation of the febrile response probably already express the necessary enzymes, such as cyclooxygenase, in the basal state. These observations suggest that PGs occupy a pivotal position in the transduction of the circulating cytokine signal via the preoptic area into a central febrile response. Whether PGs act as intracellular second messengers or as intercellular signaling molecules in this pathway remains unknown. In fact, it has not even been identified which cells in the hypothalamus have the capabilities of synthesizing PGs. Cyclooxygenase in the central nervous system

It is widely assumed that PGs are made primarily by non-neuronal cells in the CNS. Tissue culture experiments suggest that PGs are produced by astro-

42 1

cytes (Fontana et al., 1982), whereas immunocytochemical studies in mice indicate that PGD2 synthetase is produced by neurons during the perinatal period, but only by oligodendrocytes in adult rats (Urade et al., 1987). On the other hand, a recent study suggested the presence of cyclooxygenase (COX), the key enzyme in PG synthesis, in many CNS neurons in monkeys (Tsubokura et al., 1991). However, the immunocytochemical adsorption controls did not completely abolish staining with this antiserum. Although immunoblotting monkey brain with the antiserum produced a single band with electrophoretic mobility similar to COX, it is not clear that the substance being stained in formalin-fixed tissue was identical with this substance, or that it had COX activity. With these problems in mind, we examined the localization of COX in the sheep brain using a combination of immunocytochemistry, immunoblotting and enzyme assay (Breder and Saper, 1990, and unpublished observations). First, we used three different polyclonal sera antisera against sheep seminal vesicle COX; all staining with each antiserum was blocked by preadsorption with COX. The antisera recognized a single band on immunoblotting of immunoprecipitated sheep brain COX, which had electrophoretic mobility identical to that of seminal vesicle COX. Furthermore, the levels of COX enzyme activity in 15 microdissected regions of the brain correlated closely with the immunocytochemical pattern observed. Non-neuronal COX-like immunoreactive (COXir) cells were seen in the meninges (mainly macrophages) and along blood vessels (pericytes). In the parenchyma of the sheep brain, however, nearly all of the COXir cells demonstrated a neuronal morphology (Fig. 1). Small numbers of small COXir cells were seen in the OVLT. In addition, substantial numbers of COXir neurons were seen at virtually all levels of the CNS, primarily in integrative systems concerned with sensory and visceral control. Our recent preliminary observations on COXir staining in human brain, using the same antiserum, indicates some similarities in the distribution of COXir neurons in the hypothalamus and basal forebrain, but also major differences (Breder,

Malin and Saper, unpublished observations). It is not yet clear whether the staining in human brains is specific for COX; furthermore, it is possible that the prolonged agonal state of the patients may have affected the expression of COX in the brain. For this reason, the description below refers mainly to our more reliable and complete results in the normal sheep brain, fixed 10 - 20 min after death. Comparisons with human material are made where appropriate. The densest accumulations of COXir neurons in the sheep occur in the hippocampus, amygdala and basal forebrain. In the hippocampal formation, the granule cells of the dentate gyrus were heavily immunoreactive. Clusters of COXir non-pyramidal neurons were seen in the stratum oriens, particularly in the subicular and CA1 fields (Fig. 1); only occasional pyramidal cells were stained, mainly in the CA1 and CA3 fields. In the amygdala, particularly dense accumulations of COXir cells were seen in the

Fig. 1. A bright-field photomicrograph of COXir neurons in the subicular field of the hippocampal formation of the sheep. Note that most of the immunoreactive cells are of the non-pyramidal type, although a few pyramidal neurons are also COXir. Scale bar, loo gm.

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basal and medial nuclei. In the human brain, the most striking collection of COXir neurons in the medial temporal lobe was seen in the CA3 hippocampal field, and none were identified in the amygdala. In the sheep basal forebrain, numerous COXir neurons were seen in the bed nucleus of the stria terminalis and in the substantia innominata, intermixed with the magnocellular basal neurons. Large numbers of COXir neurons were also seen in the hypothalamus, in the dorsomedial and tuberomammillary nuclei; smaller numbers of COXir neurons were found in many of the medial hypothalamic nuclei, including the OVLT, and in the lateral preoptic and hypothalamic areas. In the human brain, the COXir neurons were found in the OVLT, as well as in the magnocellular basal nucleus and several hypothalamic cells groups, including the supraoptic, paraventricular and arcuate nuclei, the lateral hypothalamic area, and the sexually dimorphic intermediate nucleus (Swaab and Fliers, 1985; Allen et al., 1989; Hofman and Swaab, 1989; Saper, 1990). COXir neurons were also found in autonomic control areas of the brain-stem, such as the periaqueductal gray matter, the parabrachial nucleus and the nucleus of the solitary tract in the sheep, but these regions have not yet been examined in the human brain. COXir neurons were abundant in the cerebral cortex of the sheep, where they were found in specific laminar patterns that varied across the different cortical areas. COXir neurons were more sparse in the human cerebral cortex, and mainly seemed to be of the non-pyramidal, interneuronal type. Sensory systems at lower levels of the neuraxis have so far been examined only in the sheep. The ventral lateral geniculate nucleus was the only part of the thalamus found to contain COXir cells. In the brain-stem, COXir neurons were found in many sensory structures, such as the superior and inferior colliculi and the dorsal column nuclei. Interestingly, the COXir cells seemed to be more closely associated with the sensory regions receiving descending cortical modulation than with the areas receiving direct sensory afferents. For example, COXir neurons

were found in structures associated with descending modulation of nociception, such as the periaqueductal gray matter and the nucleus raphe magnus (Abols and Basbaum, 1981) and in parts of the superior colliculus and dorsal column nuclei receiving descending cortical afferents (Kuypers and Tuerk, 1964; Illing and Graybiel, 1986). It was also striking that COXir material was found only in the cell bodies and dendrites of labeled neurons; no fiber pathways or terminal fields were visualized. These observations suggest that PGs may be used within the brain for intracellular or perhaps local (paracrine) intercellular communication, rather than as synaptic transmitters. Although most COXir systems in the brain are probably not involved in the febrile response, one paracrine function of PGs generated at the OVLT (and possibly other circumventricular organs as well) may be to activate the central neuronal system responsible for the genesis of the febrile reaction. In this regard, it is interesting that the densest concentration of PGE2 receptors in the rat brain is located in the median preoptic nucleus, directly adjacent to the OVLT (Matsumura et al., 1990). Cytokines as neuromodulators Despite this progress in identifying the CNS sources of PGs, the effector cells within the CNS that are acted upon by PGs to produce the febrile reaction remain unknown. On the basis of analogy with other hormonal systems, in which the circulating peptide hormone also serves as a CNS neuromodulator in control of the same set of physiological responses, we hypothesized that cytokines may be present as neuroactive substances with CNS neurons regulating the febrile reaction. We first investigated the immunohistochemical staining of the human brain with antisera against recombinant human interleukin-lp (IL-1p) (Breder et al., 1988). Staining with these antisera was adsorbed by preincubation with IL-10 but not IL-la, and antisera against IL-la did not stain the human brain. Immunoreactive neurons were mainly found in the periventricular and arcuate nuclei of the

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.. . .

.-

Fig. 2. A series of line drawings illustrating thedistribution of ILloir axons and terminals in the human hypothalamus. Panels A a n d B show IL-lpir innervation at the preoptic and tuberal levels, respectively; panel A ’ demonstrates the distribution of vasopressin-like immunoreactive neurons at the preoptic level in an adjacent section from the same brain. Note that there is intense innervation of the ventrolateral quadrant of the paraventricular nucleus, which contains a cluster of vasopressin neurons, but that there is also heavy innervation of the more medial parts of the nucleus, which contain neurons that regulate autonomic function, and tuberohypophyseal cells that secrete corticotropinreleasing hormone. AC, Anterior commissure; AT, anterior thalamic nuclei; ARH, arcuate nucleus of the hypothalamus; BST, bed nucleus of the stria terminalis; DBB, nucleus of the diagonal band of Broca; DMH, dorsomedial nucleus of the hypothalamus; Fx, column of the fornix; GP, globus pallidus; IC, internal capsule; Inf, infundibulum; LHA, lateral hypothalamic area; LT, lateral tuberal nucleus; PVH, paraventricular nucleus of the hypothalamus; OT, optic tract; RT, reticular nucleus of the thalamus; SO, supraoptic nucleus; ST, stria terminalis; VMH, ventromedial nucleus of the hypothalamus.

424

hypothalamus. Immunoreactive fibers innervated the key sites in the CNS for production of the febrile responses (see Fig. 2), including the medial preoptic area (which is involved in thermoregulation), the paraventricular nucleus (which contains vasopressin and CRH cells, as well as autonomic control neurons), the ventromedial nucleus (which is involved in regulation of feeding) and the lateral hypothalamic area (which is implicated in the control of feeding, autonomic responses and arousal). Additional IL-lpir axons and terminals were seen in brain-stem sites involved in regulation of wake-sleep cycles, including the locus coeruleus and dorsal raphe nucleus, and in areas involved in autonomic regulation, including the periaqueductal gray matter, the parabrachial nucleus and the nucleus of the solitary tract. However, it was difficult to obtain adequate IL-lpir staining of human brain when the post-mortem interval until autopsy was greater than 8 h. In addition, staining was often reduced in subjects that had suffered a prolonged agonal period prior to death. In part this variability may have reflected physiological regulation of cytokine levels during illness, but it was impossible to control for the deterioration of CNS function that occurs under the same conditions. Hence, we turned to an animal model for further localization of cytokines in the CNS. Although several recent reports have indicated the presence of IL-1p in neuronal systems in the rat brain (Bandtlow et al., 1990; Lechan et al., 1990; Minami et al., 1991), our antiserum did not provide consistent staining in the rat or other experimental animals. Our recent work has involved the identification of TNFa in the mouse brain (Breder and Saper, 1988, and unpublished observations). Staining with this antiserum is adsorbed by preincubation with TNFa, and immunoblot analysis of immunoprecipitated material from mouse brain shows a single band at approximately 18 kDa M,, corresponding exactly to the position of immunoprecipitated recombinant mouse TNFa. Immunoreactive neurons were identified, in colchicine-treated mice, in the paraventricular nucleus and adjacent lateral hypothalamic area and dor-

somedial nucleus. TNFair fibers were found to innervate a unique pattern of structures that, like those receiving IL-lpir input in the human brain, are thought to be involved in the febrile reaction. The densest innervation in the hypothalamus was seen in the dorsomedial nucleus, a cell group that is involved in regulation of feeding and autonomic response (Saper et al., 1976; Ter Horst and Luiten, 1986; Bernardis and Bellinger, 1987). In addition, TNFair terminals were seen in the OVLT, the medial preoptic and paraventricular nuclei and lateral hypothalamic area (Fig. 3). The ventromedial nucleus, by contrast, received only light innervation. In the brain-

Fig. 3. A dark-field photomicrograph of the tuberal hypothalamus in a mouse stained with antiserum against TNFa, using an immunoperoxidase method. Note the dense cluster of immunoreactive fibers in the perifornical region of the lateral hypothalamic area. 3V, Third ventricle; fx, fornix; LHA, lateral hypothalamic area; VMH, ventromedial nucleus of the hypothalamus. Scale bar, 200 pn.

425

stem, TNFair axons were seen in sites involved in autonomic regulation (periaqueductal gray matter, parabrachial nucleus, nucleus of the solitary tract, ventrolateral medullary reticular formation) and control of wake-sleep cycles (dorsal raphe nucleus, locus coeruleus). However, the most intense innervation was seen in the preganglionic cell groups that contribute to gastrointestinal control, including the dorsal motor vagal nucleus (which contains stomach and intestinal preganglionic neurons) and the compact part of the nucleus ambiguus (which is involved in the control of esophageal motility) (Fox and Powley, 1985; Shapiro and Miselis, 1985; Bieger and Hopkins, 1987). It is interesting in this regard that a major activity of TNFa (and one which led to it being called “cachectin” in some laboratories) is to suppress appetite (Tracey et al., 1990). Most recently, we have used cRNA probes against the TNFa mRNA to identify TNFa-synthesizing neurons in the mouse brain. Our preliminary results to date indicate that levels of expression are probably very low in the basal state, but that after immune stimulation with lipopolysaccharide (a key component in bacterial endotoxin), TNFa mRNA is induced in neurons in the brain during the febrile reaction. However, the cell groups expressing TNFa mRNA during the febrile response may be more extensive than those visualized with immunocytochemistry, including a variety of additional sites in the hypothalamus and brain-stem involved in autonomic and endocrine control. A new model for endogenous pyrogens in the brain Our studies have demonstrated that both PG synthetic capacity and cytokine production are widely distributed among neurons in the CNS, particularly in cell groups believed to be involved in the febrile reaction. Based upon these observations, we propose the following steps in the transduction of a circulating cytokine signal to a CNS-mediated febrile reaction: ( I ) Circulating cytokines act on the OVLT to cause PG generation. Most previous studies have

focused on the OVLT as a site of cytokine action (Blatteis et al., 1987; Katsuura et al., 1990), and we have identified COXir neurons within this structure in the basal state. It is possible that at least some components of the febrile reaction may be influenced by cytokine actions at other circumventricular organs, such as the area postrema. However, these structures do not contain COXir cells, so that the initial physiological responses are not likely to depend upon PGs. (2) PGs dvfusefrom the OVL Tinto the brain. As circulating cytokines have not been found to diffuse into the brain (Hashimoto et al., 1991), and PGs are an obligate step in transducing many components of the febrile reaction (Sobrado et al., 1983; Morimoto et al., 1989;Tsagarakis et al., 1989), a parsimonious explanation would be that the PG step is proximal in the pathway. The localization of COXir activity in cell bodies and dendrites suggests that it may serve a paracrine function, as it does in the periphery, diffusing short distances to affect nearby cell groups (Campbell, 1990). This interpretation would be consistent with the demonstrated localization of dense PGE2 receptor sites in the median preoptic nucleus, adjacent to the OVLT (Matsumura et al., 1990). (3) PGs act on hypothalamic neurons that use cytokines as mediators. PGs diffusing into the brain would have the opportunity to activate nearby cell groups employing a variety of neurotransmitters. The ability of cytokines, applied at sites deep within the brain, to produce a typical febrile response suggests that some of the cells responsible for the response to PGs may use cytokines as neuromodulators. The cells that are IL-lpir in the human hypothalamus and those that are TNFair in the mouse hypothalamus are located within a few millimeters of the OVLT. These cells, and perhaps others that make these cytokines at levels too low to be detected by immunocytochemistry in the basal state, may be activated by locally produced PGs to release cytokines at distal sites within the brain. One of the sites that receives cytokine immunoreactive innervation is the OVLT, suggesting that the central cytokine system may feed back on the OVLT to amplify the circulating cytokine signal.

426

Cytokines act as neurornodulatorsto help induce the febrile reaction. Cytokines may act directly on local neurons in key sites controlling autonomic, endocrine and behavioral manifestations of the febrile response. It is likely, of course, that they are just one of the classes of neuroactive substances that participate in this response, and that some components of the febrile reaction may proceed without the intercession of central cytokines. It is also possible that central cytokines may induce PG production in neurons containing COX. Local PG action may amplify the cytokine effect, and may induce other local neuronal cell bodies that are capable of synthesizing cytokines to do so. The further investigation of this hypothesis will require combinations of methods including the injection of inhibitors of cytokine or PG synthesis or action into the CNS. However, it is not likely to be productive to inject cytokines into the CNS. Introduction of cytokines into the CNS results in activation of microglial cells, which as tissue macrophages are capable of producing cytokines of their own. Hence it is difficult, if not impossible, to control the dosage or time course of administration of cytokines by this method. Furthermore, cytokine injections result in local tissue damage that is not seen during a typical febrile reaction. Hence, it is likely that if cytokines are secreted in the brain during the febrile reaction, their action must be very localized, and largely restricted to neurons. Our data for localization of the endogenous pyrogenic substances within the brain provides a starting place for examining the roles played by cytokines and PGs of neuronal origin in the febrile reaction. Summary and conclusions The febrile reaction is an integrated endocrine, autonomic and behavioral response, coordinated by the hypothalamus, that includes certain components of the stress response, such as elevated corticosteroid secretion. It is produced by the actions of circulating cytokines, such as interleukin-1 (IL-1) and tumor necrosis factor (TNF), on the organum vasculosum of the lamina terminalis (OVLT), re-

sulting in the secretion of prostaglandin E2, which initiates a variety of responses, including elevation of body temperature and corticosteroid secretion. Although circulating cytokines apparently do not enter the brain, injections of IL-1 or TNF well within the blood-brain barrier produce identical effects. We have examined the localization of possible central sources of cytokines and prostaglandins, using immunohistochemistry, immunoblotting and enzyme assay. Our data indicate that in the brain cyclooxygenase, the key enzyme in the synthesis of prostaglandins, is found in neurons in the OVLT, but is also made by neurons in many sensory and visceral regulatory systems. We present evidence also that IL-1p in the human brain and TNFa in the mouse may be present in the central nervous system as neuromodulators that are important for producing the autonomic, endocrine and behavioral components of the febrile reaction. We propose a sequence of events in the febrile reaction involving: (1) action of circulating cytokines on cyclooxygenase containing neurons within the OVLT to produce local prostaglandin secretion; (2) local diffusion of prostaglandin E2 into the preoptic and anterior hypothalamic areas; (3) action of prostaglandin E2 on cytokine containing neurons in the preoptic and anterior hypothalamic areas; and (4) release of cytokines from neuronal terminals at distal sites involved in producing the autonomic, endocrine and behavioral components of the febrile reaction. Acknowledgements The authors thank Quan Hue Ha for technical help on the experimental studies from our laboratory. This work was supported by grants NS22835 and HD07009, the Brain Research Foundation and the William D. Mabie Research Fund. References Abols, I.A. and Basbaum, A.I. (1981) Afferent connections of the rostra1 medulla of the cat: a neural substrate for midbrainmedullary interactions in the modulation of pain. J. Comp. Neurol., 201: 285 -291. Allen, L.S., Hines, M., Shryne, J.E. and Gorski, R.A. (1989)

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reactive innervation of the ventromedial hypothalamus in the rat: possible substrate for autonomic regulation of feeding. Neurosci. Lett., 53: 289- 296. Gross, P.M., Sposito, N.M., Pettersen, S.E. and Fenstermacher, J.D. (1986) Differences in function and structure of the capillary endothelium in gray matter, white matter and a circumventricular organ of rat brain. Blood Vessels, 23: 261 - 270. Hashimoto, M., Ishikawa, Y., Yokota, S., Goto, F., Bando, T., Sakakibara, Y. and Iriki, M. (1991) Action site of circulating interleukin-I on the rabbit brain. Brain Res., 540: 217 - 223. Hofman, M.A. and Swaab, D.F. (1989) The sexually dimorphic nucleus of the preoptic area in the human brain: a comparative morphometric study. J. Anat., 164: 55 - 72. Hori, T., Shibata, M., Nakashima, T., Yamasaki, M., Asami, A , , Asami, T. and Koga, H . (1988) Effects of interleukin-1 and arachidonate on the preoptic and anterior hypothalamic neurons. Brain Res. BUN., 20: 75 - 82. Illing, R. and Graybiel, A.M. (1986) Complementary and nonmatching afferent compartments in the cat’s superior colliculus: innervation of the acetylcholinesterase-poor domain of the intermediate gray layer. Neuroscience, 18: 373 - 394. Katsuura, G . , Arimura, A., Koves, K. and Gottschall, P.E. (1990) Involvement of organum vasculosum of lamina terminalis and preoptic area in interleukin lp-induced ACTH release. A m . J. Physiol., 258: E163-EI71. Krueger, J.M., Dinarello, C.A., Shoham, S., Davenne, D., Walter, J. and Kubillus, S. (1987) Interferon a-2 enhances slow-wave sleep in rabbits. Znt. J. Zmmunopharmacol., 9: 23 - 30. Kuriyama, K., Hori, T., Mori, T. and Nakashima, T. (1990) Actions of interferon a 2 and interleukin-la on the glucose responsive neurons in the ventromedial hypothalamus. Brain Res. Bull., 24: 803 - 810. Kuypers, H.G.J.M. andTuerk, J. (1964)Thedistribution ofcortical fibers within the nuclei cuneatus and gracilis in the cat. J. Anat., 98: 143 - 162. Lechan, R.M., Toni, R., Clark, B.D., Cannon, J.G., Shaw, A.R., Dinarello, C.A. and Reichlin, S. (1990) Immunoreactive interleukin-l/3 localization in the rat forebrain. Bruin Res., 514: 135- 140. Levin, E.R., Mills, S. and Weber, M. (1989) Central nervous system mediated vasodepressor action of atrial natriuretic factor. Life Sci., 44: 1617- 1624. Lind, R.W. and Johnson, A.K. (1982) Subfornical organmedian preoptic connections and drinking and pressor responses to angiotensin 11. J. Neurosci., 2: 1043- 1051. Lind, R.W., Swanson, L.W. andGanten, D. (1984)Angiotensin I1 immunoreactivity in the neural afferents and efferents of the subfornical organ in the rat. Bruin Rex, 321: 209-215. Matsumura, K., Watanabe, Y., Onoe, H. and Hayaishi, 0. (1990) High density of prostaglandin E2 binding sites in the anterior wall of the third ventricle: a possible site of hyperthermic action. Brain Res., 533: 147- 151.

428 Milton, A.S. and Weindlandt, S. (1971) Effects on body temperature of prostaglandins of the A, E, and F series on injection into the third ventricle of unanesthetized cats and rabbits. J. Physiol. (Lond.), 218: 325 - 336. Minami, M., Kuraishi, Y., Yamaguchi, T., Nakai, S., Hirai, Y. and Satoh, M. (1991) Immobilization stress induces interleukin-lp messenger RNA in the rat hypothalamus. Neurosci. Lett., 123: 254-256. Morimoto, A . , Murakami, N., Nakamori, T. and Watanabe, T. (1988) Ventromedial hypothalamus is highly sensitive to prostaglandin E2 for producing fever in rabbits. J. Physiol. (Lond.), 397: 259- 268. Morimoto, A., Murakami, N., Nakamori, T., Sakata, Y. and Watanabe, T. (1989) Possible involvement of prostaglandin E in development of ACTH response in rats induced by human recombinant interleukin-1. J. Physiol. (Lond.), 41 1: 245 256. Nakashima, T., Hori, T., Mori, T., Kuriyama, K. and Mizuno, , K. (1989) Recombinant human interleukin-10 alters the activity of preoptic thermosensitive neurons in vitro. Brain Res. Bull., 23: 209-213. Navarra, P., Tsagarakis, S., Faria, M.S., Rees, L.H., Besser, G.M. and Grossman, A.B. (1991) Interleukins-1 and interleukins-7 stimulate the release of corticotropin-releasing hormone-41 from rat hypothalamus in vitrovia the eicasonoid cyclooxygenase pathway. Endocrinology, 128: 37 - 44. Ohgo, S., Nakatsuru, K., Oki, Y., Ishikawa, E. and Matsukura, S. (1991) Stimulation by interleukin-1 of the release of rat corticotropin-releasing factor, which is independent of the cholinergic mechanism, from superfused rat hypothalamoneurohypophyseal complexes. Brain Res., 550: 213 - 219. Plata-Salaman, C.R., Oomura, Y. and Kai, Y. (1988) Tumor necrosis factor and interleukin-I beta: suppression of food intake by direct action in the central nervous system. Brain Res., 448: 106 - 114. Raz, A., Wyche, A., Siegel, N. and Needleman, P. (1988) Regulation of fibroblast cyclooxygenase synthesis by interleukin-1. J. Biol. Chem., 263: 3022 - 3028. Samson, W.K. (1985) Atrial natriuretic factor inhibits dehydration and hemorrhage-induced vasopressin release. Neuroendocrinology, 40: 277 - 279. Saper, C.B. (1990)The hypothalamus. In: G. Paxinos(Ed.), The Human NervousSystem, Academic Press, San Diego, CA, pp. 389 - 413. Saper, C.B., Loewy, A.D., Swanson, L.W. and Cowan, W.M. (1976) Direct hypothalamo-autonomic connections. Brain Rex, 117: 305-312. Saphier, D. and Ovadia, H. (1990) Selective facilitation of putative corticotropin-releasing factor-secreting neurones by interleukin-1. Neurosci. Lett., 114: 283 - 288. Sapolsky, R., Rivier, C., Yamamoto, G., Plotsky, P. and Vale, W. (1987) Interleukin-1 stimulates the secretion of hypothalamic corticotrophin releasing factor. Science, 238: 522 - 524. Shapiro, R.E. and Miselis, R.R. (1985) The central organization

of the vagus nerve innervating the stomach of the rat. J. Comp. Neurol,, 238: 473 -488. Shibata, M., Blatteis, C.M., Krueger, J.M., Obal, F. and Opp, M. (1989) Pyrogenic, inflammatory and somnogenic responses to cytokines: differential modes of actions. In: E. Schonbaum and P. Lomax (Eds.), Thermoregulation: Research and Clinical Applications, Karger, Basel, pp. 69- 73. Sobrado, J., Moldawer, L.L., Bistrian, B.R., Dinarello, C.A. and Blackburn, G.L. (1983) Effect of ibuprofen on fever and metabolic changes induced by continuous infusion of leukocytic pyrogen (interleukin-1) or endotoxin. Infect. Zmmun., 42: 997- 1005. Standaert, D.G., Cechetto, D.F., Needleman, P. and Saper, C.B. (1987) Inhibition of the firing of vasopressin neurons by atriopeptin. Nature, 329: 151 - 153. Stitt, J.T. (1985) Evidence for the involvement of the organum vasculosum laminae terminalis in the febrile response of rabbits and rats. J. Physiol. (Lond.), 368: 501 - 51 1. Stitt, J.T. (1986) Prostaglandin E as the neural mediator of the febrile response. Yale J. Biol. Med., 59: 137 - 149. Stitt, J.T. (1991) Differential sensitivity in the sites of fever production by prostaglandin El within the hypothalamus of the rat. J. Physiol. (Lond.), 432: 99 - 110. Swaab, D.F. and Fliers, E. (1985) A sexually dimorphic nucleus in the human brain. Science, 228: 1112 - 1115. Ter Horst, G.J. and Luiten, P.G.M. (1986) The projections of the dorsomedial hypothalamic nucleus in the rat. Brain Res. Bull., 16: 231 -248. Tracey, K.J., Morgello, S., Koplin, B., Fahey, T.J., Fox, J., Aledo, A., Manogue, K.R. and Cerami, A. (1990) Metabolic effects of cechectinhmor necrosis factor are modified by site of production. 1. Clin. Invest., 86: 2014-2024. Tsagarakis, S., Gillies, G., Rees, L.H., Besser, M. and Grossman, A. (1989) Interleukin-1 directly stimulates the release of corticotrophin releasing factor from rat hypothalamus. Neuroendocrinology, 49: 98- 101. Tsubokura, S., Watanabe, Y., Ehara, H., Imamura, K., Sugimoto, O., Kagamiyama, H., Yamamoto, S . and Hayaishi, 0. (1991) Localization of prostaglandin endoperoxidase synthase in neurons and glia in monkey brain. Brain Res., 543: 15-24. Urade, Y., Fujimiya, N., Kaneko, T., Konishi, A., Mizuno, N. and Hayaishi, 0. (1987) Post-natal changes in the localization of prostaglandin D synthetase from neurons to oligodendrocytes in the rat brain. J. Biol. Chem., 262: 15132- 15136.

Discussion F.J.H. Tilders: In view of the fact that the arcuate nucleus does not exhibit a well-developed blood-brain barrier (Broadwell and Brightmann, 1976), I wonder what the evidence is of interleukin10 (IL-1) acting primarily on OVLT after which information is

429 transmitted to arcuate nucleus neurons. Is it not more likely that interleukin-10 acts directly on the arcuate nucleus? C.B. Saper: I agree that this is possible. Physiological studies to date have emphasized the role of the OVLT in fever, but I would not be surprised if cytokines had important effects at other circumventricular organs such as the median eminence. . H.P.H. Kremer: Can you speculate on the role of this IL-1 or tumor necrosis factor (TNF)-containing system in the physiology of childhood febrile convulsions related to brain development? C.P. Saper: In the absence of hard data, I can only speculate. I suspect that the febrile seizures result from effects either of cytokines or prostaglandins on the immature brain, rather than simply from an increase in body temperature. But this would have to be investigated directly. A.M.W. Van Dam: You can localize IL-lp peptide in the hypothalamus and the peptide has effects like thermoregulation in the hypothalamus. Is there also an IL-1 receptor identified in the hypothalamus? C.B. Saper: The physiological effects of IL-10 and TNFa on activity of thermoregulatory, glucoregulatory and corticotropin releasing factor (CRF) neurons indicate that there must be a receptor. However, ligand binding studies have demonstrated binding patterns that do not match very well the immunocytochemical (or physiological) data (Farrar et al., 1987). Mismatches of ligand binding with immunocytochemistry have been noted for many peptides (Herkenham, 1987). As we learn more about hormone and receptor families, the origin of this mismatch is becoming more clear. I expect that when cytokine families and their receptors have all been cloned, and their forms expressed in the brain have been mapped, that the match will be much closer. G.A. Bray: One comment and one question. In our hands peptides which stimulate food intake inhibit sympathetic nervous system and vice-versa. IL-1 is in this group since it reduces food intake and stimulates the sympathetic nervous system. My question relates to peptide transduction at the blood-brain barrier. As you noted, angiotensin and IL-1 may activate neurotransmitters in the CNS by acting on the blood-brain barrier at its perfora-

tions. Is it possible that insulin conveys its effects by a similar transduction mechanism? C.B. Saper: With respect to your comment, the effect of IL-10 and TNFa on the sympathetic system is likely to be complex. Fever, for example, requires patterned sympathetic activity to shut blood flow from the skin to deep vascular beds, to prevent heat loss. Our identification of TNFol-ir fibers innervating the sympathetic preganglionic column indicates that cytokines may be involved centrally in regulating patterned sympathetic response. To answer your question, I also have wondered for many years how insulin exerts its activity in the brain. The difficulty of many workers in identifying an insulin-ir system of neurons in the brain may indicate that a “brain insulin-like peptide” may exist, rather than the pancreatic polypeptide, in brain. It would be interesting to test whether insulin effects o n the brain require the participation of circumventricular organs. D.F. Swaab: The septum which is traditionally implicated in fever, has not beenincluded in your stainings. Does this structure not stain for cytokines? C.B. Saper: In the septa1 region, the bed nucleus of the stria terminalis contains IL-1p immunoreactive fibers in the human and TNFa immunoreactive fibers in the mouse. However, the relationship of these terminal fields to any role played by the septum in fever remains conjectural.

References Broadwell, R.D. and Brightman, M.W. (1976) Entry of peroxidase into neurons of the central and peripheral nervous systems from extracerebral and cerebral blood. J. Comp. Neurol., 166: 257 - 284. Farrar, W.L., Kilian, P.L., Ruff, M.R., Hill, J.H. and Pert, C.B. (1987) Visualization and characterization of interleukin I receptors in brain. J. Immunol., 139: 459-463. Herkenham, M. (1987) Mismatches between neurotransmitter and receptor localizations in brain: observations and implications. Neuroscience, 23: 1 - 38.

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SECTION IX

Psychiatric Diseases

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D.F. Swaab, M.A. Hofman, M. Mirmiran, R. Ravid and F.W. van Leeuwen (Eds.) Progresr in Brain Research, VoI. 93 @ 1992 Elsevier Science Publishers B.V. All rights reserved.

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CHAPTER 29

Endorphins and schizophrenia Victor M. Wiegant, Eric Ronken, Gabor Kovacs and David De Wied Depui-cment of Medical Phurmucology, Rudolf Mugnus Institute, Medical Facul?y, University of Uirecht, 3521 GD Utrecht. The Netherlands

Introduction

The quest for the endogenous ligand of the opiate receptor culminated in 1975 in the isolation from brain extracts of two pentapeptide molecules with opiate-like properties, the enkephalins (Hughes et al., 1975). Subsequently, a variety of endogenous peptides with opioid properties - now collectively termed “endorphins” - was identified. The discovery of the endorphins opened a whole new field of research and generated a tremendous number of studies on the biochemistry, pharmacology and physiology of these peptides. From this, it became clear that endorphins are produced in brain neurons, and that as neuropeptides they play a role as neuromessengers in the regulation of endocrine, autonomic and behavioral functions (for recent reviews, see De Wied and Jolles, 1982; O’Donohue and Dorsa, 1982; Olson et al., 1990; Van Giersbergen and De Jong, 1990; Van Nispen and Van Wimersma Greidanus, 1990). Soon after their discovery, similarities were recognized between pharmacological effects of opioids and certain symptoms of psychiatric illnesses. Mainly based on those similarities, the involvement of endorphins in a number of psychopathologies was hypothesized, for instance in addiction, mania, depression, autism and schizophrenia. Concerning a role of endorphins in schizophrenia, three types of hypotheses have been formulated. In the endorphin-excess hypothesis, the opioid character of endorphins stands central, whereas the 0-

endorphin deficiency and the y-type endorphin hypotheses focus on neuroleptic-like effects found with endorphins in animal experiments. The fundamental and clinical research induced by these hypotheses will be discussed in this review. Formation and biological activities of endorphins

Precursors andenzymatic generation of endorphins To date, three major families of endorphins are recognized, of which Met-enkephalin, 0-endorphin (0-E) and dynorphin are the prototype representatives. Each of these families is identified by its own high molecular weight precursor molecule, proenkephalin A, pro-opiomelanocortin (POMC) and pro-enkephalin B (or pro-dynorphin), respectively (Fig. 1). The primary structure of these precursor molecules has been elucidated by deduction from the nucleotide sequence of their respective cloned cDNA’s (Nakanishi et al., 1979; Kakidani et al., 1982;Nodaet al., 1982). Allthreeprecursors aresynthesized in neurons in the brain. Once synthesized at the rough endoplasmic reticulum in the cell body, a precursor molecule is translocated through the endoplasmic reticulum into the Golgi apparatus and then stacked in secretory vesicles. In itself, the intact precursors have no known biological activities. In order for the biologically active peptides to be expressed, the precursor must undergo a process of maturation and enzymatic processing consisting of a complex cascade of cleavage and modification steps that occur in a well-defined order and result in

434

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I' I !

I

Fig. 1 . Schematic representation of the pro-enkephalin A, proenkephalin B and pro-opiomelanocortin precursors and their main endorphin products. The Met- and Leu-enkephalin sequences are indicated by black and hatched boxes, the MSH-like sequences in POMC by dotted boxes. Asterisks mark potential Na-acetylation sites. Abbreviations: M, Met-enkephalin; MAGL, Met-Enk-Arg6-Gly7-Leu8; M-AP, Met-Enk-Arg6-Phe7; L, Leu-enkephalin; a-NE, a-neoendorphin; DN-A, dynorphinA; DN-B, dynorphin-B; ACTH, adrenocorticotropic hormone; (3-E, (3-endorphin; y-E, y-endorphin; DTyE, Des-Tyr'-y-E; DEyE, desenkephalin-y-E.

the formation of sets of biologically active neuropeptides that can be secreted from the cell (see Burbach and Wiegant, 1990). Enzymatic modifications of the precursors can already occur during translation. Subsequent cleavage by peptidases occurs primarily at sites distal to pairs of basic amino acids. These remain attached to the cleavage product and can be removed by carboxypeptidase activity (Fric-

ker et al., 1986). Several lines of evidence indicate, however, that proteolytic processing is not restricted to such sites. In particular, this appears to be the case with P-E (see below). Thus, biologically active peptides are liberated from the precursor and costored in vesicles that are transported along the axon towards the synaptic terminal. During maturing of the vesicles, the peptides can be subject to further proteolytic cleavage and to covalent modifications (e.g., acetylation). Eventually, the vesicles contain sets of highly processed biologically active endorphins, that can be released from the nerve terminal to act on specific receptors, thereby modulating the activity of neurons in the brain. The complexity of the enzymatic processing and its dramatic consequences for the biological activity of the peptide products is best exemplified in the 0-E family of endorphins that arises from the P-LPH region of POMC (Fig. 1). In POMC synthesizing cells in the brain, but also in the pituitary, the initial processing of the precursor yields, among other peptides, P-LPH-(l- 89) from which subsequently P-E-(l- 31) is cleaved. In its turn, P-E is a substrate for enzymes that catalyze the formation of a complex set of modified and shortened forms of this peptide. In vitro data evidence the presence in membrane fractions prepared from rat and human brain tissue of endopeptidase activity cleaving the Leu17 - Phel* peptide bond in 0-E, thus generating y-E (P-E-(l- 17)) (Austen et al., 1977; Austen and Smyth, 1977; Burbach et al., 1979, 1980, 1981). This y-endorphin generating endopeptidase is the rate-limiting enzyme in the formation of y-E. With a similar in vitro approach, the formation of a-E (B-E-(l- 16)) from y-E by carboxypeptidase activity was demonstrated, and both a-E and y-E are further cleaved by amino- and endo-peptidase activities present in brain fractions to yield their respective des-Tyr' and desenkephalin fragments (Burbach et al., 1979, 1980, 1981). a-E and y-E have been purified and identified from hypothalamic-hypophyseal extracts (Guillemin et al., 1976; Ling et al., 1976), and the presence of several a-and y-type endorphins as endogenous peptides has been demonstrated with var-

435

ious techniques in the anterior and intermediate lobes of the pituitary, and in the brain (Jegou et al., 1978; Fukata et al., 1979; Loeber et al., 1979; Verhoef et al., 1980, 1982; Dorsaet al., 1982; Dorsaand Majumdar, 1983; Wiegant et al., 1983, 1985, 1988; Burbach and Wiegant, 1984; Burbach et al., 1985; Dorsa and Keith, 1985). In addition to authentic y-E and a-E, we have recently found considerable quantities of 0-LPH sized, high-molecular weight products with the antigenic determinants of y-E and aE respectively in extracts of human pituitaries (Burbach and Wiegant, 1990). The elution position of these forms of y-E and a-E in size exclusion chromatography indicated that their molecular weight is only slightly lower than that of P-LPH-(l- 89). These data suggest that endoproteolytic cleavage occurs in the carboxyl terminus in 0-LPH, possibly as a relatively early step in the processing of POMC and constituting a specific post-translational pathway for the generation of a- and y-type endorphins. Another cleavage site in the 0-E sequence that does not involve paired basic amino acids, at least in the rat, is the Ala26-His27peptide bond. Following the generation of /3-E-( 1 - 27) from @E-(1 - 3 l), the His2’ residue can be removed from this peptide, resulting in the formation of P-E-(l- 26) (Smyth et al., 1981). In the rat brain, 0-E and related peptides (P-E-(1-31), P-E-(1-27), P-E-(1-26), a-E, y-E) can be Na-acetylated (Zakarian and Smyth, 1979, 1982; Smyth and Zakarian, 1980; Wiegant et al., 1983, 1985; Burbach et al., 1985). In fact, the acetylated forms of 0-E and related peptides are the major forms of these endorphins in a number of brain regions, particularly in regions of the forebrain, not only in the rat, but also in several other species (Weber et al., 1981; Smith et al., 1985; Smith and Funder, 1988). Acetylated endorphins are not detectable in the human brain (Wiegant et al., 1988; and unpublished results). Acetylation reduces the susceptibility to aminopeptidase attack thereby increasing metabolic stability, and, in addition, it modifies the bioactivity of the peptides.

Biological activities of endorphins As an endogenous opioid (Bradbury et al., 1976),

0-E is now known to influence a wide array of physiological functions. Apart from its analgesic properties, it has been implicated in the central regulation of many endocrine functions and autonomic activities, in the development of tolerance to drugs, in reward mechanisms, in learning and memory processes and in a variety of behaviors, such as sexual, social, consummatory and grooming behavior (O’Donohue and Dorsa, 1982; Olson et al., 1990). The carboxyl terminus of P-E-( 1 - 3 1) is important for the opioid character of the peptide. In the carboxyl terminally truncated forms of 0-E(1 -3l), e.g., P-E-(1-27) and &E-(1-26), the affinity for opiate receptors is markedly reduced and the ability to induce analgesia is ablated (Akil et al., 1981). In fact, P-E-(l-27) appears to be an antagonist for 0-E induced analgesia (Hammonds et al., 1984) and for the reinforcing effects of opioids (Bals-Kubik et al., 1988). The lipolytic effects of 0-E on adipocytes (Richter et al., 1987), its inhibitory action on glucose-stimulated insulin secretion (Rudman et al., 1983) and the binding of 0-E to complement (Schweigerer et al., 1982) also require the intact carboxyl terminus of P-E-( 1 - 3 1). For the expression of opioid activities of endorphins, an intact amino terminal Met-enkephalinmoiety (Tyr-Gly-Gly-Phe-Met-; e.g., in 0-E) or Leuenkephalin-moiety (Tyr-Gly-Gly-Phe-Leu-; e.g., in dynorphin) is essential. Na-acetylation or removal of the Tyr’ residue eliminates the affinity of 0-E and related peptides for opiate receptor sites (Akil et al., 1981; Wiegant et al., 1985). In addition, these modifications render 0-E inactive with respect to the induction of analgesia (Smyth et al., 1979; Deakin et al., 1980) and excessive grooming behavior (Hirsch and O’Donohue, 1986). Therefore, Nuacetylation has been proposed as an endogenous mechanism serving the bioinactivation of these peptides as opioids (Smyth et al., 1979). There are, however, biological activities residing in the 0-E sequence that do not require the intact amino terminus, in particular those associated with the free carboxyl terminus of y-E and a-E respectively. Not only are the opioid properties of 0-E partly lost in a-E and 7-E, but also specific, novel

436

central nervous system activities are exposed. These are evidenced by the effects of the peptides in a variety of neuropharmacological in vivo tests in rats, such as active and passive avoidance behavior, grip-tests and apomorphine-induced locomotor activity (for extensive reviews see: De Wied, 1987;Van Ree et al., 1987; Van Nispen and Van Wimersma Greidanus, 1990). In these experimental models, the effects of a-E resemble those of psychostimulants (e.g., amphetamine), whereas the effects of y-E resemble those of neuroleptics (e.g., haloperidol) in certain aspects. In general, these actions of a-E and y-E are independent from the opioid activities of the peptides as they are shared by the amino terminally acetylated and truncated (des-Tyr'-, des-enkephalin-) congeners of the peptides, that completely lack affinity for opioid receptors (De Wied et al., 1978a,b; GafforiandDe Wied, 1982; Van Reeet al., 1982a,b; Wiegant et al., 1985). Although the routes of formation and the biological characteristics of the peptide products have been best investigated in the POMC system, neurons producing pro-enkephalin A- or B-derived peptides may well have an organization of similar complexity. Thus, the final output of a particular endorphin producing neuron in terms of biological effects that are brought about, depends on a variety of factors. Of great importance among these are: (1) the topography of the brain neurons expressing a particular precursor. The three endorphin precursors clearly differ in this respect (Khachaturian et al., 1985b);(2) the characteristics and distribution of receptor sites in the brain that are sensitive to peptide products released. Specificclasses of opioid receptors (p, 6, H) with preference for peptides of each of the endorphin families have been described (Khachaturian et al., 1985a; Itzhak, 1988). In addition, receptor sites for non-opioid endorphins have recently been identified (Ronken et al., 1989; Ronken, 1991). These receptor systems also differ in their distribution in the brain; and (3) the characteristics of the neuropeptides that are formed and secreted. It is conceivable, that defects in a neuropeptide gene or in mechanisms governing individual steps in the enzymatic processing of the precursor molecules or

directing the (selective) secretion of certain peptides, can alter or disturb the biological signals that are broadcasted by neuropeptidergic neurons. The endorphin excess hypothesis of schizophrenia

Already before the initial identification of the enkephalins as endogenous opioids in the brain, Terenius and Wahlstrom (1974) reported on the presence of opioid activity in human cerebrospinal fluid (CSF). They demonstrated that two fractions (I and 11) recovered from CSF by chromatography over Sephadex G-10 contained material that interfered with the binding of a radiolabeled opiate (dihydromorphine) to rat brain membranes, and that this opioid activity was peptidergic in nature (Terenius and Wahlstrom, 1975). Using this radioreceptor assay, Terenius and coworkers started research on opioids in the CSF of psychiatric patients. In an initial study, they found that the levels of fraction I (but not of fraction 11) opioids were markedly elevated in a number of schizophrenics as compared to healthy controls (Terenius et al., 1976). Around the same time, Bloom et al. (1976) showed that intraventricular administration of 0-E to rats produced a naloxone-reversible, prolonged muscular rigidity reminiscent of the catatonic state sometimes observed in schizophrenic patients. Together with the notion that morphine-like drugs, in particular mixed agonists/antagonists, can induce psychotomimetic effects (hallucinations, delusions) that are not unlike those observed in schizophrenic psychoses (for references, see Van Ree and De Wied, 1981), these observations led to the hypothesis that an excessive activity of endogenous opioids in the brain is fundamental in schizophrenia (Bloom et al., 1976; Terenius et al., 1976). In further studies, Tereniusandcoworkersshowed that in particular acute schizophrenics displayed elevated levels of fraction I opioids in the CSF, and that treatment with neuroleptics significantly reduced these levels (Lindstrom et al., 1978; Rimon et al., 1980). Although the opioid activity recovered in fractions I and I1 sofar has not been fully characterized, it was found that fraction I contains a complex

437

mixture of opiate-receptor active components, with minor contributions (as assessed in radioimmunoassays) of 0-E and dynorphin-A, -B and -A-( 1 - 8). The major endorphins in fraction I1 most likely are peptides derived from pro-enkephalin A, e.g., Metenkephalin-Lys6 and -Arg6Phe7 (Nyberg and Terenius, 1982; Nyberg et al., 1983; Terenius and Nyberg, 1987). Interestingly, there is evidence that the composition of fraction I in schizophrenics differs from controls (Terenius and Nyberg, 1986). The radioreceptor assay measures the total pool of opioid activity. To investigate the possibility of more specific changes in individual endorphins in the CSF in relation to schizophrenia, radioimmunoassays (RIAs) have been used. It should be kept in mind, however, that RIAs generally detect families of peptides rather than single endorphins, and that the immunoreactive material found not necessarily represents peptides with opioid activities. Although Domschke et al. (1979) reported highly elevated levels of 0-E-like immunoreactivity (P-E-LIR) in the CSF of some patients with acute schizophrenic psychosis (and reduced levels in chronic schizophrenics), several studies including a large number of acute and chronic patients have been published that are negative in this respect (Emrich et al., 1979; Nakao et al., 1980; Naber et al., 1981; Gerner and Sharp, 1982). In addition, post-mortem analysis of brain tissue did not show differences in the concentration of 0-E-LIR in selected brain regions (hypothalamus, ventrolateral thalamus, hippocampus, septum, anterior cingulate gyrus, premotor cortex) between schizophrenic patients and control subjects (Lightman et al., 1979; Wiegant et al., 1988). There are only few data available on immunoreactive endorphins other than 0-E in this respect. A reduction in the CSF levels of immunoreactive Met-enkephalin in chronic schizophrenics versus controls was found by Wen et al. (1983). Incontrast, Kleine et al. (1984) reported higher levels of immunoreactive Met-enkephalin (and a tendency for an elevation of Leu-enkephalin) in the CSF of patients who received neuroleptic therapy. If excessive endogenous opioid activity is fundamental in schizophrenic symptomatology, then

blockade of the opioid receptor should lead to a reduction in the symptoms. Following this line of reasoning, several groups have evaluated the effects of opiate-receptor antagonists (naloxone, naltrexone) in schizophrenic patients. The first study in this respect was performed by Gunne et al. (1977), who reported a transient reduction of hallucinations in a limited number of chronic schizophrenic patients following a single injection with naloxone. In subsequent studies involving a large number of subjects, that employed open, single- or double-blind designs and single injections of naloxone (i.v., s.c.) in various doses (or daily oral administration of naltrexone), a temporary reduction of psychotic symptoms, particularly of auditory hallucinations, was found in approximately 30% of the patients (Verhoevenet al., 1981a; Pickar et al., 1982). Sofar, only a limited number of patients has been treated with repeated injections of naloxone, without apparent therapeutic efficacy (Verhoeven et al., 1984a; Pickar et al., 1989), although Schenk et al. (1978) initially reported marked beneficial effects of high doses of naloxone administered over several days in some patients with catatonic stupor. Another approach that has been used to interfere with the postulated excess opioid activity in schizophrenia is hemodialysis, which removes endorphins from the circulation. The first observations concerning effects of hemodialysis on schizophrenic psychoses were made accidentally by Feer et al. (1960). They reported an improvement of three out of five patients after one or two dialysis sessions. Later, Wagemaker and Cade (1977) reported a dramatic, long-term improvement in 10 out of 15 chronic schizophrenics who were dialysed regularly over long periods of time. In the first dialysate of some patients, a high concentration of a variant of 0-E (0-E with Leu substituted for Met at position 5 ) was found, and the level of this material was greatly reduced in subsequent dialysates (Palmour et al., 1978). In subsequent studies by others in which a large number of patients were dialysed, the presence of [Leu5]-0-E in dialysates (or blood) of schizophrenics was not confirmed, neither were the impressive effects on schizophrenic symptomatolo-

438

gy reported by Wagemaker and Cade (for references, see Van Ree and De Wied, 1981), although in some studies moderate improvements were found. Taken together, the data reviewed suggest altered activity of endorphin systems in schizophrenic patients. Although some symptoms of the disease (in particular auditory hallucinations) may relate to excess opioid activity, the evidence available to date, and more specifically the lack of therapeutic efficacy of treatment with opiate receptor antagonists, does not corroborate the hypothesis that excess opioid activity is fundamental in schizophrenia. The endorphin deficiency hypothesis of schizophrenia

Jacquet and Marks (1976) observed profound sedation and catalepsy in rats following administration of P-E into the periaqueductal grey, and noted the similarity of this effect to that seen after systemic administration of neuroleptic drugs to experimental animals. From this, they hypothesized that a disturbance in the bioavailability of 0-E in the brain is an ethiological factor in those psychopathological states for which the exogenous neuroleptics exert an ameliorative influence, for instance schizophrenia. In conjunction with this hypothesis, several studies have been performed in which patients were treated with P-E or with a potent synthetic opioid peptide that shares biological properties with P-E, the enkephalin-analogue FK 33 - 824. The results of these studies, however, do not favor this hypothesis. In an open study, Kline et al. (1977) observed an initial worsening of cognitive and mood symptoms in three out of four schizophrenics, followed by a progressive, long-lasting reduction in symptomatology. From the 29 patients treated with P-E (i.v.) in four subsequent double-blind studies, only four showed a temporary (several days) reduction of psychotic symptoms, and generally a tendency to worsening was found in the first hours following the treatment (Berger et al., 1980; Gerner et al., 1980; Petho et al., 1981; Pickar et al., 1981). Three studies including in

total 17 patients have been performed with FK 33 - 824 (Jrargensen et al., 1979; Krebs and Roubicek, 1979; Nedopil and Ruther, 1979). Nine of the patients showed a reduction of psychotic symptoms for several days, but some opiate-like side effects were observed. y-Type endorphins and schizophrenia

The hypothesis In 1978, De Wied and coworkers first reported on biological effects of endorphins that apparently are not mediated through opiate receptors. They found that both 0- and a-endorphin attenuate extinction of active avoidance (“pole jump”) behavior in rats, effects that were not blocked by the opiate antagonist naltrexone (De Wied et al., 1978a). In addition, a-endorphin attenuated extinction of passive avoidance behavior in a non-opioid fashion. Similar to classical neuroleptic drugs, y-endorphin facilitated extinction of both passive and active avoidance behavior and it was shown that the opioid-inactive y-type endorphins N“-acetyl-y-endorphin, desTyrl-endorphin (DTyE) and desenkephalin-y-endorphin (DEyE) are even more potent in this respect (De Wied et al., 1978b, 1980; Gaffori and De Wied, 1982; Wiegant et al., 1985). These y-type endorphins were also active in the “grip” test (a peptidetreated rat holds on to a pencil markedly longer than a saline-treated control), although this was not reproduced by others (Weinberger et al., 1979). A positive grasping response is not seen in opiatetreated rats, but is typical for animals treated with classical and atypical neuroleptics. The similarities in neuropharmacological profile of the various ytype endorphins suggest a common non-opioid mechanism of action of these peptides in the brain. In general, effects elicited by a-type endorphins in behavioral tests are different or opposite from those of y-type endorphins, and resemble those of the psychostimulant amphetamine (Van Ree and De Wied, 1983; Van Ree et al., 1987), a drug that can induce psychotic symptoms in humans (Meltzer and Stahl, 1976). These differences between the effects

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of a- and y-type endorphins were confirmed by Le Moal and coworkers (Le Moal et al., 1979). Based on these observations it was proposed that the endogenous a- and y-type endorphins act as functionally antagonistic neuropeptides in the brain, and that a balanced expression of their activities is of importance for behavioral homeostasis. In view of the neuroleptic-like properties of y-type endorphins in particular, De Wied hypothesized that a disturbance of this balance, leading to a relative deficiency in 7-E-type activity in the brain (or conversely a relative excess of a-E-type activity), may be an ethiological factor in psychopathologies for which exogenous neuroleptics are beneficial, e.g., schizophrenia (De Wied, 1978, 1979). Such a disturbance might arise from a defect in the formation, the metabolism or the biological activity of ytype endorphins.

Clinical studies In a number of clinical trials performed by Verhoeven and coworkers in which schizophrenic patients were treated with non-opioid y-type (DTyE, DEyE), the proposed antipsychotic activity of these peptides was evaluated. In total, these studies included 99 patients suffering from different types of schizophrenia and schizoaffective psychoses with or without neuroleptic mediation, and employed different designs (open, single blind, bould blind, double blind cross-over, double blind placebo-controlled), doses of peptides (1, 3 or 10 mg i.m. daily) and durations of treatment (8 - 20 days) (Verhoeven et al., 1979, 1981b, 1982, 1984b, 1986a,b; see also Verhoeven, 1987). The patients were classified according to the DSM I11 criteria (schizophrenia) and Research Diagnostic Criteria (schizoaffective psychosis). Response to treatment was assessed with the Brief Psychiatric Rating Scale (a decrease off the baseline score c 20% was defined as no response; 20 - 50% as a slight response; 50-80% as a moderate response; > 80% as a marked, clinically relevant improvement). In 18 of the 79 patients treated with peptide (DTyE or DEyE) a marked, clinically relevant improvement was observed, in some cases

lasting fore more than 6 months. A moderate response was found in 20 and a slight response in 25 patients, and 16 patients did not respond to peptide treatment. Positive and negative psychotic symptoms were equally affected by the peptide treatment, and no adverse side effects were seen. Of the 20 placebo-treated patients, 17 showed no response and only 3 showed a slight response. In ten studies by others, that included a total of 93 patients, 24 showed a beneficial response to yendorphins (for references, see Verhoeven and Den Boer, 1988). In one of two more recent trials, Kissling et al. (1986) found similar antipsychotic effects of treatment with DEyE and with haloperidol, suggesting equipotency with respect to the therapeutic efficacy of those substances. In the other trial (Azorin et al., 1986),patients were treated with daily doses of 1, 3 or 10 mg DEyE (i.m. during 4 weeks) or placebo, and those receiving the highest dose of DEyE showed significant improvement, while no adverse side effects were observed. Taken together, the results of these clinical studies indicate that y-type endorphins possess antipsychotic properties in some schizophrenic patients. The susceptibility to y-type endorphins clearly differs between patients, and post-hoc data analysis indicated that in particular a high intensity of negative psychotic symptoms (e.g., emotional withdrawal, motor retardation, blunted affect) is associated with poor responsiveness to the treatment (Verhoeven et al., 1986a,b; see also Van Ree et al., 1986, 1990).

Studies on post-mortem brain tissue A variety of dysfunctions of the POMC system can be envisaged, that may result in a relative deficiency in y-E-type activity in the brain. These include malfunctions in the enzymatic machinery involved in the processing of p-E, the generation and the degradation of y-type endorphins. Such defects may be reflected in altered tissue concentrations of endogenous p-E-related peptides. We have analysed post-mortem hypothalamic tissue of 28 schizophrenic patients and of 22 control subjects without known psychopathology (obtained from the MRC Brain Bank, Cambridge, U.K.), and have deter-

440

mined the levels of a-E, 0-E and y-E by specific radioimmunoassays in HPLC-fractionated extracts of the tissue (Wiegant et al., 1988). The hypothalamus was selected for this study, since it contains the cell bodies of the POMC synthesizing neurons that project to limbic and cortical brain regions (Watson et al., 1977; Bloom et al., 1978; Khachaturian et al., 1985b), and contains the highest concentrations of POMC-derived endorphins in the brain. In tissue obtained from schizophrenic patients, the concentration of both a-E and y-E was significantly higher than in controls ( + 70 and + 50%, respectively; Fig. 2). Interestingly, no difference was found in the concentration of P-E, the putative precursor of aand y-endorphins. Thus, the increase in a-E and y-E seems to be rather selective and may therefore reflect a dysfunction in the metabolism of 0-E. This contention is supported by data from others (Schoemaker and Davis, 1984), who found differences in the in vitro fragmentation of synthetic 0E by membranes isolated from brain tissue of schizophrenic patients and control subjects, suggesting that the activity of brain enzymes involved in

200 250

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the generation of endogenous y-type endorphins is indeed altered in schizophrenia. An important question pertains to the relationship of these differences to the psychopathology of the patients. As in all post-mortem studies in humans, ante-mortem pharmacotherapy is a variable that is beyond the control of the investigator. Most patients included in our study did receive medication of some type, and, in so far known, all patients but one received neuroleptics at the time of death. The possibility that the results are compromised by effects of neuroleptic therapy should therefore be seriously considered. Chronic treatment of rats with neuroleptics affects the POMC system in the rat brain (Hollt and Bergman, 1982; Nohtomi et al., 1984; Ham and Smyth, 1985; Sweep et al., 1990). However, such treatment not only increases the hypothalamic concentration of WE and y-E, but elevates that of P-E to a similar extent suggesting that under those conditions no qualitative changes in the proteolytic processing of 0-E had occurred (Sweep et al., 1990). Therefore, it seems that the selective increase of a-E and y-E in schizophrenics

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Fig. 2. Endorpin concentrations in hypothalamic tissue of diagnosed schizophrenic patients relative to controls. Post-mortem hypothalamic tissues were obtained from the MRC Brain Bank (Cambridge, U.K.). Following extraction from tissues, endorphins were fractionated by reversed-phase HPLC, the endorphin content of the fractions eluting with the retention times of synthetic WE, pE(1- 31) and y-E were quantified with specific RIAs for WE, 0-E and y-E respectively, and the endorphin concentrations were determined in ng/g tissue weight. The data shown were obtained in three separate experiments. The data of each experiment were expressed as percentage of the mean endorphin concentrations found in control subjects in that experiment. The combined results of these experiments are presented as mean + S.E.M. of 22 controls and 28 schizophrenics. Statistical significance is indicated by asterisks (P < 0.05; Student's t-test). (For futher details on subjects and methodology see Wiegant et al., 1988.)

44 1

is not simply explained by ante-mortem treatment of the patients with neuroleptics. Davis et al. (1984) have shown, however, that the in vitro fragmentation of synthetic P-E by rat brain membranes is altered by chronic treatment of the animals with neuroleptics. In particular, they found an enhanced accumulation of y-type endorphins, but no change in a-type endorphins. These data emphasize the possibility that brain enzymes involved in the processing of 6-E to y-E- and a-E-type peptides are differentially influenced by neuroleptics. The observation that the levels of y-E and a-E are elevated in brains of schizophrenic patients does not simply comply with the postulated deficiency in ytype endorphins. The activity of y-E and related peptides as neuroleptic-like principles is associated with the carboxyl-terminal (6- 17) amino acid sequence of the peptide, as evidenced by structure activity studies in animals (De Wied et al., 1980). Modifications in that part of the molecule generally destroy its biological activity. Thus, the possibility that the biological activity of y-E in schizophrenics

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is impaired, for instance as a consequence of an alteration in the structure of the peptide, was investigated. To that end, the HPLC fractions from post-mortem tissue of schizophrenic and control subjects that contained y-E were assayed for bioactivity in rats using the one-trial passive avoidance test. In this paradigm, neuroieptics and peptides possessing neuroleptic-like activity such as y-E, DTyE and DEyE attenuate the retention of the avoidance response (De Wied et al., 1978a; De Wied, 1987). This effect is not only observed after peripheral (s.c.) administration, but also when the peptides are injected in pg doses topically in the brain, into the nucleus accumbens (Kovacs et al., 1982). A bioassay with such extreme sensitivity was a prerequisite for these studies since only small amounts of y-E can be recovered from human hypothalamic tissue. When animals were treated (by injection into the nucleus accumbens) with synthetic y-E or with y-E fractions obtained from hypothalamic tissue of controls (pooled from six subjects) and tested for passive avoidance retention, a similar

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Fig. 3. Effects of y-endorphin (7-E) isolated from post-mortem hypothalamic tissue of control subjects and schizophrenic patients on rat passive avoidance behavior. Fractions containing immunoreactive y-E and eluting in reversed-phase HPLC with the retention time of synthetic y-E were prepared as described in Wiegant et al. (1988). Pools were prepared from fractions of controls and of schizophrenics, rechromatographed by HPLC, dried, dissolved in saline, calibrated with a specific y-E RIA (Wiegant et al., 1988), and tested for bioactivity in a one-trial passive avoidance test in rats (Kovacs et al., 1982). Aliquots of the pools containing 10 or 30 pg immunoreactive 7-E, saline and synthetic y-E were injected unilaterally into the nucleus accumbens through a permanent cannula (injection volume 1 PI), 1 h before the retention test (i.e., 24 h after the learning trial). The median entrance latencies of the treatment groups (n = 7) are shown. Statistical analysis was performed with a Kruskall-Wallis non-parametric ANOVA followed by Mann-Whitney Utests. *Significantly different ( P < 0.05) from saline-treated animals; and 9, from animals treated with the same dose of y-E from control subjects.

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dose-dependent inhibition was found with both treatments. However, y-E containing HPLC fractions isolated from hypothalami of schizophrenics (pooled from seven patients) did not display such activity, but were either inactive or induced effects opposite to those of synthetic and control y-E (Fig. 3). Similar results were obtained with pooled y-E fractions from nine other controls and seven schizophrenic patients. In a following series of experiments, y-E containing HPLC fractions prepared from hypothalami of 12 other patients were tested individually and compared to synthetic y-E. Again, a deviant biological activity was found (Wiegant et al., in preparation). In these experiments, HPLC was used to fractionate and collect y-E from tissue extracts. The fractions containing y-E were identified based on reactivity in a specific y-E RIA and HPLC retention time as compared with that of synthetic y-E (Wiegant et al., 1988). There is no doubt, however, that such fractions contain other tissue constituents in addition to immunoreactive y-E. They may even be contaminated with drugs taken by the patients prior to death. Additional experimental evidence, however, indicates that the deviant bioactivity is indeed associated with a y-E-like peptide, and that it is unlikely that other substances (for instance contaminating drugs or metabolites of drugs) are responsible for the deviant bioactivity found in schizophrenics. The following observations were made (Wiegant et al., in preparation). Firstly, y-E fractions prepared by HPLC from hypothalamic extracts of 12 individual patients, of which at least two did not receive neuroleptics at the time of death, were tested and each of those displayed deviant (or no) bioactivity. Secondly, y-E fractions prepared from individual pituitaries of two of these patients and of four other schizophrenics displayed no or deviant bioactivity, similar to the fractions isolated from hypothalamus. At least one of these patients was not on neuroleptics. Thirdly, from HPLC profiles of hypothalamic and pituitary extracts, the fractions containing immunoreactive P-E and eluting at the position of synthetic P-E-(l- 31) were collected and digested in vitro with Cathepsin D.

This enzyme cleaves the Leu16-Phe17 bond in 0-E, thereby generating y-E. Subsequently, this “in vitro generated” y-E was recovered from the digest by HPLC, calibrated by RIA and tested in the rat behavioural bioassay. Preparations originating from 0-E of schizophrenic patients displayed no or deviant bioactivity, whereas those originating from pE of control subjects and from synthetic P-E were active. Fourthly, y-E purified to homogeneity from a single schizophrenic pituitary by consecutive Sephadex G-50 chromatography, reverse phase HPLC and paired ion HPLC, did not display significant yE-type bioactivity when assayed in the passive avoidance paradigm. Thus, the deviant biological activity remained associated with y-E under various experimental and chromatographical conditions, and was found in two tissues that express POMC (and y-E) to very different extents (pituitary, hypothalamus). In addition, the aberrant y-E appeared to be contained in the molecular structure of P-E, the putative precursor for endogenous y-E, from which it could be artificially liberated in vitro. Taken together, these data are strong indication that the deviant bioactivity indeed reflects intrinsic properties of the y-E molecule and is related to the structure of the peptide. A structural defect in y-E could arise either from a mutation in the y-E coding region of the POMC gene and then should be detectable in the amino acid composition or sequence of the peptide, or from a post-translational modification. Although amino acid substitutions, in particular those in the Cterminal region of y-type endorphins generally render the peptides biologically inactive, they do not necessarily affect their retention time on HPLC and/or their properties in the RIA systems used in this study (Wiegant et al., unpublished results). Therefore, it remains possible that, with the methodology used to isolate authentic y-E, a structurally abnormal form of y-E is recovered from tissue extracts, and that this aberrant y-E is reponsible for the observed deviant bioactivity in schizophrenics. To further investigate this possibility, the amino acid composition of biologically inactive y-E, purified from a single schizophrenic

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pituitary (Burbach and Wiegant, 1984) was determined. No differences were found with the composition of y-E similarly purified from a control pituitary and with synthetic y-E. In addition, analysis of the nucleotide sequence of the y-E coding region of pituitary POMC mRNA from two other pituitaries of schizophrenics (that contained y-E with deviant bioactivity) did not show differences as compared to controls (Bovenberg et al., 1986). These results rule out the first possibility, namely that a mutation in the POMC gene is responsible for the biologically defective y-E in schizophrenia. Post-translational modifications of the peptide, however, are neither detected after hydrolyzing conditions such as used for instance for the analysis of the amino acid composition, nor are they detectable at the level of the POMC mRNA. Thus, the possibility that such a modification is responsible for the deviant bioactivity remains open, and is presently under investigation. The deviant biological activity of y-E appeared to be expressed in two ontogenetically different tissues, the hypothalamus and the pituitary, and the POMC cells in these tissues are differentially regulated. This suggests that the defect may be genetically determined. Interestingly, the expression of a beneficial effect of y-type endorphins in schizophrenic patients is associated with a higher incidence of certain antigens of the major histocompatibility complex, the human lymfocyte antigen (HLA) system, in these patients (De Jongh et al., 1982; Van Ree et al., 1986), indicating that genetic factors encoded within the HLA genomic region are associated with the therapeutic effect of the peptides. Summarizing, the data available to date provide evidence that: (1) y-type endorphins exert neuroleptic-like actions in rats; (2) y-type endorphins display antipsychotic activity in certain schizophrenic patients; (3) the levels of y-E and a-E in brain tissue of schizophrenics are elevated, possibly as a consequence of a disturbance in the metabolism of 0-E; and (4) the bioactivity of y-E from schizophrenics is abnormal. It is tempting to speculate that, in absence of y-E with normal bioac-

tivity, altered activity of feedback mechanisms in the brain of schizophrenics is responsible for an increased formation of a-E and (bio-inactive) y-E (Wiegant et al., 1988). The effects induced by a-E in animals resemble those of amphetamine (De Wied, 1978; Van Ree et al., 1980), a drug with psychotogenic properties in humans (Meltzer and Stahl, 1976). If the bioactivity of a-E is not disturbed in schizophrenics, (part of) the symptomatology might be related to the excess of this peptide in the brain of such patients. Together these results support the hypothesis that a deficit in y-E-type activity underlies schizophrenic psychopathology in a category of patients.

Putative mechanism of action of y-type endorphins interaction of y-type endorphins with dopaminergic neurotransmission in vivo. It has been hypothesized, that dopaminergic hyperactivity in the brain underlies schizophrenia (see Iversen, 1978; Stevens, 1979). This hypothesis finds its basis in observations that drugs that enhance dopaminergic activity in the brain exacerbate, and drugs that inhibit dopamine function reduce schizophrenic symptomatology. Although the “dopamine hypothesis” has received much attention over the years, and a wealth of supportive neuropharmacological evidence is available, direct and convincing evidence from clinical studies and biochemical analysis of brain tissue substantiating a dopamine dysfunction in schizophrenia is largely lacking (Costal1 and Naylor, 1986). Several distinct dopamine neuronal systems have been identified in the brain (Bjorklund and Lindvall, 1984). These systems, the nigro-striatal, tubero-infundibular, mesocortical and mesolimbic dopamine systems are not only anatomically distinct but differ also in functional aspects. The mesocortical and mesolimbic systems consist of dopamine neurons that are located in the ventral tegmental area, and project to cortical and limbic regions of the forebrain. These include thalamic, septa1 and amygdaloid nuclei, the nucleus accumbens and the nucleus of the stria terminalis (mesolimbic system), and the prefrontal, piriform, orbital and entorhinal

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cortical regions (mesocortical system; Bjorklund and Lindvall, 1984). Through intricate feedback and feedforward interconnections, the mesocorticolimbic areas form a complex network. Its functions are associated with attentional, emotional and cognitive processes (Le Moal and Simon, 1991). Generally, it is assumed that coherent changes in the activity of the components of the mesocorticolimbic system are essential for adequate responses to environmental challenges, e.g., for (behavioral) adaptation. It is thought, that in schizophrenia neurotransmission is disturbed particularly in this system, and that neuroleptic drugs exert their antipsychotic activity by counteracting dopamine hyperactivity in mesocorticolimbic brain regions. The extrapyramidal side effects often induced by neuroleptics would arise from interaction of the drugs with striatal dopaminergic activity (Marsden et al., 1986). In view of this, studies on the neuropharmacological actions of y-type endorphins in the brain have focused mainly on limbic brain regions and dopamine. They have yielded evidence that in rats projection areas of the mesocorticolimbic dopamine systems contain target sites for (non-opioid) ytype endorphins. For instance, local administration of such peptides into the nucleus accumbens inhibits ACTH-induced excessive grooming (Gispen et al., 1980). In addition, it attenuates passive avoidance behavior, and extremely low doses of y-type endorphins (10 pg or less) are sufficient to produce this effect (Kovacs et al., 1982; Gaffori, Wiegant and De Wied, unpublished data). Administration of y-type endorphins into the piriform cortex blocks the sniffing response induced by injection of apomorphine in this area (Van Ree et al., 1989). Moreover, several lines of experimentation indicate that y-type endorphins indeed interfere with dopamine activity in these brain regions. Low doses of the dopamine agonist apomorphine (administered systemically or into the nucleus accumbens) reduce motor responding in a novel environment (Van Ree and Wolterink, 1981; Van Ree et al., 1982a,b). These doses are thought to be insufficient for stimulation of postsynaptic receptors and it is assumed that they act on

presynaptic dopamine (auto)receptors to decrease the release and/or synthesis of dopamine from the presynaptic element (Strombom, 1976). The hypolocomotor response induced by apomorphine is antagonized both by neuroleptics and y-type endorphins (Van Ree et al., 1982a,b; Serra et al., 1983; Kiraly and Van Ree, 1984). The peptides also prevent the decrease in microdialysable dopamine induced by low doses of apomorphine in the nucleus accumbens (Vulto et aI., 1985; Radhakishun et al., 1988). High doses of apomorphine induce stereotypies and hyperlocomotion. These effects are thought to arise through interaction of apomorphine with postsynaptic dopamine receptors, and can be blocked by classical and atypical neuroleptic drugs but are not influenced by y-type endorphins (Van Ree et al., 1982a; Serra et al., 1983; Kiralyand Van Ree, 1984). These data suggest that the peptides act at presynaptic dopamine terminals, and that their mechanism of action differs from that of neuroleptics. Brain binding sites specific for y-type endorphins. A common feature of most modern neuroleptic drugs is their antagonistic affinity for dopamine receptors. Therefore, the molecular interaction of y-type endorphins with brain dopamine receptors has been studied. It was found, that these peptides do not display affinity for brain membrane binding sites for [3H]-haloperidol, [3H]-spiperone and [3H]-apomorphine (Van Ree et al., 1978; Pedigo et al., 1979; Wiegant and Ronken, unpublished results). They do not only lack affinity for dopamine receptors, but also for binding sites of a number of other neurotransmitters and psychoactive drugs, including naloxone and fentanyl (except for the opioid y-E), diazepam, PCP and NMDA (Tonnaer, personal communication; Ronken and Wiegant, unpublished data). Thus, it was postulated, that the central effects of y-type endorphins are mediated through a unique type of receptor that is specific for these peptides. Using the autoradiographic approach on rat brain cryosections in vitro with [35S]Met-DEyEof high specific activity as the radioligand, we have recently identified sites in the

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rat brain that specifically bind y-type endorphins with high (nanomolar) affinity (Ronken et al., 1989; Ronken, 1991). These binding sites appeared to be located preferentially in regions of the forebrain that receive projections of mesocorticolimbic dopaminergic neurons, for instance in the nucleus accumbens, the amygdala, the cingulate and the piriform cortex. This topography is particularly interesting in view of the neuroleptic-like properties of y-type endorphins, and the pharmacological interactions of these peptides with limbic dopamine activity. The contention that the binding sites identified relate to the non-opioid biological effects of ytype endorphins is supported by evidence obtained with the monoclonal anti-idiotypic DEyE antibody CR 14.1. This antibody blocks the in vivo effects of DEyE on apomorphine-induced hypolocomotion (Van Ree et al., 1988) and is an extremely potent displacer of [35S]Met-DEyEfrom its brain binding sites in vitro (Ronken, 1991), while it neither has affinity for opioid receptors nor for a-,0-and yendorphins (Rust et al., in preparation). y-Type endorphins inhibit brain dopamine reuptake

in vitro. The efficiency of dopaminergic neurotransmission is not only dependent on biosynthesis and release of dopamine and its interaction with receptors, but also on the concentration of the neurotransmitter in the synaptic cleft. Thus, in addition to enzymatic inactivation, carrier-mediated

transport of dopamine from the extracellular compartment back into the cytosol of the presynaptic element (reuptake) is of importance, and modulation of the activity of reuptake carriers could be a mechanism for y-type endorphins to influence dopamine activity. To investigate this possibility, we studied the uptake of [3H]-dopamine in slices of rat brain tissue in vitro. It was found, that DEyE and other y-type endorphins inhibit the uptake of dopamine in the nucleus accumbens, a region that is relatively rich in binding sites for these peptides (Ronken et al., 1989), whereas the uptake of dopamine in the striatum, a region where binding sites for y-type endorphins are relatively scarce, was not affected by the peptides (Ronken, 1991). The observation that the number of binding sites for [3H]-GBR 12935 - a selective inhibitor of the dopamine transporter - is increased following coincubation of nucleus accumbens membranes with DEyE suggests that a direct interaction between the occupied receptor for y-type endorphins and the dopamine transporter may underly the inhibition of dopamine uptake by the peptides (Fig. 4). The molecular weights of the y-type endorphin binding site (60 kDa; Ronken, 1991) and the GBR-sensitive dopamine transporter (58 kDa; Lew et al., 1991) are very similar. It remains to be investigated, however, whether the binding site is a separate entity or represents (a subunit of) the transporter. Inhibition of the presynaptic reuptake

Fig. 4.Schematic representation of the proposed mechanism of action of desenkephalin y-endorphin (DErE) and other y-type endorphins in the brain. Binding of the peptide to its receptor results in inhibition of the activity of dopamine transporters present in the presynaptic membrane of mesolimbic dopaminergic nerve terminals, and in an increased exposure of sites sensitive to GBR-12935, a specific inhibitor of the transporter. As a result, the extracellular concentration of synaptic dopamine is increased.

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mechanism will result in a slower disappearance of dopamine from the synaptic cleft, and thereby prolong interaction of the transmitter with (post- and pre-)synaptic receptors. It can be envisaged that reuptake inhibition requires ongoing release of transmitter in order to effectively alter the signal at the receptor. In this respect, it is of interest that behavioral effects of y-type endorphins are only observed when the experimental conditions induce arousal in the animals, for instance by exposure to an electric footshock or a novel environment as is the case in avoidance, locomotion and grip tests. Such “stressors” are known to stimulate the release of monoamines in the brain (Thierry et al., 1976; Hofmann et al., 1988), including that of dopamine in terminal regions of the mesolimbic dopamine system (Dunn, 1988; Roth et al., 1988; Scatton et al., 1988). In addition it can be expected that in stressful1 situations endogenous y-type endorphins are released from the projections in those regions of hypothalamic POMC neurons, since it has been shown that endogenous opioids including P-E are released in the brain under those conditions (Akil et al., 1976, 1986), and that P- and y-type endorphins are colocalized in the same cells (Vaudry et al., 1980; Stoeckel et al., 1985; Van der Beek et al., 1990). In view of the importance of the mesocorticolimbic dopamine system for attentional, emotional and cognitive brain functions, it is feasible that a change in the neurochemical status of this system induced through inhibition of dopamine reuptake by endogenous or exogenous y-type endorphins can result in altered interpretation of environmental stimuli and, consequently, in altered behavioral responses. Although neuroleptics induce some acute responses in schizophrenics, it generally takes several days before some improvement is seen and stable antipsychotic effects need at least several weeks of treatment to establish. Thus, it has been argued that simple antagonism of dopaminergic transmission is insufficient to ameliorate the core symptoms of schizophrenia, but that more complex secondary neurochemical changes are required (for instance in receptor populations, sensitivity of signal transduction systems, etc.) that take many days or longer to

fully develop, thus accounting for the delayed appearance of the antipsychotic action of neuroleptics (see Bradley, 1986). In the case of y-type endorphins, the inhibition of dopamine reuptake in mesolimbic (and mesocortical) areas might trigger such adaptive mechanisms. Indeed, it has been shown recently that (sub)chronic treatment of rats with DEyE (administered systemically or into the nucleus accumbens) enhances the induction of hypomotility by low doses of apomorphine (Van Ree et al., 1982b), results in a decreased locomotor response to novelty (Van Ree et al., 1982c), reduces the basal release of dopamine in the nucleus accumbens in vivo (microdialysis) and in vitro (tissue slices) and lowers the dopamine levels in the nucleus accumbens but not in the striatum (Radhakishun, 1988). Moreover, chronic treatment of rats with an antiserum against y-type endorphins (injected into the nucleus accumbens) markedly increases locomotor activity in a novel environment (Van Ree et al., 1982c; seealsoVanReeet al., 1986,1987). Although these data clearly indicate that chronic excess or deficiency of y-type endorphins can result in longterm and persistent alterations in dopamine neurotransmission events in limbic structures, the exact nature of the underlying neurochemical changes sofar has not been established and is presently under investigation. Malfunction of y-type endorphins - as postulated in schizophrenia - could thus lead to disturbances in brain functions (e.g., cognition, emotion and attention), that can be restored by substitution with exogenous, bioactive y-type endorphins. The apparent specificity of these peptides as inhibitors of mesolimbic, but not of striatal dopamine uptake may not only explain their antipsychotic properties in schizophrenic patients, but also the absence in these peptides of the adverse (extrapyramidal) side effects that are often induced by neuroleptics. Conclusions

Since it was first appreciated in the sixties that pituitary peptide hormones influence behavioral

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adaptation in animals and carry information to the brain, it has been shown that similar peptides are also produced inside the brain. The concept that they function as neuromessengers serving the communication between neurons is now widely accepted. To date, over a hundred neuropeptides are known to occur in the brain, and it is thought that modulation of neurotransmission events is their prime action. Among other neuropeptides, the endorphins have been implicated particularly in brain functions that are of importance for adaptation to environmental challenges. It is feasible therefore, that malfunctions of endorphin systems can lead to diseases of adaptation that involve inadequate behavior as is seen for instance in schizophrenia. In view of the enormous complexity of neuropeptide systems, the molecular levels of malfunction can be manifold. Evidently, the therapy of choice for such diseases then should aim at the normalization of the disturbed endorphin functions. The antipsychotic actions of y-type endorphins in schizophrenic patients may relate to the deficiency in y-E-type activity that has been postulated as an ethiological factor in schizophrenia. This postulate finds additional support in the findings that the levels of y-E and a-E are elevated in post-mortem hypothalamic tissue of schizophrenic patients as compared to control subjects and that the biological activity of y-E isolated from post-mortem hypothalamic tissue of schizophrenic patients is disturbed. Animal experiments have indicated that y-type endorphins interfere with brain dopamine activity. The antipsychotic actions of the peptides in schizophrenics, however, are not accompanied by the extrapyramidal side effects often seen withneuroleptic drugs. This suggests a more selective action of ytype endorphins in the brain. The preferential localization of receptors for y-type endorphins in mesocorticolimbic regions of the brain, and the properties of these peptides as inhibitors of dopamine uptake that act preferentially in mesolimbic regions but not in the striatum, may account for this greater selectivity. Clearly, our understanding of the actions of ytype endorphins in the brain and of the possible role

of these peptides in schizophrenia is only beginning to emerge. Yet, the data that are now available provide a basis for further fundamental and clinical research that may eventually lead to the development of more causal therapies for such devastating diseases as schizophrenia based on peptide- or peptidomimetic drugs. Acknowledgements

Part of the research presented in this review was supported by the Dutch Organization for Scientific Research (N.W.O.), MEDIGON Grant no. 900 - 543 - 063. G.K. was a recipient of a stipend from the Pharmacologisch Studiefonds Utrecht. The authors thank Mrs. Pauline Mettrop for typing the manuscript. References Akil, H., Madden, J., Patrick, R.L. and Barchas, J.D. (1976) Stress-induced increase in endogenous opiate peptides: concurrent analgesia and its partial reversal by naloxone. In: H.W. Kosterlitz (Ed.), Opiates and Endogenous Opioid Peptides, North Holland, Amsterdam, pp. 63 -70. Akil, H., Young, E., Watson, S.J. and Coy, D.H. (1981) Opiate binding properties of naturally occurring N- and C-terminus modified beta-endorphins. Peptides, 2: 289 - 292. Akil, H., Young,E., Walker, J.M. and Watson, S.J. (1986)The many possible roles of opioids and related peptides in stressinduced Analgesia. In: D.D. Kelly (Ed.), Sfress-induced Analgesia - Ann. N . Y. Acad. Sci., 467: 140- 153. Austen, B.M. and Smyth, D.G. (1977) Specific cleavage of lipotropin C fragment by endopeptidases; evidence for a preferred conformation. Biochem. Bjophys. Res. Commun., 77: 86-94. Austen, B.M., Smyth, D.G. andSnell, C.R. (1977) y-Endorphin, or-endorphin and Met-enkephalin are formed extracellularly from lipotropin C fragment. Nature, 269: 619- 621. Azorin, J.M., Charbaut, J., Granier, F., Huber, J.P., Metzger, J.Y .,Richou, H., Van Amerongen, P., Blum, A. and Dufour, H. (1986) Des-enkephalin-gamma-endorphin in exacerbation of chronic schizophrenia: a double-blind, placebo-controlled study. Abstracts Symposium on Neuropeptides and Brain Function, May28 -30,1986, Utrecht, TheNetherlands, p. 52. Bals-Kubik, R., Hertz, A. and Schippenberg, T.S. (1988) pEndorpin-(1 -27) is a naturally occurring antagonist of the reinforcing effects of opioids. Naunyn Schmiedebergs Arch. Pharmacol., 338: 392 - 396. Berger, Ph.A., Watson, S.J., Akil, H., Elliott, G.R., Rubin,

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453 Krul, J.M., Brouwer, G.J., Thijssen, J.H.H., De Wied, D., Van Praag, H.M., Ceulemans, D.L.S. and Kahn, R.S. (1984b) Clinical, biochemical and hormonal aspects of treatment with des-Tyr'-y-endorphin in schizophrenia. Psychiat. Res., 11: 329 - 346. Verhoeven, W.M.A., Van Ree, J.M. and De Wied, D. (1986a) Neuroleptic-like peptides in schizophrenia. In: G.D. Burrows, T.R. Normanand G. Rubinstein (Eds.), Handbookon Studies in Schizophrenia, Elsevier, Amsterdam, pp. 253 - 274. Verhoeven, W.M.A., Westenberg, H.G.M. and Van Ree, J.M. (1986b) A comparative study on the antipsychotic properties of desenkephalin-y-endorphin and ceruletide in schizophrenic patients. Acta Psychiat. Scand., 37: 372 - 382. Vulto, A.G., Sharp, T. and Ungerstedt, U. (1985) The nonopioid des-Tyrl-y-endorphin (P-endorphin(2 - 17)) stimulates dopamine release as studied by in vivo dialysis of the rat nucleus accumbens. Netherlands Arch. Pharmacol. Sci., 7: 236. Wagemaker, H. and Cade, R. (1977) The use of hemodialysis in chronic schizophrenia. Am. J. Psychiatry, 134: 684 - 685. Watson, S.J., Barchas, J.D. and Li, C.H. (1977) 6-Lipotropin: localization of cells and axons in rat brain by immunocytochemistry. Proc. Natl. Acad. Sci. U.S.A., 74: 5155 - 5158. Weber, E., Evans, C.J. and Barchas, J.D. (1981)Acetylated and non-acetylated forms of P-endorphin in rat brain and pituitary. Biochem. Biophys. Res. Commun., 103: 982 - 989. Weinberger, S.B., Arnsten, A. and Segal, D.S. (1979) Des-Tyr'y-endorphin and haloperidol: behavioral and biochemical differentiation. Life Sci., 24: 1637- 1644. Wen, H.L., Lo, C.W. and Ho, W.K.K. (1983) Met-enkephalin level in the cerebrospinal fluid of schizophrenic patients. Clin. Chim. Acta, 128: 367 - 371. Wiegant, V.M., Verhoef, J . , Burbach, J.P.H. and Van Amerongen, A. (1983) Characterization of Nm-acetyl-aendorphin from rat neurointermediate lobe and its distribution in pituitary and brain. Life Sci., 33: 125 - 128. Wiegant, V.M., Verhoef, J., Burbach, J.P.H., Van Amerongen, A., Gaffori, O., Sitsen, J.M.A. and De Wied, D. (1985) NuAcetyl-y-endorphin is an endogenous non-opioid neuropeptide with biological activity. Life Sci., 36: 2277 -2285. Wiegant, V.M., Verhoef, C.J., Burbach, J.P.H. and De Wied, D. (1988) Increased concentrations of a-and y-endorphin in post-mortem hypothalamic tissue of schizophrenic patients. Life Sci., 42: 1733 - 1742. Zakarian, S. and Smyth, D.G. (1979) Distribution of active and inactive forms of endorphins in rat pituitary and brain. Proc. Natl. Acad. Sci., U.S.A., 76: 5972-5976.

munocytochemical studies to show whether the endorphins are localized not only in the arcuate nucleus but also in the pars tuberalis of the pituitary? V.M. Wiegant: We have no data on the immunocytochemical localization of endorphins in the human pars tuberalis and I am not aware of reports on this subject in the literature. The hypothalamic aliquots included in our studies (Figs. 2, 3) were obtained as finely diced, frozen tissue from the MRC Brain Bank, Cambridge, U.K. According to the specifications of the Brain Bank, the hypothalamus was dissected from 5 mm coronal brain cryosections according to the atlas of Roberts and Hanaway (1971) as the tissue bordered frontally by the optic chiasm, laterally by the mediolateral edge of the globus pallidus and the ansa lenticularis, caudally by the mammillary bodies (included) and dorsally by the third ventricle. C.B. Saper: It has been said that schizophrenia is the graveyard of neuropathology. Nevertheless, I wonder if you know of any chemical neuroanatomical studies examining the proopiomelanocortin (POMC) neuron system in the brains of schizophrenics immunocytochemically? V.M. Wiegant: To my knowledge there are no studies in the literature on this subject. G.A. Bray: In our studies acetylation of melanocyte-stimulating hormone (MSH) markedly changed its function. We have pursued this to examine the effect of desacetyl- and N--acetylendorphin. The acetylated 0-endorphin does not affect food intake, nor does it suppress sympathetic activity. Could you give comment on whether the y-endorphin you have studied is the acetylated or non-acetylated form? What form is present in the brains of schizophrenics? Could the lack of effect of your yendorphin of schizophrenic brain reflect its acetylation? V.M. Wiegant: In all our experiments the tissue extracts were fractionated by HPLC prior to quantification of a-E, P-E and yE (by RIA) and estimation of the biological activity of y-E by in vivo bioassay inrats. In the HPLC system used the acetylated and non-acetylated forms of a-,y- and &endorphins are effectively separated, and the bioassays were performed with the fractions containing IR-y-E eluting as non-acetylated y-E. Thus, the deviant bioactivity found in fractions from schizophrenic tissues does not relate to presence of acetylated y-E in the fractions, but most likely to authentic -y-E. This contention is supported by observations that the deviant bioactivity remained associated with non-acetylated y-E under various chromatographical conditions. Finally, it should be kept in mind, that in line with previous results (Burbach and Wiegant, 1984), we found no evidence for the occurrence in the human brain or pituitary of acetylated endorphins.

Discussion

References

D.F. Swaab: There are strong parallels in your findings concerning the activity of endorphins in the hypothalamus and pituitary. In the area of the median eminence, the human hypothalamus always contains a part of pars tuberalis of the pituitary which is very tightly connected to the median eminence. Do you have im-

Burbach, J.P.H. and Wiegant, V.M. (1984) Isolation and characterization of @-endorphinand y-endorphin from single human pituitary glands. FEBS Lett., 166: 267 - 272. Roberts, M. and Hanaway, J. (1971) Atlas of the Human Brain in Section, Lee and Fibiger, Philadelphia, PA.

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CHAPTER 30

Neurohypophyseal peptides and psychopathology Jean Jacques Legros’ and Marc Ansseau2 I

’

Neuroendocrine Unit, Endocrine Service, and Psychiatric Unit, University of Liege, 8-4000 Liege, Belgium

Introduction Neurohypophyseal function interests the biological psychiatrist for three major reasons: (1) The magnocellular hypothalamo-neurohypophyseal system is influenced directly, without peptide intervention, by various cerebral neurotransmitters. Thus, release into the peripheral bloodstream of neurohypophyseal peptides, i.e., oxytocin (OT), vasopressin (AVP) and the inactive portion of their precursors, the neurophysins, allows a direct approach to the estimation of the degree of central activation or inhibition. (2) Outside the magnocellular system, there are several parvocellular cerebral zones capable of synthesizing the same peptides, with biological actions apparently entirely independent of their peripheral effects. (3) In certain cerebral areas, there are specific receptors for AVP and OT which could be responsible for the behavioral effects of these peptides (see Goudsmit et al., this volume). In the present paper, we will first summarize the effects of exogenous AVP and OT on certain human behavioral and cognitive characteristics. After that, we will review the clinical aspects of modifications of neurohypophyseal function in various neuropsychiatric disorders. Influence of exogenous AVP and OT on human behavior Following the pioneering work of David de Wied (1965) in the rat, many studies have been devoted to

the effects of AVP, OT and their analogs or antagonists on animal behavior. Our studies in normal middle-aged men (Legros et al., 1978) and in four patients suffering from posttraumatic amnesia (Oliveros et al., 1978) confirmed these effects in humans and initiated many clinical studies regarding the stimulatory influence of AVP or its structural analogs on various cognitive parameters (see review in Legros and Timsit-Berthier, 1988). In general, this stimulatory action has been confirmed in the absence of major psychological or neuronal deterioration and in the presence of “average” baseline memory performance, leaving room for a significant improvement in psychometric test scores. This action on memory could be due to better focusing of attention, as has recently been shown for normal middle-aged men (Jennings et al., 1986). Unexpected hypomanic episodes, consistent with the stimulatory effect of this peptide, have also been observed in several cases (M. Timsit-Berthier, personal communication). Furthermore, the stimulatory influence of AVP has been confirmed in studies using the paradigm of slow cerebral evoked potentials, and more specifically the Contingent Negative Variation (CNV). AVP infusion prevented the spontaneous diminution of CNV amplitude during the testing session (Timsit-Berthier et al., 1982). The influence of OT was studied several years after that of AVP. The effect of OT on maternal behavior in the rat (Pederson and Prange, 1979)and its inhibitory action on cognitive parameters, the opposite of that of AVP (Bohus et al., 1978), were

456

already well demonstrated. In humans, the first studies of Ferrier et al. (1980) confirmed this inhibitory action, also found to a modest but significant extent in certain psychometric tests in normal men (Geenen et al., 1988). The same study showed that OT infusion induced a diminution in CNV amplitude and an increase in the post-imperative positivity, which persisted 1week after the day of infusion (Geenen et al., 1988). This relatively longterm action was the basis of various attempts at treatment of obsessive compulsive disorder by OT (Ansseau et al., 1987; Charles et al., 1989). Neurohypophyseal function in psychiatric disorders Anorexia nervosa Anorexia nervosa is probably the disorder in which psychological, neurological and endocrine factors are most interlinked. Disturbances of water regulation are frequently found in these patients: delayed elimination of a water load is recognized by the clinician. This phenomenon appears secondary to a diminution of renal filtration due to hypovolemia caused by the malnutrition of these patients (Russell and Bruce, 1966). Some patients, however, present a picture of polyuria/polydipsia suggestive of diabetes insipidus. This is most commonly due to psychogenic polydipsia, which reflects the desire of these patients to consume a minimum of calories and to battle their hunger by consuming up to 10 1 of water daily! More recent work by Gold and Robertson’s group (Gold et al., 1983; Demitrack and Gold, 1988) has shown that there may be a central dysregulation of AVP secretion, since the normal positive correlation between plasma osmolality and AVP concentration was not observed in the anorectic. This anomaly might also be attributed to a disturbance in effective intravascular volume due to weight loss. However, in the same study the authors showed the same patients, having normalized their weight but conserving their psychopathology, showed the same absence of correlation and thus the same disturbance. By contrast, in patients cured psychiatrically and having returned to normal weight, this significant

correlation returned, reflecting the fact that the equilibrium between the hypothalamo-neurohypophyseal system and the state of hydration of the organism had been re-established. Thus, this study suggests a link between dysfunction of AVP secretion and a psychiatric disturbance in these cases. However, to our knowledge, this has not yet been confirmed in the literature. The concentration of OT in the cerebrospinal fluid (CSF) is reduced in anorectics as compared with controls, while the opposite is found in bulimia. The psychopathological consequences of these anomalies are, however, unknown (Demitrack et al., 1992). Bulimia Separate from hormonal causes of hypoglycemia (particularly hyperinsulinemia), which may explain abnormal hunger sensations, there is “bulimia nervosa”, a true compulsive behavior sometimes associated with stress. Studies by Gold’s group (Demitrack et al., 1991) showed alterations of AVP secretion similar to those observed in anorexia nervosa (which one must remember is often associated with bulimia), associated, however, with an increase in CSF OT which contrasts to the findings in anorexia nervosa. TABLE I Mean k S.D. hNpI (vasopressin-neurophysin) plasma levels (ng/ml) at two times of the day in controls and depressed patients Controls (n = 16)

Patients (n = 26)

P

Day 1 8 a.m. 8p.m.

0.34 i 0.06 0.35 k 0.13

0.20 f 0.11 0.19 k 0.11

< 0.05 < 0.05

Day 2 8 a.m. 8 p.m.

0.33 0.20 0.30 k 0.15

Day 3 8 a.m. 8 p.m.

0.31 0.15 0.28 f 0.11

0.20 0.19

0.10

<

0.05

Day 4 8 a.m. 8 p.m.

0.35 f 0.15 0.29 f 0.12

0.18 f 0.10 0.19 f 0.10

< <

0.05 0.05

*

*

0.24 0.19

+ 0.12

<

0.05

* 0.10

< 0.05

* 0.10

< 0.05

After Laruelle et al. (1990), with permission.

451

These findings could represent an effect of obesity or of associated metabolic abnormalities since we have previously shown an increase in OT-neurophysin (hNpII) in obese patients (Legros and Franchimont, 1972)andin alcoholics (Legros et al., 1983a). Here again, the causes and consequences of this hypersecretion of OT remain unknown.

patients than in a group of age-matched controls (Table I; Laruelle et al., 1990). Moreover, in a recent study comparing 12 male depressed patients to 14 normal males, we not only confirmed a lower basal hNpI level but also a reduced response to apomorphine (unpublished results) (Fig. 1). Studies performed on the CSF confirmed this diminution of AVP levels in depressed patients, although no correlation existed between the intensity of the depressive symptomatology and the reduction in AVP levels (Gjerris et al., 1985). We have also been able to confirm a reduction in the CSF concentration of hNpI in a group of eight patients with unipolar depression (aged 37 - 50) as compared with a group of 12 patients without neu-

Major depression Infusion of hypertonic saline is accompanied by AVP into the blood: this secretion is diminished in depressed patients as compared with controls, although the basal peptide levels are similar (Gold et al., 1981). We have recently been able to show that the basal level of AVP-neurophysin (hNpI) was significantly lower in a group of major depressive 25 20

APO ( 0 5 m g s c )

p-+,

1

-

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,

0 - 4 NORMAL .

*

MtSD p
**

-

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\ 7

MALES ( n = l l l

c . . DEPRESSED (n=121

15-

< Gl

C

10-

(3

5 -

-20

0

I

I

I

+20

+LO

+60

,

I

+120

T

25-

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--,

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\

~ 1 5 C

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I

I

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+60 +120 TIME ( M i n u t e s ) Fig. 1. Mean k S.D.plasma level of growth hormone (GH) and hNpI (vasopressin-neurophysin) before ( - 20,O) and after ( + 20, + 40, + 6 0 , + 120) subcutaneous injection of 0.5 mg apomorphine in 14 normal male volunteers and 12 endogenously depressed patient9 (RDC criteria) (unpublised data). -20

0

+20

+LO

458

ropsychiatric disorders (aged 29 - 61) (Linkowski et al., 1984) (Table 11).

Mania In contrast to observations in depression, the first studies of Gold (Gold et al., ‘1981) showed hyperreactivity of AVP secretion into the blood in manic patients. We also found an elevation in the concentration of neurophysins in the CSF in patients with bipolar affective disorder as compared with patients with unipolar depression and controls (Legros et al. 1983b) (Table 11). Later, we were able to test a patient during a major depressive episode and subsequently during his first manic episode, both basally and during a test of neurohypophyseal activation by apomorphine. The basal plasma level of hNpI as well as the hNpI response to apomorphine were

TABLE I1 CSF hNp1 (vasopressin-neurophysin) and hNpI1 (oxytocinneurophysin) values (ng/ml, mean + S.D.) in patients and controls. Significantly lower hNpI values were observed in unipolar when compared to controls (P < 0.001); significantly higher hNpI ( P < 0.001) and hNpII (P < 0.005) values were found in bipolar compared to controls Unipolar depression (n = 8)

Bipolar depression (n =8)

Controls (n = 12) ~

hNpI (ng/ml) hNpII (ng/ml)

0.19 k 0.07 0.75 k 0.43 3.7 L 0.53 7.2 k 1.5

0.34 k 0.19 3.05 + 1.4

After Linkowski et al. (1984), with permission.

TABLE 111 Basal plasma levels of hNpI (vasopressin-neurophysin) in five manic patients compared to controls (mean k S.D.)

hNpI (ng/ml)

Controls (n = 120)

Manic (n = 5)

0.44 c 0.05

0.76 k 0.15

~

After Legros and Ansseau (1989), with permission.

P < 0.02

TABLE IV CSF hNpI (vasopressin-neurophysin) and hNpII (oxytocinneurophysin) (mean k S.D.) values in schizophrenic patients as compared with controls. Significantly lower hNpI values were observed in schizophrenics when compared to controls ( P < 0.05) and higher hNpII in schizophrenics compared to controls ( P < 0.05)

hNpI (ng/ml) hNpII (ng/ml)

Schizophrenia (n = 12)

Controls (n = 12)

0.23 c 0.11 4.5 k 1.12

0.34 k 0.19 3.05 1.4

After Linkowski et al. (1984), with permission.

much higher when this patient was in the manic phase (Legros and Ansseau, 1986). This increase in basal AVP-neurophysin was also noted in a larger study of 50 psychiatric patients including five manic patients (Legros and Ansseau, 1989) (Table 111).

Schizophrenia There is no consensus concerning AVP secretion in schizophrenia. Van Kammen et al. (1981) found reduced levels in the CSF while Beckmann et al. (1985) found normal levels; we ourselves found reduced levels of hNpI (Linkowski et al., 1984) in agreement with the former authors. By contrast, other studies have shown that psychotic schizophrenic patients excrete less urine following water loading than controls, which could be secondary to AVP hypersecretion (Raskind et al., 1987; Goldman et al., 1988; Emsley et al., 1989). Oxytocin concentrations in the CSF have been studied by Beckmann et al. (1985), who showed elevated levels in schizophrenic patients, regardless of neuroleptic treatment, as compared with controls. The previous year, we had also shown an elevation in the levels of hNpII in the CSF as compared with a control group (Table IV; Linkowski et al., 1984). We recently confirmed the CSF findings at the peripheral level. Nine schizophrenic males (age 28.4 k 3.5 years) showed a definite elevation in plasma hNpII as compared with 14 age- (24.3 f 0.9

459

years) and sex-matched volunteers: 2.8 f 0.7 ng/ml and 1.1 f 0.2 ng/ml, respectively. By contrast, apomorphine stimulation, which significantly elevated both types of neurophysins in the controls, was ineffective in the patient group (Legros et al., 1990). Thus, in contrast to the results regarding AVP, there seems to be a consensus regarding hypertonicity of the OT system in schizophrenic patients. Conclusions and perspectives

The review of the literature clearly outlines the presence of central AVP and OT pathways. The presence of specific receptors for AVP and OT in many cerebral areas involved in the regulation of metabolic or behavioral homeostasis tends to confirm that these neuropeptides possess important properties for the survival of the individual and the species. Furthermore, various neuropsychiatric disorders are accompanied by modification in the central or peripheral secretion of these two neuropeptide systems as summarized in Table V. It is possible that this represents merely one of the numerous neuroendocrine consequences of the biochemical perturbations underlying psychiatric illness. According to this hypothesis, the study of neurohypophyseal function is interesting, on the one hand, as a marker of these anomalies (a “window to the brain”) and, on the other hand, to understand certain anomalies of electrolyte and water balance observed in our patients. TABLE V Summary of the general modifications of vasopressinergic and oxytocinergic functions in depression, mania and schizophrenia as obtained through AVP, hNpI, OT and hNpII assay AVP

OT

Depression

\\

=

or4

Mania

f

=

orf

Schizophrenia

=

or\

f

It is also possible that the perturbations observed peripherally may be an indirect reflection of certain states of central hyper- or hyposecretion of neuropeptides which might, then, be closely linked to the genesis of the behavioral abnormality. It is, in fact, interesting to note that the anomalies of endogenous function described here are generally in agreement with our knowledge of the actions of the exogenous peptides on behavior, i.e., global stimulant effect of AVP and global inhibitory effect of OT. We are currently developing a conceptual frame work which tries to integrate the various changes in AVP and OT levels observed in several psychopathological conditions. According to this model, AVP and OT could independently act on two different psychological dimensions. Vasopressin levels could relate to ageneral concept of stimulation, particularly of mood and cognition, and OT levels to a general concept of dissociation, with delusions, hallucinations, incoherence or loosening of associations as particular symptoms. We are currently testing this model in an ongoing study which assesses the relationships between on the one hand AVP and OT functions and on the other hand stimulation and dissociation as two independent dimensions evaluated by specific rating instruments in a large sample of various psychopathological conditions. The study of those perturbations could form part of an avenue of research devoted to the possibility of modulating the endogenous release, perhaps even of blocking the central action of these neuropeptides by the use of peptide antagonists or by intervening at the level of biodegradation using enzyme antagonists. References Ansseau, M., Legros, J.J., Mormont, C . , Cerfontaine, J.L., Papart, P., Geenen, V., Adam, F. and Franck, G. (1987) Intranasal oxytocin in obsessive-compulsive disorder. Psychoneuroendocrinology, 12: 231 - 236. Beckmann, M., Lang, R.E. and Gattaz, W.F. (1985) Vasopressin-oxytocin in cerebrospinal fluid of schizophrenic patients and normal controls. Psychoneuroendocrinology, 10: 187- 191. Bohus, B., Kovacs, G.L. and De Wied, D. (1978) Oxytocin,

460

vasopressin and memory: opposite effects on consolidation. Brain Res., 157: 414-417. Charles, G., Guillaume, R., Schittecatte, M., Pholien, P., Van Wettere, J.P. and Wilmotte, J . (1989) L’ocytocine dans le traitement du trouble obsessionnel: un rapport nCgatif a propos de deux cas. Psychiat. Psychobiol., 4: 111 - 115. Demitrack, M.A. and Gold, P.W. (1988) Oxytocin: neurobiologic considerations and their implications for affective illness. Prog. Neuropsychopharmacol., 12: S23 - S51. Demitrack, M.A., Lesem, M.D., Listwak, S.J., Brandt, H.A., Jimerson, D.C. and Gold, P.W. (1992) Cerebrospinal fluid oxytocin in anorexia nervosa and boulimia nervosa: clinical and pathophysiological considerations. Am. J. Psychiatry, in press. De Wied, D. (1965) The influence of the posterior and intermediate lobe of the pituitary and pituitary peptides on the maintenance of a conditioned avoidance response. Znt. J. Neuropharmacol., 4: 157 - 167. Emsley, R., Potgieter, A., Takjaard, F., Joubert, G. and Gledhill, R. (1989) Water excretion and plasma vasopressin in psychiatric disorders. Am. J. Psychiatry, 146: 250 - 253. Ferrier, B.M., Kenett, D.J. andDevlin, M.C. (198O)Influenceof oxytocin on human memory processes. Life Sci., 27: 2311-2317. Geenen, V., Adam, F., Baro, V., Mantanus, H., Ansseau, M., Timsit-Berthier, M. and Legros, J.J. (1988) Inhibitory influence of oxytocin infusion on contingent negative variation and some memory tasks in normal men. Psychoneuroendocrinology, 13: 367 - 375. Gjerris, A., Hummer, M. , Vendsborg, P., Christiensen, N. J . and Rafaelson, D. J. (1985) Cerebrospinal fluid vasopressin changes in depression. Br. J. Psychiatry, 147: 696 - 701. Gold, P.W., Goodwin, F.K., Post, R.M. and Robertson, G.L. (1981) Vasopressin function in depression and manic. Psychopharmacol. Bull., 17: 7- 9. Gold, P.W., Kaye, W., Robertson, G.L. and Ebert, M. (1983) Abnormalities in plasma and cerebrospinal fluid arginine vasopressin in patients with anorexia nervosa. N . Engl. J. Med., 308: 1117-1123. Goldman, M.B., Luchins, D. J. and Robertson, G.L. (1988) Mechanism of altered water metabolism in psychotic patients with polydipsia and hyponatremia. N . Engl. J. Med., 318: 397 - 403. Jennings, J.R., Nebes, R.D. and Reynolds, C.F. (1986) 111 Vasopressin peptide (DDAVP) may narrow the focus of attention in normal elderly. Psychiat. Rex, 17: 31 - 39. Laruelle, M., Seghers, A., Goffinet, S., Bouchez, S. and Legros, J.J. (1990) Plasmatic vasopressin neurophysin in depression: basic levels and relations with HPA axis. Biol. Psychiatry, 27: 1249- 1263. Legros, J.J. and Ansseau, M. (1986) Vasopressin-neurophysin and bipolar depression: a case report. Biol. Psychiatry, 21: 1212- 1216. Legros, J.J. and Ansseau, M. (1989) Increased basal plasma

vasopressin-neurophysin in mania. Horm. Res., 3 1: 81 - 84. Legros, J.J. and Franchimont, P. (1972) Human neurophysin blood level under normal, experimental and pathological conditions. Clin. Endocrinol., 1: 99- 113. Legros, J.J. and Timsit-Berthier, M. (1988) Vasopressin and vasopressin analogues for treatment of memory disorders in clinical practice. Prog. Neuropsychopharmacol. Biol. Psychiatry, 12: S71- S86. Legros, J.J., Gilot, P., Seron, X., Claessens, J., Adam, A., Moeglen, J.M., Audibert, A. and Berchier P. (1978) Influence of vasopressin on learning and memory. Lancet, i: 41 - 42. Legros, J.J., DeConinck, I., Willems, D., Roth, B., Pelc, O., Brauman, J. and Verbanck, M. (1983a) Increase of neurophysin I1 serum levels in chronic alcoholic patients: relationship with alcohol consumption and alcoholism blood markers during withdrawal therapy. J. Clin. Endocrinol. Metab., 56: 871 - 875. Legros, J.J., Geenen, V., Linkowski, P. and Mendlewicz, J. (1983b) Increased neurophysins I and I1 cerebrospinal fluid concentration from bipolar versus unipolar depressed patients. Neuroendocrinol. Lett., 5: 201 - 205. Legros, J. J., Ansseau, M., Gazotti, C., Carvelli, T., Von Frenckell, R. and Timsit-Berthier, M. (1990) Neurophysins as markers in neuropsychiatric disease: increased oxytocinneurophysin basal level but decreased sensitivity to apomorphine in schizophrenics. Neuroendocrinol. Lett., 12: 287. Linkowski, P., Geenen, V., Kerkhofs, M., Mendlewicz, J. and Legros, J. J. (1984) Cerebrospinal fluid neurophysins in affective illness and in schizophrenia. Eur. Arch. Psychiat. Neurol. Sci., 234: 162 - 165. Oliveros, J.C., Jandali, M.K., Timsit-Berthier, M., Remy, R., Benghezal, A , , Audibert, A. and Moeglen, J.M. (1978) Vasopressin in amnesia. Lancet, i: 42. Pederson, C.A. and Prange, Jr., A.J. (1979) Induction of maternal behavior in virgin rats after intracerebroventricular administration of oxytocin. Proc. Natl. Acad. Sci. U.S.A., 76 (12): 6661 -6665. Raskind, M.A., Courtney, N. and Murberg, M.M. (1987) Antipsychotic drugs and plasma vasopressin in normal and acute schizophrenic patients. Biol. Psychiatry, 22: 453 - 462. Russel, G.F. and Bruce, J.T. (1966) Impaired water diuresis in patients with anorexia nervosa. Am. J. Med., 40: 38 - 48. Timsit-Berthier, M., Mantanus, H., Devos, J.E. and Spiegel, R. (1982) Action of lysin vasopressin on human electrophysiological activity: night-sleep pattern, auditory evoked potential, contingent negative variations. NeuropsychobiolOgy, 8: 248 - 258. Van Kammen, D.P., Waters, R.N. and Gold, P . (1981) Spinal fluid vasopressin, angiotensin I and 11, beta-endorphin and opioid activity in schizophrenic: a preliminary evaluation. In: C. Penis, G. Strume and B. Jansson (Eds.), Biological Psychiatry, Elsevier North Holland, Amsterdam, pp. 339- 344.

46 1

Discussion D.F. Swaab: You mentioned a difference in oxytocin (OXT) levels in cerebrospinal fluid (CSF) obtained either from ventricular or lumbar punctures. Can you tell us how exactly these samples were obtained (from the same patient at the same moment?). And how d o you explain these differences? J.J. Legros: The data were published previously (Born et al., 1982). CSF samples were obtained from the same patients at the same time in the course of a monitoring of CSF pressure (Neurosurgery Service, University of Liege). The difference observed could be due to the richness of OXT fibers in the medulla. C.B. Saper: I am interested in your observation that levels of neurophysins were higher in lumbar than in ventricular fluid. We recently reported that in the rat more than half the neurons in the paraventricular nucleus that project to the spinal cord stain with antisera against either OXT or AVP, after colchicine treatment (Cechetto and Saper, 1988). Do you think that OXT and AVP in lumbar CSF may reflect hypothalamic control of spinal sympathetic preganglionic neurons? If so, are there any data relating CSF OXT and A W to serum norepinephrine or epinephrine for example? J.J. Legros: This hypothesis is fully consistent with our data. To my knowledge, there is no study which correlates CSF oxytocin and vasopressin and serum catecholamines in the human. H.P.H. Kremer: Were you able to obtain sequential observation

(CSF samples and apomorphine testing) of your patients during and between the episodes of illness? J.J. Legros: No, wedit not obtainsequential CSFsamplingin the same psychiatric patients because it has been rejected by our Ethical Committee. As far as apomorphine testing is concerned, our initial observation of a decreased response during the depressic phase and an increased response during the manic phase was affirmed in the same individual at a 6 months interval (Legros and Ansseau, 1986). D. De Wied: What could be the meaning of the increased OXT levels in schizophrenia? J.J. Legros: As discussed in the presentation, the increased OXT levels could represent an activation of a dopaminergic hypothalamic drive. We also hypothesized that OXT could, by itself, contribute to the behavioral disturbances of schizophrenia.

References Born, J., Geenen, V. andLegros, J.J. (1982)NeurophysinIl- but not neurophysin I-concentrations are higher in lumbar than in ventricular cerebrospinal fluid in neurological patients. Neuroendocrinol. Lett., 4: 31 - 35. Cechetto, D.F. and Saper, C.B. (1988) Neurochemical organization of the hypothalamic projection to the spinal cord. J. Cornp. Neurol., 272: 579 -604. Legros, J. J. and Ansseau, M. (1986) Vasopressin-neurophysin and bipolar depression: a case report. Biol. Psychiatry, 21: 1212- 1216.

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463

Subject Index A4/&protein in amorphous plaques, 258 abnormalities in temperature control, 334 abolition of slow wave sleep related GH release, 25 acanthosis nigricans, 335 accessory neurosecretory nucleus, 6, 12, 237 accessory supraoptic cells, 15 acetyl-L-carnitine, 377 acetylated endorphins, 435 acetylation, 435 acetylcholine esterase, 61 acidophilic granules, 11 acquired hypothalamic diabetes insipidus causes, 284 acquired immunodeficiency syndrome (AIDS), 212, 254 acromegalic somatotroph cells, 26 ACTH, 33, 34, 376 ACTH(4-9), 376 ACTH-secreting microadenoma, 398 activation of the vasopressin cells in senescence, 241 activity rhythms, 143 AD patients, 156 adaptation to environmental challenges, 447 r to repeated stress, 368 adaptive behavior, 112 Addison’s disease, 289 adenosine, 12 adenosine A,, 60 adinazolam, 394 adipsia (hypodipsia), 37 adrenal, 33, 123 androgen production, 335 in premature aging, 382 responses to stress, 22 steroid hypersecretion, 373 steroid receptor level in hippocampal cortisol, 371 steroids, 365 steroids and the dentate gyms, 375 adrenal medulla, 30 adrenalectomy, 406 adrenarche, 30 adrenergic a-1 activity, 191 a-adrenergic blockade with thymoxamine, 20 0-adrenergic hormones, 295 @,-adrenergic receptor, 57 adrenergic receptor family, 57

adrenocorticotropic hormone (ACTH), 76, 385 affective disorders, 110, 111, 394, 400 age-related changes, 83 in kidney function, 244 in rodent HNS function, 240 in the sexually dimorphic nucleus, 207 age-related decrease in vasopressin secretion, 238 age-related deficits in HPA function, 367 age-related effects on binding sites, 60 age-related loss in the number and affinity of renal binding sites for vasopressin, 241 age-related memory decline, 244 age-related neuronal degeneration, 376 aged animals, 241 agenesis of the corpus callosum, 37 aggressive behavior, 344, 347, 354, 355 aggressivity and sexuality, 347 aging, 9, 15, 16, 21, 45, 83, 87, 151, 221, 251, 276, 366, 404 aging and dementia (AD), 110 agonal state, 85, 92, 187 agonistic interactions, 348 agony, 60 Ahlstrom’s syndrome, 339 AIDS, 213, 255 akinetic mutism, 37 alcoholism, 399, 457 aldehyde fuchsin, 15, 251 alertness, 36 allometric analysis of the volume of the hypothalamus, 135 allometric scaling, 139 allometry, 139 alprazolam, 394 Alz-50 staining, 11, 253, 261 globose tangles, 255 of the NTL, 258, 261 Alzheimer’s disease (AD), 8, 11, 12, 15, 16, 83, 86, 87, 112, 116, 151, 156, 180, 241, 253, 254, 258, 261, 263, 266, 374, 382, 404, 406 Alzheimer pathology, 11, 265 Alzheimer-related pathology, 258 Alzheimer-related cytoskeletal pathology, 11 ambulatory activity monitor, 162 amenorrhea, 35, 353 amnesia, 455 AMPA receptor family, 57

464 amplitude, 156 amygdala, 62, 256, 370, 386 lesions, 347 amyloid, 253 analbuminemic rat, 279 androgen insensitivity syndrome, 217, 280 levels and aggression, 354 androgen receptor-deficient testicular feminised (tfm) mouse, 329 androgen and estrogen receptors, 345 androgen-sensitive neurones, 326 androgenic anabolic steroids, 351 androgenic gonadal hormones, 353 androgenized females, 351 androgens, 354 - 355 A-4 androstenedione, 331 anergia, 351 aneurysm, 284 Angelman’s syndromes, 340 angiogenesis, 378 angiotensin cascade, 274 angiotensin I converting enzyme, 275 angiotensin 11, 80, 104, 274, 279, 295, 419 angiotensin I1 immunoreactivity in the rat SCN, 116 angiotensin I1 neurons, 116 angiotensin 111, 274 angiotensin-converting enzyme level (ACE), 32 angiotensinogen, 274 anhedonia, 351 animal models of genetic obesity, 337 for osmoregulatory disturbances, 273 for pituitary gonadal disturbances, 321 annual cycle, 140 of vasopressin synthesis in the SCN, 143 annual oscillations, 88 annual periods, 141 annual timing system, 142 annual variations, 142 anorexia (aphagia), 37, 39, 419 anorexia-like states, 257 anorexia nervosa, 21, 22, 261, 351, 399, 456 anosmia, 198, 202, 255 ansa peduncularis, 252 ansa lenticularis, 249 ante-mortem factors, 45, 59 anterior cingulate gyrus, 437 anterior hypothalamus, 110, 119, 344 - 346 anterior hypothalamic area, 104, 107 anterior hypothalamic plexus, 110 anterior periventricular area, 274 anthropoid primates, 136 anti-A4/@-protein, 253

anti-CD4 treatment, 329 anti-T monoclonal antibody, 255 antidepressants, 394, 403 antidiuretic hormone, 295 antiglucocorticoid RU 38486, 388 antiglucocorticosteroid, 397 antimineralocorticoid RU 28318, 388 antipsychotic activity of gamma-type endorphins in schizophrenic patients, 447 of peptides, 439 antisense probe, 194 anxiety disorders, 394, 403 anxiogenic effect of CRH, 394 anxiolytic drugs, 394 aphagia, 37, 39, 419 apomorphine, 457 arachidonic acid, 420 arcuate nucleus, 222, 229, 250, 328, 422, 423, 428 area postrema, 419 area lateralis hypothalami, 61 arginine, 24 arginine-vasopressin (AVP), 45, 54, 89, 154, 158, 295, 309, 386 arginine-vasopressin rnRNA, 47 argyrophilic grains, I1 aspiny neurons, 54 aspirin, 420 astrocytes, 116 ataxia with normal gonadotrophins, 41 ATP, 89 atrial natriuretic peptide, 104, 387, 419 atrophic autoimmune thyroiditis, 284 atrophy of the AVP-containing neurons, 307 atropine, 24 atypical familial polydipsia and polyuria, 297 autoantibodies to hypothalamic vasopressin cells, 287 in serum, 284 autoantigen@) in the hypothalamus, 290 autoimmune diseases, 283 general features, 284 autoimmune hypothalamic diabetes insipidus, 283 autoimmune Addison’s disease, 284 autoimmune mechanism, 30 autonomic function, 134 autonomic nervous system, 334 autopsy, 31 autoradiography, 90, 12i autosomal recessive, 297 autosomal dominant central diabetes insipidus, 296, 300 autosornal dominant pattern of diabetes insipidus, 296 AVP, 158 AVP mRNA, 90 AVP-NP I1 DNA probe, 300

465 AVP-NP I1 gene, 299, 300, 310 AVP-NP I1 gene base change, 304 AVP-NP I1 glycoprotein precursor protein, 304 AVP/OT locus, 309 cosegregates with the DI phenotype, 303 AVP/OT region of human chromosome, 20, 297 AVP secretion in schizophrenia, 458 axospinous synapses in the dentate gyrus, 372 Bardet-Biedel syndrome, 339 basal forebrain, 102, 192, 194 basal ganglia, 52, 53 basal hypothalamus, 39 basal nucleus of Meynert (NBM), 252 basophilic pituitary adenoma, 335 bed ,nucleus of the medial forebrain bundle, 249 of the stria terminalis (BNST), 6, 84, 208, 345, 366, 422 - 423 behavior, 3, 343 behavioral rhythms in humans, 101 in mammals, 139 behavioral effect of centrally administered CRH, 395 belligerence, 35 benzodiazepine, 62, 65, 389, 394 receptors, 57, 60, 252, 258 receptor subtype, 62 biological activities of endorphins, 433, 435 biological aging, 156 biological clock, 119, 144, 151 in infants, 127, 154 in utero, 120 biological rhythms, 8, 88, 137, 144, 151 bipolar depression, 458 bisexuality, 218 biventricular hydrocephalus, 33, 34 blindness, congenital or acquired, 110, 111 blood-brain barrier, 284, 419, 428 blood coagulation, 238 blood flow, 419 blood pressure, 237, 419 body weight disturbances, 23 in insectivores, 135 in primates, 135 body temperature, 36, 39, 151, 153, 155, 156, 222, 344, 419 of elderly and AD patients, 156, 157 and corticosteroid secretion, 426 brain banking, 83 brain hypoxia, 92 brain insulin-like peptide, 429 brain volume in insectivores, 134, 135, 136

in primates, 134, 135, 136 Brattleboro rat, 276, 297, 304, 323 bronchopneumonia, 92 brown fat, 334 bulimia, 399, 456 C-terminal glycopeptide of propressophysin (CPP), 89, 277 CA1, 421 CA3, 421 cachexia, 11,261 Callithricidae, 253 Callithrix jacchus, 253 caloric restriction, 21 carbocyanine dye (DiI), 155 carbohydrate intolerance, 335 carbohydrate metabolism in the liver, 238 cardiac arrhythmias, 37 cardio-respiratory control, 187 Carpenter syndrome, 339 castration, 326, 346 castration-induced obesity, 333, 335 catecholaminergic neurons, 9 caudal infundibular nucleus, 224 caudate nucleus, 54,406 CCK, 60, 227 CD4 and CD8 antigens, 328 CD4 monoclonal antibody, 326 CD4' T-cells, 328 cell adhesion molecule, 198 cell division, 202, 206 cell growth, 190 cell loss, 10 during aging, 240 cell migration, 323 cell number in the suprachiasmatic nucleus, 148 cellular metabolism of brain cells, 190 central nervous system distribution of CRH, 385 central diabetes insipidus, 295 Centre d'Etude Polymorphisme Humain (CEPH), 309 cerebellar and spinocerebellar syndromes, 40 cerebellum, 386 cerebral cortex, 12, 15 cerebral gigantism, 37 cerebral thrombosis, 284 cerebrospinal fluid, 69, 78, 121, 396, 406,436 ceroid lipofuscinoses, 15 chiasmatic gray, 9 chiasmatic hypothalamus, 108 chiasmatic region, 3, 6, 10, 12, 14, 133 of the rhesus monkey, 7 childhood febrile convulsions, 429 chlorpropamide, 276, 307 cholecystokinin, 51, 52, 54,227, 387, 419 cholecystokinin mRNA, 53

466 choleratoxin, 386 cholinergic magnocellular nuclei of the basal forebrain, 12 cholinergic mechanisms modulate somatostatin release, 29 cholinergic muscarinic blockade abolishes the GH responses, 25 cholinergic-somatostatinergic hypothalamic unit, 24 , cholinergic-somatostatinergic system, 30 chromosomal translocations at chromosome 15, 336 chromosome 4, 338 chromosome 7, 338 chromosome 8, 338, 385 chromosome 15, 336 chromosome 20, 297, 309, 314, 317 chronic stress, 405 chronic disease states, 87 chronic respiratory failure, 86 chronically elevated levels of androgens in women, 353 chronobiological variations of thyrotropin-releasing hormone, 167 circadian changes, 88 in peptide content, 116 in neurotensin, 116 circadian cycle, 115 circadian disorders, 111 circadian function, 111 circadian information to the fetus, 121 circadian oscillations, 121 circadian pacemaker, 102, 119, 128,242 circadian pattern, 111 circadian phase, 125 circadianrhythm, 9, 31, 101, 117, 119, 139, 140, 151, 180, 242, 395 in AD, 155. 159, 243 in aging, 159 of body temperature, 153, 158 of cortisol, 20 in early human development, 152, 155 in fetal life, 132, 158 of fetal heart rate variability, 152 of homosexuals, 218 of newborn infants, 152 of rest-activity, 157 in sexual behavior, 211 circadian rhythm-generating system, 151 circadian timing, 101, 110 circadian timing defect, 187 circadian timing system, 101, 112, 119, 129 circadian timing system during aging, 155 circadian variation, 88 in basal TSH, 19 of pituitary hormones, 167 circulating cytokines, 425 circumventricdar organs, 284, 419, 425 cleavage of glycoprotein from NP 11, 298

clock, 137 clomiphene, 337 clonidine, 24 CNS lymphoma, 34 co-release of peptides, 396 cognitive processes, 349 Cohen’s syndrome, 339 cohybridization experiments, 50 cold water stress, 367 cold-responsive neurons, 420 collection of human brain samples, 46 coma, 37 comparative psychology, 343 complement system, 293 complement-fixing ability of autoantibodies, 293 compulsive water drinking, 37 computer-assisted quantitative morphological analyses, 265 computerized tomography (CT),31 confusion, 35 consciousness, 3 constant conditions, 119 control of reproduction in the human, 223 coolness-sensitive neuron, 177 copulatory behavior, 343, 348 corpus callosum, 37 corpus mamillare, 61 cortex, 12, 89 cortical projection of the tuberomamillary nucleus, 12 corticosteroids, 63, 389, 419 corticosteroid receptor down-regulation in aged rats, 382 corticosteroid receptors in the rat SCN, 163 corticosterone, 123, 366 elevation by repeated stress, 373 responses to stress, 367 rhythms, 122 3H-corticosterone uptake with age, 373 in the hippocampus, 369 corticotrophic anterior pituitary cells, 385 corticotropin hormone-releasing hormone (CRH), 197, 338, 424 corticotropin deficiency, 290 corticotropin-releasing factor, 163, 227, 252, 258, 340, 367, 385, 429 corticotropin-releasing hormone, 19, 237, 385, 419 in pathogenesis of affective disorders, 385 in pathogenesis of alcoholism, 385 in pathogenesis of anorexia nervosa, 385 in pathogenesis of Cushing’s disease, 385 in pathogenesis of dementia, 385 cortisol, 34, 35, 398, 401 hypersecretion, 374 response to CRH in AD patients, 406 COXir neurons, 421, 422, 425

467 COXir non-pyramidal neurons, 421 craniopharyngeoma, 38, 284,288 CRH and its negative feedback control, 386 CRH gene, 385 promoter activity, 391 transcription, 403 CRH-induced ACTH release from pituitary adenoma cells, 393 CRH intron, 390 CRH, a mediator of anxiety, 394 CRH promoter sequences, 390 CRH receptors, 393 CSF concentration of hNpI, 457 OXT and AVP, 461 pressure, 461 CSF-somatostatin-like immunoreactivity, 407 CTB proteoglycan, 198 CTS, 102 CT scan, 39 Cushing’s disease, 33, 333, 335, 340, 398 cyclic variations, 141 cyclical light-dark environment, 154 cyclooxygenase (COX), 420, 421, 426 cynomolgus monkeys, 252 cytoarchitectural plan of the human hypothalamus, 134 cytokines, 419, 422, 424,425, 426, 429 cytokines act as neuromodulators, 422, 426 cytoplasmic vasopressin cell antibodies, 285 cytoskeleton, 253 cytotactin, 198 cytotoxic T-cells, 328 daily cycle of sleep and wakefulness, 101 dark pulses, 103 day-night rhythm, 121 DDAVP, 292 de Morsier’s syndrome, 37 death of AVP-producing neurons, 307 degeneration in retina and optic tract in AD, 243 degenerative brain disorders, 9, 10, 16, 111 degenerative effects of glucocorticoids on the hippocampus, 374 degradation of mRNA, 46 dehydration, 241, 248 delta sleep-inducing peptide, 168, 180, 182 dementia, 12, 86, 88, 155- 157, 264, 404, 407 argyrophilic grains, 10, 15 Parkinson’s disease, 264 dendritic spine, 169 dentate gyrus, 405 2-deoxyglucose autoradiography, 120 deoxyglucose analysis, 346 depressive episode, 383,458

depressive illness, 255 depressed mood, 395 depression, 20,39, 143,264, 355, 382, 394,400.405 depression in Parkinson’s disease, 264 DES, 218, 353 desacetylated form of MSH, 338 desamino-D-arginin vasopressin (DDAVP), 34 desmethylimipramine (DMI), 90 destructive effects of glucocorticoids, 371 desynchronization, 122 developing biological clock, 120, 132, 153 of the human infant, 154 developing circadian system in rats, 121 development of the human SCN, 154, 155 of the human SDN, 209 of the hypothalamic biological clock, 119 of the olfactory bulb, 199 developmental disorders, 111 dexamethasone, 367, 392, 396 dexamethasone depression, 406 diabetes, 33 diabetes insipidus, 31, 37, 38, 288, 456 with aging, 241 diabetes mellitus, 22, 30 diabetic autonomic neuropathy, 30 diabetic non-obese mouse, 290 diagonal band of Broca, 173, 176 diencephalic epilepsy, 37 diencephalic pathology, 356 diencephalic syndrome of infancy, 37 diencephalon, 133 dietary restriction, 377 diphtheria toxin A-chain, 329 disorders of autonomic nervous system, 37 disorders of circadian rhythm, 116 disorders of entrainment, 111 disorders of food intake, 37 disorders of psychic function, 37 disorders of sleep and consciousness, 37 disorders of sleep-wake cycle, 111 disorders of sleep-wake schedule, 110 disorders of thermoregulation, 37 in SIDS, 168 disorders of water intake, 37 disruption of migration, 198 disturbance of pacemaker function, 111 of sleep, 111 diurnal circadian variation in plasma levels of corticosterone, 382 diurnal light intensity, 140 diurnal variations in human SCN, 140 DNA polymorphisms, 310

468 dopamine (DA), 20,66,72, 88,225,446 agonists, 20 agonists for treatment of hyperprolactinaemic states, 19 carrier-mediated transport, 445 hyperactivity in mesolimbic brain regions, 444 levels, 88 receptor, 72, 444 receptor-mediated mechanism, 72 dopaminergic activity, 352 dopaminergic hyperactivity in schizophrenia, 443 dopaminergic nerve cells, 9 dopaminergic paranigral nucleus, 12 dopaminergic pathways in circadian TSH changes, 20 dopaminergic receptor, 57 dopaminergic system, 9 dorsal bundle, 389 dorsal hypothalamic area, 107 dorsal lateral geniculate, 103, 106 dorsal nucleus, 168 dorsal vagal area, 9 dorsomedial nucleus, 9, 12, 61, 65, 107, 173,423, dorsomedial and tuberomammillary nuclei, 422 Down’s syndrome, 86, 255 down-regulation of type I1 glucocorticoid receptors, 373 drinking behavior, 115, 116, 344, 419 Drosophila melanogaster, 310 drowsiness, 35, 39 DST non-suppression in Alzheimer’s disease, 407 dwarf mice, 329 dynorphin, 225, 227, 317, 433 dysgenesis or degeneration of the neurons that produce AVP, 297 dysphoria, 351 dyspnea, 86 dystrophic neurites, 253 eating behavior, 116 ectopic cells, 6 ectopic neurosecretory nerve cells, 8 ectopic neurosecretory supraoptic neurons, 9 efferent projections of the SCN, 109, 112 ejaculation, 238 electrolyte balance, 134 emotion, 343, 350, 355, 356 emotional-affective states, 134 emotional behaviors, 344 emotional disturbances, 387 encephalitus, 38 endocrine autoantibodies, 290 endocrine cells of the pituitary, 10 endocrine disturbances, 334 endocrine effects of CRH, 397 endocrine evaluation, 31 endocrine functions of the hypothalamus, 19, 137, 144

endocrine obesity, 335 endogenous circadian rhythms, 152, 153 endogenous clock, 8 endogenous depression, 221, 457 endogenous free-running rhythm, 152 endogenous generation of circadian rhythms, 101 endogenous opioid, 435 endogenous pyrogens in the brain, 425, 426 endogenous rhythmicity, 123, 152 endoplasmic reticulum, 276, 298 0-endorphin @END), 71, 72, 225, 376, 401, 433 deficiency, 433 release, 72 pulsatile release, 77 0-and a-endorphin, 438 endorphin families, 436 endorphin concentrations in hypothalamic tissue, 440 endorphins and schizophrenia, 433 energy metabolism, 134, 256 endorphin deficiency hypothesis of schizophrenia, 438 endorphin excess hypothesis of schizophrenia, 433, 436 enhanced central production of CRH in depression, 401 enhancement of memory processes by vasopressin, 238 ENK mRNA, 52 enkephalin (ENK), 53, 54, 104, 227,433 enkephalin-producing neurons, 103 enkephalins as endogenous opioids, 436 entrained fetal clock, 125, 126, 129, 132 entraining effects, 110, 111, 119 entrainment in human infants, 153 pathways, 102, 111 of circadian rhythms, 102, 153 environmental causes of adrenocortical hyperfunction, 382 environmental light-dark cycle, 153 environmental masking effect, 156 environmental Zeitgeber effects, 162 ependymal lining of the third ventricle, 6, 8, 9 epiphysis, 136 epithalamus, 133 essential hypernatremia, 37 estr(o)us cycle, 143, 332, 334 estradiol, 354 estradiol injections, 351 estrogen feedback, 210, 228 receptor, 202, 227, 229 receptor gene transcripts, 202, 224 replacement therapy, 225 sensitive cells, 326 withdrawal 223 ether stress, 367 euthyroid sick syndrome, 22 evoked potentials, 455

469 excessive endogenous opioid activity, 437 excessively drinking mice (STR/N), 273 excitatory amino acids, 405 neurotoxicity, 374 excitotoxic neurotransmitters, 256 excitotoxin-mediated neuronal damage, 255 exon B of the W-neurophysin gene, 276 experimental obesity, 333 experimentally induced autoimmune encephalomyelitis, 292 expression of CRH mRNA in the PVN, 392 extracellular calcium, 8 1 F8f amide, 317 failure of DNA repair mechanisms in the AVP-NP I1 gene, 307 familial hypothalamic DI, 288 familial neurogenic diabetes insipidus, 276 fatigue, 395 fat mass, 30 febrile response, 419, 420, 422, 425, 426 feedback by glucocorticoids, 392 feeding, 122, 154,256, 344, 419 female sexual behaviour, 322, 325 fetal biological clock, 158 fetal brain, 127 fetal circadian rhythm, 152 fetal clock, 119 fetal development of SON and PVN, 243 fetal human SCN, 155 fetal human hypothalamic or pituitary tissue, 69 fetal hypothalamic tissue, 72 fetal mouse olfactory pit, 197 fetal pituitary, 76 fetal rhythm, 152 fetal rhythmicity in the adrenalectomized mother, 163 fetal SCN, 116, 125, 154, 159 fever, 429 fibronectin, 198 fixation, 85, 91 [3H]-flunitrazepam binding sites, 252 flushing, 230 follicle stimulating hormone, 322 folliculogenesis, 325 food ingestion, 39, 134 food intake, 11, 36, 257, 395, 429 in AD patients, 257 rhythm, 132 food restriction, 377 forgetfulness, 31 fornix, 11, 249, 250, 254 forskolin, 386 frame shift mutation, 277 free-running environment, 128 free-running period, 15 1

free-running rhythms, 110, 122 freezing artifacts, 235 freezing procedures, 91 Frisch hypothesis, 30 frontal cortex, 370 FSH, 221 release, 353 functional neuroanatomy of the CRH neuron, 385 G protein, 170 GABA, 15, 155, 191 GABA-producing SCN neurons, 104 GABA,, 57, 191 GABA,, 191 GABA, receptor, 202 GAD-containing neurons, 106 galanin, 12, 60, 62, 263, 317, 325 binding sites, 65 cDNAs, 227 mRNA, 65 immunoreactive fibers, 252 tuberomammillary neurons, 263 gallocyanin, 251 gamma-amino butyric acid (GABA), 86 gamma-endorphin generating endopeptidase, 434 gamma-type endorphin, 438, 446 hypotheses, 433 inhibits dopamine, 445 gamma-type endorphins and schizophrenia, 438 gastrin releasing peptide, 104 gastroenteropancreatic tumors, 19 geldings, 345 gender, 45, 60, 141, 205 dysphoria, 214 identity, 209, 210, 214 role, 209 genetic obesity, 333, 336 geniculate complex, 105 geniculate neuropeptide Y neurons, 112 geniculohypothalamic projection, 112, 242 geniculohypothalamic tract (GHT), 103, 112, 121 genomic DNA probes, 3 15 germinoma, 284 gestation, 152, 167, 244 GFAP, 235 GH-deficient children, 23 GH-releasing hormones, 23 G H response to a-adrenergic agonism, 24 G H response to L-dopa administration, 24 GHRH, 227 receptors, 24 GHT (NPY) fibers, 103 ginsenoside, 377 glia cells, 155

470 glial fibrillary acidic protein, 224 glioma, 38 of the midbrain-hypothalamic region, 347 gliosis, 15, 224, 255 of hypothalamic nuclei, 296 glucagon secretion from the pancreas, 340 glucocorticoid, 22, 335, 365, 389 binding globulin, 368 cascade hypothesis, 365 exposure in stress, 371 in the generation of neural damage, 365 hypothesis of stress and aging, 365 induced neural damage in the hippocampus, 365 output in anticipation of food, 377 receptors, 338, 369, 388 replacement in adrenalectomized obese animals, 338 glucose-excited neurons, 420 glucose-inhibited neurons, 420 glucose utilization, 102 [3H]-glutamate binding, 252 glutamate receptor, 57 glutamic acid decarboxylase (GAD) levels, 86 glycopeptide, 309 preparation, 278 glycoprotein, 20, 297 glycosylation site, 298 GnRH, 189 associated peptide (GAP), 33 1 deficiency, 331 probe, 225 promoter, 323 pulse generator, 74, 229 GnRH cDNA, 225 GnRH mRNA, 225 Golgi apparatus, 192, 277, 298, 307 Golgi-impregnated neurons, 235 Golgi study on neurons in parietal cortex, 377 gomori-positive neurosecretory material, 274 gonadal atrophy, 40,229 gonadal axis, 350 gonadal hormones, 143, 343, 348, 351 gonadal hormonal activation, 345, 348 gonadal hormonal axis, 349 gonadal steroids, 137, 348 gonadotropic hormone-releasing hormone, 321 gonadotropin deficiency, 222 gonadotropin elevation, 167 in post-menopausal women, 221 releasing hormone (GnRH), 19, 71, 72 secretion, 221, 353 grafting placodal tissue, 328 grafts, li6, 326 granulomata, 32

granulomatous diseases, 284 Grave’s thyrotoxicosis, 289 growth hormone, 19, 23, 334 deficiency, 37, 290 hypersecretion, 19 growth hormone-releasing hormone, 19, 225, 310, 334, 407 GTP binding protein, 57 guanosine triphosphate, 170 hallucinations, 35, 37 Hashimoto’s thyroiditis, 289 heart rate, 152, 153 heat shock protein, 190 hemangiomas, 38 hemodialysis, 437 hemodynamic regulator, 397 hereditary hypothalamic disease, 47 herpes virus thymidine kinase gene, 329 heterologous probes, 49 heterosexual men, 210 high affinity binding techniques, 57 hippocampus, 256, 370, 386, 389, 437 adrenal steroid receptors with aging, 369 control of the hypothalamus, 402 formation, 421 lesions made with kainic acid, 366 hirsutism, 335, 353 histamine, 12, 15, 252, 263, 366 histamine-immunoreactive perikarya, 252 histiocytosis X,284, 292 with DI, 288 history of linking analysis, 310 hNpII in the CSF, 458 HNS activity, 240 function during aging, 238 homeostatic regulatory processes, 112, 134 homologous probes, 48 homosexuality, 210, 343, 344,437 homozygous Brattleboro rat, 307 hormonal activation, 349 hormonal milieu, 112 hormonal rhythm, 119 hormone substitution, 346 hormone-concentrating pyramidal neurons in the hippocampus, 369 hormones, 351, 356 hormones and emotion, 355 hostility, 343 hot flush, 221 hot-spot recombinational area, 278 HPA-hyperdrive hypothesis of anxiety and depression, 395 5-HT, 88 human autoantibodies, 285

47 1 human autosomal dominant central diabetes insipidus, 296 human brain, 422 human circadian system, 129 human CTS, 106 human genome, 31 1 human hereditary central diabetes insipidus, 295 human hippocampus, 373 human hypothalamic diabetes insipidus, 276 human hypothalamic-pituitary system, 73 human neurogenic diabetes insipidus, 276 human newborn circadian rhythms, 154 human post-mortem material, 58, 59 human SCN in early development, 154 humoral factors, 115 humoral signal from the SCN, 116 Huntington chorea basal ganglia, 53 Huntington disease, 10, 12, 52, 254-256, 258, 311 Huntington gene, 258 hydrocephalus, 38 hydrostatic pressure, 198 110-hydroxylase inhibition with metyrapone, 20 hyperactivity of the HPA system, 365, 403 hyperadrenalism, 335 hypercorticism, 163, 398, 399,406 in elderly human subjects, 374 hypercortisolemic primates, 404 hyperfecundity of Huntington, 188 hyperglycemia, 32, 39 hyperinsulinemia, 456 hyperosmolality during fetal and early postnatal life, 248 hyperphagia (bulimia), 37, 39, 333, 347 hypersecretion of OT, 457 hypersomnia, 395 hyperthermia, 37 hyperthyroid patients, 20 hyperuricemia, 335 hypervasopressinemia, 280 hypnotics, 155 hypocorticism, 163 hypoglyc(a)emia, 25, 404, 456 hypogonadal (hpg) mouse, 322 hypogonadal disorder, 198, 221 hypogonadism, 19, 31, 37,40,201,290, 336-337 hypogonadotrophic hypogonadism, 23, 199,255, 323, 337 with ataxia, 41 hypomanic episodes, 455 hypomentia, 336 hyponatremia, 35 hypoparathyroidism, 289 hypophyseal stalk, 267 hypophyseal portal microvascular arrangements, 26 hypophysectomy, 238 hypopituitarisrn, 333

hypo(poikilo)thermia, 39 hypothalamic circadian pacemaker, 153 hypothalamic control of spinal sympathetic preganglionic neurons, 461 hypothalamic diabetes insipidus dominant, recessive, acquired, idiopathic, 283, 289 hypothalamic disease, 31 hypothalamic dopamine, 88 hypothalamic 5-HT, 87 . hypothalamic glucocorticoid receptors, 392 hypothalamic gray, 4, 6, 7, 11, 12, 237 hypothalamic injury, 333 hypothalamic lesions, 39 hypothalamic obesity, 333 hypothalamic POMC neurons, 446 hypothalamic portal system, 229 hypothalamic pulse generator, 20 hypothalamic regulatory peptides, 19 hypothalamic sarcoidosis, 39 hypothalamic stimulation, 346 hypothalamic sulcus, 3 hypothalamic tumors, 39, 111 hypothalamic volume in anthropoid primates, 134 chimpanzees, 134 insectivores, 134 monkeys, 134 prosimians, 134 hypothalamic-hypophyseal extracts, 434 hypothalamic-pituitary control, 19 hypothalamic-pituitary-adrenal axis (HPA axis), 365, 385 hypothalamic-pituitary, ovarian and adrenal dysfunction, 353 hypothalamic-pituitary-thyroid function, 20 hypothalamo-autonomic projection, 6 hypothalamo-hypophyseal-gonadal system, 6, 167, 275, 307, 321,455 hypothalamo-hypophyseal tract, 6 hypothalamo-neurohypophyseal system and aging, 237 hypothalamo-neurohypophyseal system and Alzheimer’s disease, 237 hypothalamo-neurohypophyseal system and development, 237 hypothalamo-pituitary adrenal axis, 167 hypothalamo-pituitary axis, 167 hypothalamo-pituitary gonadal axis, 348, 351 hypothalamo-pituitary ovarian axis, 353 hypothermia, 37 hypothyroidism, 20, 34 hypotonia, 336 hypotonic polyuria and polydipsia, 283 hypoxia, 92, 402, 404 idiopathic diabetes insipidus, 287 idiopathic hypothalamic diabetes insipidus, 288 IGF-1, 23

472 IL-1 receptors, 429 IL-lpir, 424 IL-lpir axons and terminals, 423 imipramine, 90, 403 immune response, 134 immunohistochemistry, 33 of receptors, 65 impaired diurnal rhythms, 334 impairment of cognitive function, 242 a2-interferon, 419 in situ hybridization histochemistry, 45, 62 of receptor mRNAs, 66 in utero environment, 123 in vitro perifusion of pituitary tissue, 69 of hypothalamic tissue, 69 inappropriate VP secretion, 280 indomethacin, 116, 420 inferior olive, 15 influence AVP and OT on human behavior, 455 infundibulum, 6,9, 33,423 neuronal hypertrophy, 222 nucleus, 3, 9, 12,40, 62, 86, 170, 179, 223, 235 nucleus of post-menopausal women, 221, 222, 225 initiation of parturition, 126, 132 insomnia, 155, 395 insulin, 24, 32,429 resistance, 334 insulin-dependent diabetes mellitus, 292 insulin-induced hypoglycaemia, 24 interferon gamma, 387 intergeniculate leaflet (IGL), 103, 106, 112, 119, 242 interleukin, 387 interleukin-1 (IL-1), 1, 2, 6, 387, 419, 426 interleukin-lp (IL-lp), 422, 428 interleukin-6, 419 intermediate nucleus, 7, 14, 15, 137, 206 intermediolateral column of the spinal cord, 6 internal capsule, 249 internal desynchronization, 156 internal synchronization, 151 interstitial nucleus of the anterior hypothalamus, 1, 14, 207 intraneuronal argyrophilic grains, 255 intrapituitary T4 and T, conversion, 21 intraventricular hemorrhage, 38 irregular sleep-wake syndrome, 111 ischemia, 89

jet-lag syndrome, 111 Kallmann fetus, 202 Kallmann syndrome, 37, 189, 198, 202, 255, 323 Kleine-Levin syndrome, 37

Klinefelter syndrome, 210 Kliiver-Barrera stain, 252 L-dopa, 4 labor, 238 lactate level, 86 infusion, 404 lactation, 238 lactic acidosis, 92 lamina terminalis, 108, 109, 137, 173 laminin, 198 lateral area, 62 lateral eminence, 10, 250 lateral geniculate, 103 lateral habenulae, 329 lateral hypothalamic area, 10, 15, 104, 105, 115, 117, 249, 256, 344 - 346,420, 424 lateral mammillary nucleus, 173 lateral and medial tuberal nuclei, 252 lateral preoptic area, 173, 422 lateral septum, 104, 329, 345 lateral sulcus, 326 lateral tuberal hypothalamic region, 249 lateral tuberal nucleus, 9, 10- 12, 15, 172, 188, 249, 251, 253, 261,423 in Huntington’s disease, 261 lateral tuber cinereum, 249 lateralization, 89 for cholineacetyltransferase, 168 for dopamine, 168 for GABA, 168 for LHRH, 168 for noradrenaline, 168 Laurence-Moon-Biedl syndrome, 37, 38 lean body weight of fatllean ratio, 30 learning, 343 lectin, 104 Lemuriformes, 253 lesioning, 347 Lewy bodies, 9, 12, 255, 256, 258, 263 in the NTL, 255 LH, 221 secretion, 353 LH and FSH synthesis, 324 LHRH administration, 351 in cerebrospinal fluid, 179 deficiency, 199 fibers, 252 gene, 192, 194, 199 neuronal migration, 194 LHRH fiber in the hypothalamus of SIDS, 187 LHRH immunoreactive fiber in human infant, 181

473 LHRH immunoreactive neuron in human brain, 178, 190 LHRH-expressing cells in hypogonadal disease (Kallmann syndrome), 199 libido, 31 light therapy, 143 light-dark cycle, 101, 119, 137, 140, 242, 151 light-dark entrainment, 119, 125 light-dark information, 142 light-dark pulses, 117 limbic forebrain, 112 limbic inhibitory control over the HPA system, 388 limbic structures, 345 limbic system, 355 limitations of the autoradiographic technique, 65 linkage analysis, 309, 3 11, 3 15 linkage maps, 312 linkage strategy, 299 lipofuscin, 3, 6, 15, 251, 253 deposits, 8, 9, 372 granules, 4, 6, 10, 11 P-lipotropin, 401 locomotor activity, 116, 395 locomotor circadian behavior, 116 locus coeruleus, 9, 386 LOD score, 301, 312 lordosis, 322 Lorisiformes, 253 loss of ovarian follicles, 221 loss of rhythmicity, 111 loss of V P neurons, 276 lumbar puncture, 32, 34 luminous flux, 102 lupus erythematosus, 292 lutehizing hormone (LH), 76, 210, 321, 322, 348 luteinizing hormone-releasing hormone (LHRH), 167, 182, 189, 201, 331, 345, 349, 350, 352 lymphoma, 34 M2 cholinergic muscarinic antagonists, 30 magnetic resonance imaging (MRI),31, 39 magnocellular basal forebrain nuclei, 12 magnocellular neurons, 237, 276 in the SON, 239 magnocellular neurosecretory complex, 6 magnocellular neurosecretory nuclei, 3, 6 magnocellular nuclei, 9 magnocellular oxytocin neurons, 237 magnocellular subdivisions, 237 magnocellular vasopressin neurons, 237 major depression, 395, 457 male rats, 346 male and female sexual behavior, 348 mammillary bodies, 3, 9, 34, 60, 62, 72, 171, 249, 250, 266 mammillary complex, 3

mammillary nucleus, 171, 263, 267 mammillary region, 3, 10- 12, 133 mammalian circadian timing system, 112 mammalian diencephalon, 133 mammalian preoptic area, 16 mammalian species, 137 mania, 458 manic episode, 458 masculine copulatory behavior, 345 masculinization, 351 masturbation frequency, 355 maternal behavior, 344, 455 maternal biological clock, 121 maternal circadian signals, 158 maternal circadian system, 152 maternal entraining signal@), 123, 125 maternal rhythms, 125 maternal SCN, 122 maternal and fetal rythms, 121 maternal-fetal communication, 121 maternal-infant synchrony, 129 mating behaviour, 332 maturation of the HNS, 244 mechanisms of hypothalamic obesity, 333 medial basal hypothalamus, 230 medial forebrain bundle, 249 medial mammillary nucleus, 173, 175 medial preoptic area, 104, 107, 137, 345, 424 medial preoptic nucleus, 137, 420, 424, 425 medial tuberal nucleus, 173 median eminence, 29, 171, 237, 267, 324, 325, 429 of an hpg mouse, 326 rnediobasohypothalamus (MBH), 70, 72 mediolateral region of the posterior hypothalamus, 170 meiosis I, 310 melanin, 9, 337 melanocyte-stimulating hormone, 337 melatonin, 8, 20, 123, 125, 141, 143, 155, 187 binding to the SCN, 126 melatonin receptors, 125 in the human fetus, 132 rhythmicity, 139 melting curve analysis, 49 membrane receptor, 57 memory, 33, 343, 356 decline and impaired HNS function, 239 disorder, 356 performance, 455 loss, 35, 39 menarche, 30 meningioma, 38 meningitis, 38 menopausal flushes, 222, 230 menopause, 221

474 menstrual cycle, 334 mesolimbic dopamine system, 446 messenger RNA for AVP-NP 11, 298 Met-enkephalin, 84, 433 metabolic activity, 120 rhythm, 120 of male SCN neurons, 138 metabolic changes, 15 metyrapone, 396 midbrain tegmentum, 3 mid-tuberal area, 249 migrating LHRH neuron, 192 migration, 199 of primordial GnRH cells, 322 mineralocorticosteroid receptor, 388 molecular biology of the AVP gene, 297 of hereditary central DI in the rat, 297 of human AVP gene in central DI, 298 monkey RHT, 105 monkey SCN, 104 monkey SCN efferent?,, 106 monkey SCN organization, 106 monoamines, 83, 88 5’ monodeiodinase, 21 monosodium glutamate, 326 moonface, 340 Morris water maze, 368 motivation, 343, 356 mRNA transcripts, 47 a-MSH, 91, 337 Mullerian duct inhibitory substance, 322 multiple sclerosis, 255 muscarinic M1 antagonist, 29 muscarinic cholinergic receptors, 57, 61, 62, 252, 258 muscarinic receptor blockade, 26 muscarinic receptor protein, 57 mutant precursor accumulated in neurons, 306 mutated VP precursor, 276 mutation of chromosome 20 in the AVP/OT region, 304 myasthenia gravis, 289 myelin, 4 myxoedema, 289 N-methyl-maspartate (NMDA) receptors, 252, 258, 405 naloxone, 437 narcolepsy, 37,40 nasal epithelium, 202 nasal septum, 199 nasal stress, 202 NCAM in LHRH cell migration, 198 scaffolding, 199 neonatal intensive care, 154

neoplasm, 347 nephrogenic diabetes insipidus, 273, 288, 295, 296 nervus terminalis, 198, 202 neural cell adhesion molecule (NCAM), 199, 323 neural lobe of the pituitary, 237 neural transplantation, 323 neuritic plaques, 265, 268 neurodegenerative diseases, 91, 249, 256, 258 neuroendocrine deficits, 37 1 neuroendocrine function in humans, 73 neuroendocrine varieties of obesity, 338 neurofibrillary degeneration of Alzheimer’s disease, 263 neurofibrillary tangles, 9, 11, 12, 34, 253, 265, 268 neurogenesis in the mammalian SCN, 148 of the rat SCN, 120 neurogenic diabetes insipidus, 273 neurohypophyseal peptides and psychopathology, 455 neurohypophysis, 3, 6, 9 neurokinin B, 227 neuroleptic, 436, 444, 446 neuroleptic syndrome, 255 neurologic abnormalities, 347 neurologic manifestations of hypothalamic disease, 37 neuromodulator, 190 neuron sensitive to warmth, 177 neuronal activity within the SCN, 155 neuronal clock, 139 neuronal degenerative changes during aging, 372 neuronal enlargement in the subventricular nucleus, 222 neuronal hypertrophy, 222, 229 neuronal loss, IS, 258 neuronal loss in aging, 235 neuronal migration, 189 neuronal oscillators, 139 neuropeptide gene, 90 neuropeptide gene expression in post-mortem tissue, 90 neuropeptide messenger RNA (mRNA), 90 neuropeptide Y (NPY), 45, 97, 115, 227, 317 neuropeptide Y neurons, 86, 112, 242 neuropeptide Y-producing neurons, 103 neuropeptide receptor, 57 neurophysin (NP), 89, 277, 309 in the CSF, 458 levels in men, 238 neurophysin 11, 297 neurophysin gene, 280 neuropil threads, 11, 12 neurosecretory granules, 276, 298, 309, 317 neurosecretory neurones, 237 neurosecretory paraventricular cells, 6 neurosecretory vasopressin cells, 406 neurosensory epithelium, 202 neurotensin (NT), 57, 107, 112, 115, 171

475 neurotransmitter control of CRH neurons, 389 neurotransmitter regulation of hypothalamic CRH release, 389 neurotransmitter receptor, 57, 59 neurotropic virus-based DNA vector, 332 nicotinic receptor, 66 NMDA receptor, 57, 255 content, 258 nocturia, 31 nocturnal elevation in TSH, 21 nocturnal wandering, 155 non-acetylated form of MSH, 337 non-24-h sleep-wake syndrome, 111 noradrenaline (NA), 88, 89, 389 noradrenergic axons projecting from the LC to the PVN, 403 noradrenergic locus coeruleus, 12 normal gonadotrophins with ataxia, 41 normetanephrine (NM) levels, 89 Northern blot analysis, 45, 48 novelty stress, 368 NPY, 51, 106- 108 NTL, 252- 253 in Alzheimer disease, 254 afferent and efferent connections, 252 changes in aging, 253 changes in neurological diseases, 253 in Huntington's disease, 255, 258 of infant brains, 252 neuron number counts, 254 neuronal loss and gliosis, 258 neuropil, 252 of non-primates, 257 in Parkinson's disease, 255 pathology, 258 nuclear estrogen receptors, 190 nuclear grays, 3 nuclear progesterone receptor, 190 nucleus of the solitary tract, 238 nucleus amygdala, 386 nucleus of the diagonal band of Broca, 423 nucleus intermedius, 16 nucleus parabrachialis pigmentosis, 12 nucleus paraventricularis, 386 nucleus periventricularis hypothalami, 61 nucleus posterior hypothalami, 61 number of vasopressin cells of the human SCN, 140 nursery unit, 152 obese patients, 457 obese yellow mouse, 337 obesity, 39, 333, 336, 353 occipital cortex, 406 olfactory bulb, 197, 386 olfactory epithelism, 202

olfactory involvement in LHRH, 198 olfactory nerves, 199 olfactory pit, 200 olfactory pit-derived LHRH, 192 olfactory placode, 189, 192, 194, 328 oligonucleotides as hybridization probes, 46 ontogeny of peptides in the human hypothalamus, 167 Onuf's nucleus, 168 opiate-like properties, 433 opiate receptor, 73, 75, 433, 435 - 437 opioid peptidergic activities, 436 opioids, 433 opioids in the CSF of psychiatric patients, 436 optic chiasm, 6, 7, 72, 103, 107, 137, 250 optic glioma, 38 optic nerve glioma, 111 optic tract, 6, 249, 254 Org 2766, 376 organ-specific autoimmune diseases, 289 organum vasculosum of the lamina terminalis, 207, 419,426 osmolality, 238 osmo-receptive elements, 238 osmotic challenge, 278, 316 osmotic stimulation, 238 of vasopressin secretion, 248 OT in cerebrospinal fluid, 456 OT mRNA, 50 OT-neurophysin, 457 OT-NP I alleles, 301 OT-NP I genomic clone, 300 OT-NP I RFLP allele sizes and frequencies, 301 ovarian failure, 221 ovaries, 123 overeating, 333 overt rhythms, 152, 155 OVLT, 422, 425,428 ovulatory function, 334 OXT fibers in the medulla, 461 OXT levels in schizophrenia, 461 oxytocin, 6, 15, 45, 48, 54, 191, 197, 237, 238, 293, 300, 309, 386,455 oxytocin cells in senescence, 240 oxytocin concentrations in the CSF, 458 oxytocin mRNA, 48 oxytocin neurons, 340 oxytocin neuron numbers, 116 oxytocin staining, 91 oxytocin-neurophysin, 458

'2

P-creatine, 89 pacemaker, 101, 10 104, 112, 158 pacemaker circadian activity, 155 pallidum, 3, 6 pancreatic polypeptide, 429

476 panhypopituitarism, 3 1 panic anxiety, 387 panic attacks, 403 papal system, 263 paracrine factors, 80 paracrine regulation or direct cell-cell communication, 8 1 paraventricular nucleus (PVN), 6, 12, 14, 15,49, 54, 61, 86, 89, 106-108, 116, 139, 142, 168, 171, 206, 237, 247, 274, 295, 296, 306, 309, 333, 340, 366, 406,422-424 Parkinson disease, 9, 12, 16, 19, 52, 53, 254, 255, 258, 263, 266 pars tuberalis, 131 parvocellular neurons, 237, 276 parvocellular vasopressin neurons, 237 passive avoidance behavior, 441 pathological changes in the NTL, 255 pathways in neuroregulation of GH, 25 PCO syndrome, 344, 354, 355 pedigrees of autosomal dominant central Di families, 298 pedophilia, 347 peptidase inhibitors, 71 peptides at presynaptic dopamine terminals, 444 peptides in schizophrenia, 447 pericentrometric loci, 312 perifusion chamber, 70 perinatal development, 151 perinatal hormonal treatment, 348 24-h period by daily environmental time cues, 119 periodic discharge syndrome of Wolff, 37 periodic disease of hypothalamic origin, 37 periodicity of free-running rhythm, 132 periodicity of LH and PEND rhythms, 78 periodicity of pulsatile GnRH release, 74 periventricular nucleus, 9, 12, 170, 173, 422 periventricular organs, 238 pernicious anemia, 289 PG, 425 PGD2 synthetase, 421 PGE2 receptors, 420 PGs produced by astrocytes, 420 pH, 86, 87 phase advance, 111 phase-response curve, 103 phorbol esters, 386 phosphate-activated glutaminase, 86 phosphodiesterase, 275 phospholipase C activation, 386 photic information, 136, 140 photoperiod, 133, 137, 143 photoperiodic information, 142 photoperiodic influences on SCN, 142 photoreceptive elements, 102 photoreceptors, 101, 112 physiological rhythms, 101

in mammals, 139 Pick disease, 255 pigment, 15 pigment-NissI preparations, 10, 16 pigment stains, 251 pigmento-architectonic studies, 15 pineal gland, 8, 125, 136, 139 in patients with SIDS, 187 pineal tumor, 187 pineal volume, 141 pinealectomy, 123 pinealocytes, 141 pinealoma, 284 pirenzepine, 24 and nocturnal GH surge, 26 pituitary, 123, 370 pituitary adenoma, 36 pituitary apoplexy, 38 pituitary cells, 80 pituitary and ovarian dysfunction, 353 pituitary of schizophrenics, 443 pituitary tumors, 38, 39 pituitary-adrenal axis, 348 pituitary-adrenocortical and pituitary gonadal axes, 356 pituitary-gonadal axis, 348 plaques, 265 plasma LH and T levels, 349 plasma osmolality, 237, 248, 274, 456 plasma corticosterone levels, 368 plasma cortisol levels, 374 plasma cortisol surges, 400 plasma growth hormone levels, 457 plasma and urine osmolality, 248 plasticity of the aging brain, 376 platelet aggregation, 238 POA, 331 POA grafts, 324 of hpg female mice, 325 POA implant, 329 poikilothermia, 37 poly A-tail of VP mRNA, 282 polycystic ovary (PCO) syndrome, 333, 352, 353 polydipsia, 39, 298, 456 polyendocrine autoimmunity, 289 poly-lysine tail, 298 polymerase chain reaction (PCR), 90 polymorphism typing, 314 polymorphisms information content (PIC), 31 1, 313 polyuria, 31, 241, 298, 456 POMC gene, 395 POMC synthesizing cells in the brain, 434 pons, 33 portal system, 10, 19, 175, 180, 324, 386 positive estrogen feedback effect, 21 1

477 positron emission tomography, 58 post-menopausal women, 221, 223 post-mortem analysis of brain tissue, 437 post-mortem delay, 45, 46, 53, 86, 89-91, 168, 169 post-mortem delay and binding sites, 169 post-mortem factors, 60, 66 post-mortem hypothalamic tissue, 400, 439, 447 post-mortem material, 58 post-mortem neurotransmitter studies, 265 post-mortem stability of mRNA, 46 post-natal period, 125, 167 post-partum hypopituitarism, 222 post-partum necrosis, 284 post-partum pituitary necrosis, 40 post-translational processing enzyme, 307 posterior hypothalamus, 34, 347 posterior lobe of the pituitary, 237 posterior nucleus, 61, 62, 171, 252 posterior pituitary, 309 posterodorsal medial nucleus of the amygdala, 345 posteromedial nucleus, 9, 12 Prader-Labhart-Willi syndrome, 38 Prader-Willi syndrome, 37, 180, 201, 208, 209, 211, 255, 331, 333, 336 precocious puberty, 31, 37 prefrontal cortex, 386 pregeniculate nucleus, 106 pre-mammillary hypothalamus, 253 premature infants and neonates, 38 premature ovarian failure with Addison’s disease, 284 pre-menopausal and post-menopausal women, 222 pre-menopausal women, 223 pre-mortem anoxia, 45 pre-mortem factors, 66 pre-natal male differentiation of the external genitalia, 202 pre-natal biological rhythm in human, 132 pre-natal maternal entrainment, 123 pre-natal rhythms, 127 premotor cortex, 437 preoptic-anterior region, 137 preoptic area, 136, 137, 252, 322, 346, 420 preoptic LHRH neurons, 332 preoptic recess, 4 preoptic and anterior hypothalamic areas, 426 preproenkephalin, 45, 51 preproenkephalin A, 53 pre-term infants, 152 primary polydipsia, 273 primate hippocampus, 374 primate scale, 15 PRL response to dopamine blockade, 20 proceptivity, 348 processing and secretion of hypothalamic peptides, 71 prodynorphin (PDYN), 309

pro-enkephalin, 436 pro-enkephalin A, 433 pro-enkephalin B, 433 progesterone, 345, 354 progesterone receptor, 202 programmed ceIl death, 209 prolactin(e), 34, 123, 353 prolonged hypercoticism in depression, 383 prolonged stress, 371 proopiomelanocortin (POMC), 227, 337, 433 proopiomelanocortin-derived compounds, 385 prosimiae, 136,253 prostaglandin (PG), 115, 420, 426 prostaglandin E2, 426 prostaglandin synthesis, 115, 116 prostaglandins as mediators in the febrile reaction, 420 prostatic carcinoma, 235 psychiatric illnesses, 408 psychoactive drugs, 444 psychoendocrine functions of the hypothalamus, 356 psychological theories of emotion, 356 psychomotor agitation, 395 psychoneuroendocrinology, 348 psychosocial stressor, 387 psychostimulants, 436 pubarche, 30 puberty, 321 pulmonary edema, 37 pulsatile GnRH release from these human MBHs, 75 pulsatile GnRH secretion, 325 pulsatile pattern of hormone release, 80, 395 pulsatile release of LH, 75 pulsatile and sleep-related patterns of GH, 26 putamen, 54 PVN, 47,240 during fetal development, 248 lesions, 340 neurons, 91 in senescent subjects, 240 pyknosis and desintegration of neurons in NTL, 255 pyramidal neuron loss in the hippocampus, 366 pyramidal neurons are COXir, 421 pyrogenic cytokines, 419 pyrogens in the CNS, 419 radiation therapy, 36 rage behavior, 37 raphe nucleus, 139, 389 rat growth hormone promoter, 329 rat SCN, 103 rate of brain aging, 378 receptor, 61 receptor autoradiography, 58, 60, 62 receptor changes, 91

478 receptor localization, 57 receptor protein, 57 receptor visualization, 65 receptors in human hypothalamus, 60 recessively inherited obesity, 338 recombinants in a pedigree, 312 recombination between genes, 312 reflex ovulators, 332 regulation human CRH, 390 rejection, 326 renal sensitivity to vasopressin, 241 repair mechanisms for DNA breaks, 307 repression of the hCRH gene promoter by glucocorticoids, 392 reproduction, 112, 134, 137, 189, 191, 223 reproductive cycles, 143 reproductive behavior, 190 rest-activity, 151, 152, 155 rest-activity rhythm in demented patients, 162 restoration of circadian function, 116 restriction endonuclease digestion, 3 14 restriction enzyme analysis, 313 restriction fragment length polymorphisms (RFLPs), 300, 309, 313 reticular formation, 112 reticular nucleus of the thalamus, 106 retina, 103, 242 retinal ganglion cells, 103 retinal projections, 8, 115, 117 retino- and/or geniculohypothalamic projections, 116 retino- and geniculohypothalamic pathways, 117 retinohypothalamic pathway, 112, 151 retinohypothalamic projection (RHT), 102 retinohypothalamic tract, 119, 139, 242 retinopathy, 26 retrochiasmatic area, 102, 105, 110 retrochiasmatic nerve cells, 9 retrochiasmatic nucleus, 9, 12 rheostatic regulatory processes, 134 rhesus monkey hippocampus, 373 RHT projection, 105, 108 RHT and GHT input, 10 rhythmicity, 123, 189 rhythms in pre-term infants, 162 riboprobes, 192 role of oxytocin produced by the fetus during labor, 244 rosy locus of Drosophila melagogaster, 279 salinity, 198 sarcoidosis, 32, 284 sarcoma, 38 satiety, 168 scale of the hypothalamus, 134 schizophrenia, 382,433, 440, 458

SCN annual cycle, 142 SCN changes during aging and in AD, 158 SCN during aging and in Alzheimer’s disease, 243 SCN innervations of the paraventricular nucleus, 141 SCN neurons, 155 SCN total cell number, 140, 141 season of death, 148 seasonal affective disorders, 143 seasonal changes in total cell number, 148 seasonal control of reproductive and metabolic phenomena, 139 seasonal light intensity, 140 seasonal rhythm, 140, 148 seasonal timing, 143 seasonal variations, 137, 140, 141, 143, 148 seasonal variations in the human SCN, 140 secretory pulses of GH, 19 self-stimulation and sexual behavior, 346 sella, 33 senescence, 238 septal-preoptic area, 230 septa1 region, 429 septum, 194, 429, 437 septum in fever, 429 sequence analysis of AVP gene, 303 serotonin, 20 serotonin IA, 57, 66 serotonin IB, 57 serotonin IC, 57 serotonin lD, 57 serotonin 2, 57 serotonin 5-HT3 receptor, 57 sex differences in brain morphology, 137, 168, 205 sex differences in neurotransmitters, 328 sex hormones, 190, 209, 353 sex-specific differences in recombination rates, 3 12 sexual arousal, 348, 349 sexual arousal and behavior, 345, 350 sexual behavior, 137, 206, 218, 344, 345, 395 sexual differentiation of the brain, 210, 321, 322 sexual differentiation of the human hypothalamus, 205, 209 sexual differentiation in the human SDN, 207, 208 sexual dimorphism, 138 sexual excitement, 355 sexual impotency, 202 sexual interaction, 351 sexual maturation, 351 sexual motivation, 346, 348, 354 sexual orientation, 205, 210, 343, 348, 354 sexual preference, 347 sexual and aggressive behaviors, 355 sexuality, 343, 344, 347, 349, 356 sexually differentiated functions, 137 sexually dimorphic intermediate nucleus, 12, 422

479 sexually dimorphic nucleus of the preoptic area, 137, 141, 144, 168 Shapiro syndrome, 40 Sheehan disease, 40 sheep brain, 421 shift-work, lq6 shivering, 419 short-term stress, 386 signal which initiates puberty, 30 silver staining coiled bodies containing straight filaments, 255 silver staining neurofibrillary tangles, 256 silver and thioflavin-S stains neurofibrillary tangles, 253 simian virus, 323 single photon emission tomography, 58 sister chromatids, 310 size of the hypothalamus, 134 Sjogren syndrome, 289 sleep, 3, 19, 39, 344, 398, 419 sleep abnormality in infants at risk of SIDS, 183 sleep disturbance, 156 in elderly people, 155 sleep patterns, 395 sleep-rhythm reversal, 37 sleep-wake cycle, 128, 152 sleep-wake disturbances, 155 sleep-wake patterns of children, 129 slow wave sleep, 23 solar cycle of light and dark, 101 somatostatin, 45, 51, 52, 62, 104, 155, 167, 174, 182, 197, 258, 391, 403, 407 somatostatin analogues, 19 somatostatin antibodies, 21 somatostatin binding sites, 60,252 somatostatin-immunoreactive cell bodies, 252 somatostatin receptor, 61 somatostatinergic neurons of the hypothalamus, 407 somnolence, 37, 334 SON, 47,240 SP, 227 spatial learning task, 368 spatial memory capacity during aging, 405 spermatogenesis, 324 sphincter disturbance, 37 spiny projection neurons, 54 splanchnic vasodilator, 397 SRIF binding sites, 170, 173, 175 SRIF immunoreactive neuron, 171 starvation-induced decline in TSH, 21 Stein-Leventhal syndrome, 335 steroid, 32, 35 steroid effects on circadian rhythms, 143 steroid hormone, 190, 351 steroid hormone receptors, 345 steroid molecules, 346

steroid negative feedback, 229 steroid receptors in hippocampus, 366 stress, 45, 122, 344, 388, 394 in aging, 382 stress-induced CRH and AVP gene expression, 388 stress-induced opioid peptide effect, 191 stria terminalis, 325 striatum, 54, 252 structure and organization of the hCRH gene, 390 subarachnoid hemorrhage, 38 subfornical organ, 238, 274, 419 subjective day, 121 subjective night, 121 substance P, 171, 225, 227,230 substance P immunoreactivity, 62 substantia innominata, 422 subthalamic nucleus, 3 sudden infant death syndrome (SIDS), 167, 181, 182 suicide, 395 suprachiasmatic nucleus (SCN), 12, 20, 102, 112, 119, 137-140, 142, 143, 151, 170, 171, 208, 210, 242, 247, 275, 306,332 supraoptic nucleus (SON), 6, 12, 49, 54, 65, 116, 139, 142, 173, 206, 237, 247, 250, 274, 287, 295, 306, 309, 406, 422, 423 suprasellar craniopharyngioma, 40 suprasellar cysts, 284 suprasellar mass, 35 supratrochlearis nucleus, 173 sympathetic preganglionic nucleus, 340 sympathetic system, 429 symptomatic hypothalamic diabetes insipidus, 288 synaptic sprouting, 372 synchronization of the infant’s behavioral and hormonal rhythms, 154 syndrome of inappropriate antidiuretic hormone secretion (SIADH), 33 T lymphocytes, 328 T4to T, conversion, 21 tachykinin, 57, 230 tachykinin gene expression, 227 tachykinin neurons, 229 tangles, 265 tanycyte, 179 Tarsiformes, 253 telomeric loci, 312 temper tantrums, 337 temperature regulation, 69 temporal cortices, 406 temporal isolation, 155, 156 terminalis nerves, 199 testicular feminization, 217 testosterone, 33, 345, 348, 351, 354

480 testosterone levels, 355 testosterone suppletion, 346 thalamic nuclei, 386 thalamus, 61, 133 thelarche, 30 thermal stability, 49 thermoregulation, 134, 177, 222, 424, 429 third ventricle, 34 [3H]-thymidine grain, 202 thyroid hormone economy, 22 thyroid hormone levels, 33 thyroid-parathyroids, 123 thyroid stimulating hormone (TSH), 19 thyrotropin, 19 thyrotropin-releasing hormone, 167, 182 binding site, 170, 176, 177 circadian rhythm, 167 in post-mortem tissues, 177 time of day of birth, 126 time-of-day information, 125 tissue fixation, 45, 47 and in situ hybridization histochemistry, 46 TM galanin neuron, 268 TM neuron, 253 TNFa, 424 transcortin, 368 transgenic animals, 329 transient DI, 284 transmitter input on LHRH mRNA, 191 transsexuality, 214 transsexuals, female-to-male, 355 triazolobenzodiazepines,394 tricyclic antidepresant therapy in depression, 22 TSH release is pulsatile, 19 TSH response to cold, 21 TSH response to TRH, 21 TSH response to stress and illness, 22 TSH rhythms, 20 tuber cinereum, 9, 12, 249, 250 tuber nucleus, 173, 177 tuberal hypothalamus, 20, 253 tuberal nucleus, 60 - 62 tuberal region, 3, 12, 133, 252, 263 tuberomammillary neurons, 251 - 253, 258 tuberomammillary nucleus (TM), 10- 12, 173, 251, 253, 263 tuberomammillary region, 266 tumor necrosis factor (TNF), 387, 419, 426, 429 Tupaiiformes, 253 Turner syndrome, 235 two-point linkage, 315 type I diabetes mellitus, 30 tyrosine hydroxylase, 227, 403 tyrosine hydroxylase probe, 227

unipolar depression, 458 urine osmolality, 241 urine volume, 241 vagal gastric motor neurons, 340 vagus nerve, 337 vas deferens, 238 vasoactive intestinal polypeptide, 158, 387 vasoconstrictor, 237 vasopressin, 6, 48, 138, 197, 208, 237, 273, 277, 283, 293, 317, 329, 385, 393, 419,423, 424, 455 in senescence, 240 - 242 vasopressin cell antibodies, 287,288 vasopressin cell autoantigen, 287 vasopressin cells in Alzheimer disease, 242, 244 in human SCN, 154, 212,243 in suprachiasmatic nucleus, 140 vasopressin excretion, 241 vasopressin gene, 277 vasopressin levels in age-related memory decline, 242 in aging rodents, 240 in Alzheimer disease, 242 in the umbilical cord, 244 vasopressin mRNA, 121 vasopressin neurons in autoimmune hypothalamic diabetes insipidus, 292 in the human SCN, 213 immunoreactive, 144, 148 vasopressin secretion upon osmotic stimulation, 240 vasopressin synthesis and release in Alzheimer disease, 244 vasopressin and neurophysin, 458 in aging humans and rodents, 240 plasma levels, 456 vasopressin and oxytocin cell numbers in premature and mature fetuses, 243 in the fetal period, 243 vasopressin-containing cell group, 142 vasopressin-containing cell line, 293 vasopressin-containing neurons, 140 vasopressin-immunoreactive cells in the PVN of Alzheimer patients, 242 vasopressinergic and oxytocinergic functions in depression, 459 in mania, 459 in schizophrenia, 459 vasopressinergic fibre network, 329 ventral lateral geniculate nucleus, 139 ventral and medial mammillary nuclei, 252 ventral medial nucleus of the hypothalamus, 39 ventricular or lumbar punctures, 461 ventrolateral thalamus, 437

481 ventromedial hypothalamus, 333, 347 ventromedial hypothalamic neuron, 190 ventromedial hypothalamic stimulation, 345 ventromedial nucleus of the hypothalamus, 62, 168, 171, 206, 333, 340, 345, 420, 423, 424 vertebrates, 133 visualization of receptors, 58 volume of the oxytocin cell population, 243 volume of the human SCN, 140, 141 vomeronasal nerves, 199 VP neurons, 276 VP-neurophysin moiety of the ostrich, 278 vulnerability to stress-induced loss of hippocampal neurons, 378 wake-sleep, 424 wakefulness reaction during sleep, 188

warm-responsive neurons, 420 water balance, 39 water intake, 241 wear and tear, 375 of glucocorticoids during adult life, 369 weight loss, 257, 395 weight gain, 395 Wernicke’s disease, 38 xenografts, 328 X-linked inheritance of diabetes insipidus, 296 Z score, 312 Zeitgebers, 140, 142, 154 zona incerta, 3, 173 Zurich study, 264

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